FR3095817A1 - Genetically modified bacteria for improved production of lactate from CO2 - Google Patents

Abstract

The invention relates to a bacterium which naturally oxidizes hydrogen and genetically modified to produce lactate from CO2, said bacterium being genetically modified to overexpress at least one gene encoding a lactate dehydrogenase and to inhibit the expression of an operon. lut and a method of producing lactate from CO2 using such a bacterium.

Description

Genetically modified bacteria for improved production of lactate from CO2

FIELD OF THE INVENTION.

The invention relates to a bacterium which naturally oxidizes hydrogen and is genetically modified to produce lactate from CO ₂ . The invention also relates to a method for producing lactate, or lactic acid, from CO ₂ using such a genetically modified bacterium.

BACKGROUND OF THE INVENTION.

Lactic acid finds applications in many industries. For example, lactic acid is used as a precursor to polylactic acid (PLA) which is a fully biodegradable polymer, used for example in food packaging. Lactic acid can also be used as an additive, as an antioxidant, acidifier or flavor enhancer by the food industry. In cosmetics, lactic acid is generally used as a bacteriostat or as a peeling agent.

Currently, the production on an industrial scale of lactic acid is mainly based on the fermentation of carbohydrates, and in particular of glucose, lactose, sucrose or maltose. If many bacteria can cause the transformation of these sugars into lactic acid, only lactic bacteria can exclusively produce lactic acid, the other bacteria generally producing a mixture of several organic acids.

In addition, the production of lactic acid from fermentable sugars, such as glucose or lactose, raises the question of competition between food and the production of convenience products such as lactic acid.

At the same time, emissions of carbon dioxide (CO ₂ ) into the atmosphere are constantly increasing. Biological systems that fix carbon through natural biochemical metabolic processes, such as algae, have already been used to produce molecules of interest by photosynthetic reactions. However, the production yields remain insufficient and limit the economic interest of these systems.

Similarly, methods using genetically modified bacterial cells have been developed to transform sugars into molecules of interest in heterotrophic fermentation systems. Angermayr et al ., 2012 describes the overexpression of Bacillus subtilis lactate dehydrogenase in the cyanobacterium Synechocystis . WO 2014/205146 and WO 2015/155790 describe methanotrophic bacteria whose energy source comes from their carbonaceous substrate. Such systems have several drawbacks. Indeed, heterotrophic fermentation systems are sensitive to contamination, which considerably impacts production yields. In addition, these heterotrophic systems do not solve the problems of competition with food, since fermentable sugars remain necessary, nor of negative environmental impacts.

There is therefore still a need for microbiological processes to allow the production of molecules, such as lactic acid, in large quantities from CO ₂ as the only carbon source, so as not to compete with food by reducing greenhouse gas emissions.

By working on this problem, the inventors have discovered that it is possible to force bacteria that naturally oxidize hydrogen, and capable of producing organic matter from CO ₂ , into a lactate synthesis pathway, possibly to the detriment other synthetic routes. In particular, the inventors have discovered that it is possible to promote an endogenous lactate synthesis pathway, which is not or only slightly expressed naturally in the bacterium, by acting on the expression of the gene(s) associated with this synthesis pathway . Thus, the inventors have discovered that hydrogen-oxidizing bacteria can be genetically modified so as to overexpress an endogenous lactate dehydrogenase or a heterologous one, and/or so as to repress the expression of genes involved in a pathway for the synthesis of molecules from in competition with the production of lactate, in order to produce lactate from CO ₂ . More particularly, the inventors have discovered that the bacterium Cupriavidus necator (also called Hydrogenomonas eutrophus, Alcaligenes eutropha, Ralstonia eutropha , or Wautersia eutropha) can be genetically modified so as to overexpress an endogenous lactate dehydrogenase, by acting on the promoter and/or the copy number of the gene coding for this particular lactate dehydrogenase, so as to produce lactate from CO ₂ . Alternatively or additionally, the production of lactate can be improved by introducing one or more genes expressing a heterologous lactate dehydrogenase and/or by repressing the expression of genes involved in a pathway for the synthesis of molecules that compete with the production of lactate .

These modifications, although effective, reach limits when it is sought to promote the production of lactate by prolonging the culture over time. The inventors then noted after a few hours of culture that the bacteria re-consumed the lactate produced. Contrary to what is generally expected, the inventors have observed that the deletion of the genes encoding the lactate ferricytochrome C reductases (lldD and lldA) described in the literature as making it possible to avoid the consumption of lactate, in particular in Escherichia coli (Woo et al. 2014 ; Niu et al. 2014; Mazumbar et al. 2013; Wang et al. 2012; Chai et al. 2009) and in Issatachenkia orientalis (Suominen et al. 2012; Millet et al. 2012) does not make it possible to suppress the re-consumption of lactate in bacteria that naturally oxidize hydrogen, and capable of producing organic matter from CO ₂ . In these bacteria, they identified the genes (lutA, lutB and lutC) united in an operon coding for the components of an enzymatic complex using lactate, the inhibition of which makes it possible to reduce substantially or even completely eliminate the re-consumption of lactate. product.

BRIEF DESCRIPTION OF THE INVENTION.

The invention therefore relates to a bacterium which naturally oxidizes hydrogen and which is genetically modified to produce lactate from CO ₂ , said bacterium being genetically modified to overexpress at least one gene coding for a lactate dehydrogenase and to inhibit the expression of an operon called lut, said operon comprising lutA, lutB or lutC genes coding for the components of an enzymatic complex using lactate.

According to the invention, the bacterium can be genetically modified to overexpress an endogenous and/or exogenous lactate dehydrogenase.

A subject of the invention is also the use of a bacterium according to the invention for the production of lactate from CO ₂ , preferably for the production exclusively of L-lactate, or for the production exclusively of D-lactate.

The invention also relates to a process for producing lactate from CO ₂ , comprising the steps of culturing the bacterium with CO ₂ as the sole carbon source, then recovering the lactate.

DESCRIPTION OF FIGURES.

Figure 1 shows a bacterium naturally oxidizing hydrogen according to the invention capable of producing lactate from CO2 and H2.

Figure 2 shows the different metabolic pathways naturally present in Cupriavidus necator, and in particular, glycolysis and the Calvin cycle.

Figure 3 shows the different genetic modifications (overexpression and/or inhibition of gene expression) that can be carried out in Cupriavidus necator to promote the production of L-lactate from CO2.

Figure 4 shows the different genetic modifications (overexpression and/or inhibition of gene expression) that can be carried out in Cupriavidus necator to promote the production of D-lactate from CO2.

The various abbreviations used in the description and figures 2, 3 and 4 are given in Table 4.

Figure 5 represents the production of lactate by the bacterium CN0002 from fructose.

Figure 6 represents the production of lactate by the naturally occurring hydrogen-oxidizing bacterium CN0002 and genetically modified to produce lactate from CO2.

figure 7 represents the comparison of cell growth and lactate consumption in two strains of Cupriavidus necator, CN0004 and CN0009, one of which includes the deletion of the lldD and lldA genes (CN0009).

Figure 8 represents the comparison of cell growth and lactate consumption in four strains of Cupriavidus necator, with or without deletion of the lut1 and lut2 operons.

DETAILED DESCRIPTION OF THE INVENTION.

The subject of the invention is a bacterium which naturally oxidizes hydrogen, genetically modified to produce lactate from CO ₂ , said bacterium being genetically modified to overexpress at least one gene coding for a lactate dehydrogenase

In the context of the invention, a bacterium " naturally oxidizing hydrogen " designates a bacterium capable, without prior genetic manipulation, of using gaseous hydrogen as an electron donor and oxygen as an electron acceptor, and capable of fixing carbon dioxide. These bacteria are also called “ knallgas ” bacteria. Naturally hydrogen-oxidizing bacteria require carbon dioxide as a carbon source and hydrogen as an energy source.

According to the invention, the bacterium which naturally oxidizes hydrogen is preferentially selected from Ralstonia sp, Cupriavidus sp., Hydrogenobacter sp., Rhodococcus sp ., Hydrogenovibrio sp.; Rhodopseudomonas sp. , Rhodobacter sp., Aquifex sp., Corynebacterium sp., Nocardia sp., Rhodospirillum sp., Rhizobium sp., Thiocapsa sp., Pseudomonas sp., Hydrogenomonas sp., Hydrogenobacter sp., Hydrogenophilus sp., Hydrogenautresmus sp ., Helicobacter sp., Xanthobacter sp., Hydrogenophaga sp., Bradyrhizobium sp., Alcaligenes sp., Amycolata sp.; Aquaspirillum sp., Arthrobacter sp., Azospirillum sp., Variovouax sp., Acidovouax sp., Bacillus sp., Calderobacterium sp., Derxia sp., Flavobacterium sp ., Microcyclus sp., Mycobacterium sp., Paracoccus sp., Persephonella sp. ., Renobacter sp., Thermocrinis sp., Wautersia sp., and cyanobacteria such as Anabaena sp.

In particular, the bacterium is selected from Rhodococcus opacus, Rhodococcus jostii, Hydrogenovibrio marinus, Rhodopseudomonas capsulata, Rhodopseudomonas palustris , Rhodobacter sphaeroides , Aquifex pyrophilus , Aquifex aeolicus, Cupriavidus necator, Cupriavidus metallidurans, Corynebacterium autotrophicum, Nocardia autotrophica, Nocardia virodieus opaca, sulfoviridis, Rhodopseudomonas blastica, Rhodopseudomonas sphaeroides, Rhodopseudomonas acidophila, Rhodospirillum rubrum , Rhizobium japonicum, Thiocapsa roseopersicina, Pseudomonas facilis, Pseudomonas flava, Pseudomonas putida, Pseudomonas hydrogenovoua, , Pseudomonas palleronii, Pseudomonas pseudoflava, Pseudomonas saccharophila, Pseudomonas thermophila, Pseudomonas hydrogenothermophila, Hydrogenomonas pantotropha , Hydrogenomonas eutropha, Hydrogenomonas facilis , Hydrogenobacter thermophilus, Hydrogenobacter halophilus, Hydrogenophaga flava, Hydrogenophaga palleronii, Hydrogenophilus thermoluteolus , Hydrogenautremus marinus, Helicobacter pylori, Xanthobacter autotrophicus, Xanthobacter flavus, Hydrogenophaga flava, Hydrogenophaga palleronii, Hydrogenophaga eutrophia, Hydrogenophaga pseudobflava, Roughenophagia japonica eutrophus, Alcaligenes facilis, Alcaligenes hydrogenophilus, Alcaligenes latus, Alcaligenes paradoxus, Alcaligenes ruhletii, Amycolata sp., Aquaspirillum autotrophicum , Arthrobacter strain 11/X, Azospirillum lipoferum, Variovouax paradoxus , Acidovouax facilis , Hydrogen bacillus schlegelii, Bacillus tusciae, Calderobacterium hydrogenophilum gummosa, Flavobacterium autautremophilum, Microcyclus aquaticus, Mycobacterium goudoniae, Paracoccus denitrificans, Persephonella marina, Persephonella guaymasensis, Renobacter vacuolatum, Thermocrinis ruber, Wautersia sp., Anabaena oscillarioides, Anabaena spiroides and Anabaena cylindrica .

The terms " recombinant bacterium " and " genetically modified bacterium " are used interchangeably herein and refer to bacteria which have been genetically modified to express or overexpress endogenous nucleotide sequences, to express heterologous (exogenous) nucleotide sequences, or which have an alteration in the expression of an endogenous gene. By "alteration" is meant that the expression of the gene, or level of an RNA molecule or equivalent RNA molecules encoding one or more polypeptides or polypeptide subunits, or the activity of one or more polypeptides or polypeptide subunits is regulated, such that the expression, level or activity is higher or lower than that observed in the absence of this modification. It is understood that the terms " recombinant bacterium " and " genetically modified bacterium " refer not only to the particular recombinant bacterium, but also to the progeny or potential progeny of such bacterium. Since some changes may occur in subsequent generations, due to mutation or environmental influences, this offspring may not be identical to the parent cell, but is still included within the scope of the term as used herein.

According to the invention, the term " overexpression of a gene " means the fact that said gene is more expressed in the bacterium considered than in a bacterium not genetically modified to overexpress said gene, leading to a production or a greater production of the corresponding protein (and more particularly lactate dehydrogenase) and in particular an increase greater than 20%, more preferably 30%, 40%, 50%, 60%, 70%, 80%, 90%. According to the invention, such an overexpression means the expression of an endogenous lactate dehydrogenase, which is not or only slightly expressed in the non-genetically modified bacterium, or the expression of an exogenous lactate dehydrogenase.

In a particular embodiment, the bacterium is selected from naturally hydrogen-oxidizing bacteria possessing an endogenous lactate dehydrogenase.

Advantageously, the bacterium is selected from Cupriavidus sp . , such as Cupriavidus necator , Hydrogenobacter sp . , such as Hydrogenobacter thermophilus , Rhodococcus sp., such as Rhodococcus opacus , and Pseudomonas sp., such as Pseudomonas hydrogenothermophila .

The terms “ endogenous ” or “ native ” designate a gene which is normally or naturally present in the genome of the bacterium considered. Conversely, the terms “ exogenous ” or “heterologous” as used here with reference to a gene (nucleotide sequence) designate a gene which is not normally or naturally present in the genome of the bacterium considered.

In the case of a bacterium possessing an endogenous lactate dehydrogenase, it is possible to genetically modify said bacterium, so as to allow overexpression of said gene. In particular, it is possible to modify the bacterium so that at least one gene coding for an endogenous lactate dehydrogenase is under the control of a promoter allowing such overexpression. A promoter designates the sequence at the 5′ end of the structural gene under consideration and which directs the initiation of transcription. In general, according to the invention, the promoters which can be used include constitutive promoters, namely promoters which are active in most cellular states and environmental conditions, as well as inducible promoters which are activated or repressed by stimuli exogenous physical or chemical properties, and which therefore induce a variable level of expression depending on the presence or absence of these stimuli. Alternatively or additionally, it is possible to genetically modify the endogenous promoter, for example to remove potential inhibitions of its induction. It is also possible to overexpress an endogenous lactate dehydrogenase by multiplying the number of copies of the gene in the genome of the bacterium, by means of plasmids and/or by introducing nucleotide sequences at other loci on the chromosome(s) of the bacterium. bacteria, etc.

In one embodiment, the bacterium according to the invention is genetically modified to overexpress an endogenous lactate dehydrogenase. Advantageously, the bacterium is genetically modified to overexpress only endogenous L-lactate dehydrogenase or only endogenous D-lactate dehydrogenase.

Tables 1 and 2 below list, by way of example, sequences encoding L-lactate dehydrogenase (Table 1: Examples of L-lactate dehydrogenase (EC 1.1.1.27)) and D-lactate dehydrogenase (Table 2: Examples of D-lactate dehydrogenase (EC 1.1.1.28)) from different bacteria, together with the corresponding protein sequences. According to the invention, these sequences can be the target of genetic modifications, including multiplication, to lead to a genetically modified bacterium capable of producing lactate from CO ₂ .- a polyhydrosiloxane II of formula X(R) ₂ SiO -((R) ₂ SiO) _m -(RXSiO) _n -Si(R) ₂ X with R as defined above and X corresponding to H or R,

Microorganism Embarrassed GenBank protein sequence Cupriavidus necator H16 Ldh CAJ91814.1 SEQ ID No. 1 Pediococcus acidilactici ldhA 1082254718 SEQ ID No. 2 Streptococcus equinus (Streptococcus bovis) Ldh KFN85486.1 SEQ ID No. 3 Bacillus coagulans Ldh AGU00860.1 SEQ ID No. 4 Lactobacillus casei Ldh CAQ65818.1 SEQ ID No. 5 Lactobacillus helveticus Ldh ABX26516.1 SEQ ID No. 6 Lactobacillus delbrueckii subsp . bulgaricus Ldh KRN37463.1 SEQ ID No. 7 Lactobacillus plantarum Ldh EFK28653.1 SEQ ID No. 8 Lactobacillus pentosus Ldh EIW14906.1 SEQ ID No. 9 Lactococcus lactis subsp. lactis Ldh BAL51029.1 SEQ ID No. 10

Microorganism Embarrassed GenBank protein sequence Cupriavidus necator H16 ldhA1 CAJ92810.1 SEQ ID No. 11 Lactobacillus delbrueckii subsp. bulgaricus ldhA CAI96942.1 SEQ ID No. 12 Escherichia coli str. K-12 substr. MG1655 ldhA 16129341 SEQ ID No. 13 Bacillus coagulans ldhA ADV02473.1 SEQ ID No. 14

In a particular embodiment, the bacterium is a Cupriavidus necator bacterium, genetically modified at the level of the promoter associated with the gene(s) coding for endogenous L-lactate dehydrogenase and/or endogenous D-lactate dehydrogenase, in order to promote the expression of one and/or the other gene. Indeed, the inventors have discovered that Cupriavidus necator has genes encoding an endogenous L-lactate dehydrogenase (ldh) and D-lactate dehydrogenase (ldhA1), the expression of which depends on the culture conditions and which is generally almost nil in limitation of nutrients. According to the invention, this bacterium can advantageously be genetically modified at the level of the nucleotide sequence coding for said promoter, in order to lift this negative regulation and allow the expression of the corresponding genes, even with a limitation of nutrients. It is in particular possible to modify Cupriavidus necator so as to associate the sequence(s) coding for endogenous L-lactate dehydrogenase and/or D-lactate dehydrogenase with a constitutive promoter or with an inducible promoter.

In one embodiment, the genes encoding endogenous L-lactate dehydrogenase and/or D-lactate dehydrogenase are modified so as to be under the control of a constitutive (non-downregulated) recombinant promoter or inducible by presence of a particular molecule. By way of example, it is possible to use constitutive promoters such as pLac, pTAC, pJ5 (Gruber et al. , 2014), an inducible promoter such as the pBAD promoter, inducible to arabinose (Grousseau et al. , 2014), the pPHAP promoter inducible under phosphate limitation (Barnard et al. 2015), the pCBBL promoter inducible under autotrophic conditions (Lütte et al. , 2012) or the rhamnose-inducible pKRrha promoter (Sydow et al. , 2017).

In a particular embodiment, the bacterium according to the invention is genetically modified to overexpress a gene coding for the expression of a protein exhibiting at least 50% homology with SEQ ID No. 1 (sequence of L-lactate dehydrogenase from Cupriavidus necator H16), preferably at least 75%, 80%, 85%, 90%, 95%, 99%. In another embodiment, the bacterium according to the invention is genetically modified to overexpress a gene coding for the expression of a protein exhibiting at least 50% homology with SEQ ID No. 11 (sequence of a D- lactate dehydrogenase from Cupriavidus necator H16), preferably at least 75%, 80%, 85%, 90%, 95%, 99%.

Alternatively or cumulatively, the bacterium can be genetically modified to overexpress at least one gene encoding an exogenous or heterologous lactate dehydrogenase.

According to the invention, the genome of the bacterium is then modified so as to integrate a nucleic sequence coding for such an exogenous lactate dehydrogenase. Said nucleic sequence may have been introduced into the genome of the bacterium or one of its ancestors, by means of any suitable molecular cloning method. In the context of the invention, the genome of the bacterium means all the genetic material contained in said bacterium, including the extrachromosomal genetic material contained for example in plasmids, episomes, synthetic chromosomes, etc. The nucleic acid sequence introduced may be a heterologous sequence, that is to say which does not exist naturally in said bacterium, or a homologous sequence. Advantageously, a transcriptional unit containing the nucleic sequence of interest, placed under the control of one or more promoter(s) and one or more ribosome binding sites, is introduced into the genome of the bacterium. Such a transcriptional unit also advantageously comprises the usual sequences such as transcriptional terminators, and where appropriate other transcriptional regulatory elements.

A gene encoding an exogenous lactate dehydrogenase can for example be derived from a bacterium, a fungus, a yeast or a mammal. Preferably, such a gene is derived from a bacterium and in particular from E. coli, Bacillus coagulans, Pediococcus acidilactici, Streptococcus bovis ( Streptococcus equinus ), from a lactic acid bacterium, and in particular from Lactobacillus casei, Lactobacillus helveticus, Lactobacillus bulgaricus , Lactobacillus delbrueckii , Lactobacillus plantarum or Lactobacillus pentosus, Lactococcus lactis subsp. lactis . The genes listed in Tables 1 and 2 can also be used to genetically modify a bacterium according to the invention.

In a particular embodiment, the sequence of the heterologous lactate dehydrogenase used to genetically modify the bacterium according to the invention has at least 50% homology with the sequence of the L-lactate dehydrogenase of Pediococcus acidilactici (SEQ ID No. 2 ), preferably at least 60%, 70%, 75%, 80, 85%, 90%, 95%, 99%, 100%.

In a particular embodiment, the sequence of the heterologous lactate dehydrogenase used to genetically modify the bacterium according to the invention has at least 50% homology with the sequence of the L-lactate dehydrogenase of Streptococcus equinus (Streptococcus bovis) (SEQ ID No. 3), preferably at least 60%, 70%, 75%, 80, 85%, 90%, 95%, 99%, 100%.

In a particular embodiment, the sequence of the heterologous lactate dehydrogenase used to genetically modify the bacterium according to the invention has at least 50% homology with the sequence of the L-lactate dehydrogenase of Bacillus coagulans (SEQ ID No. 4 ), preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80, 85%, 90%, 95%, 99%, 100%.

In a particular embodiment, the sequence of the heterologous lactate dehydrogenase used to genetically modify the bacterium according to the invention has at least 50% homology with the sequence of the L-lactate dehydrogenase of Lactobacillus casei (SEQ ID No. 5 ), preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80, 85%, 90%, 95%, 99%, 100%.

In a particular embodiment, the sequence of the heterologous lactate dehydrogenase used to genetically modify the bacterium according to the invention has at least 50% homology with the sequence of the D-lactate dehydrogenase of Lactobacillus delbrueckii subsp . bulgaricus (SEQ ID No. 12), preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80, 85%, 90%, 95%, 99%, 100%.

In a particular embodiment, the sequence of the heterologous lactate dehydrogenase used to genetically modify the bacterium according to the invention has at least 50% homology with the sequence of the D-lactate dehydrogenase of Escherichia coli (SEQ ID No. 13 ), preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80, 85%, 90%, 95%, 99%, 100%.

In a particular embodiment, the sequence of the heterologous lactate dehydrogenase used to genetically modify the bacterium according to the invention has at least 50% homology with the sequence of the D-lactate dehydrogenase Bacillus coagulans (SEQ ID No. 14) , preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80, 85%, 90%, 95%, 99%, 100%.

In one embodiment, the bacterium is genetically modified to overexpress at least one gene encoding an L-lactate dehydrogenase (endogenous or heterologous), so as to be able to produce L-lactate. In the case of a bacterium possessing a gene encoding an endogenous D-lactate dehydrogenase, the expression of said gene is advantageously at least partially inhibited, so as to promote the production of L-lactate only.

Alternatively, the bacterium can be genetically modified to overexpress at least one gene encoding a D-lactate dehydrogenase (endogenous or heterologous) so as to be able to produce D-lactate. In the case of a bacterium possessing a gene coding for an endogenous L-lactate dehydrogenase, the expression of said gene is advantageously at least partially inhibited, so as to promote the production of D-lactate only.

According to the invention, the term " inhibition of the expression of a gene " means the fact that said gene is no longer expressed in the bacterium considered or that its expression is reduced, compared to the wild bacterium (not genetically modified for inhibit the expression of the gene), leading to the absence of production of the corresponding protein or to a significant drop in its production, and in particular to a drop of more than 20%, more preferentially 30%, 40%, 50%, 60 %, 70%, 80%, 90%. In one embodiment, the inhibition may be total, that is to say that the protein coded by said gene is no longer produced at all. The inhibition of the expression of a gene can in particular be obtained by deletion, mutation, insertion and/or substitution of one or more nucleotides in the gene considered. Preferably, the inhibition of the expression of the gene is obtained by total deletion of the nucleotide sequence. According to the invention, any method of inhibiting a gene, known per se by those skilled in the art and applicable to a bacterium can be used. For example, the inhibition of the expression of a gene can be obtained by homologous recombination (Datsenko et al., 2000; Lodish et al., 2000); random or directed mutagenesis to modify the expression of a gene and/or the activity of the encoded protein (Thomas et al., 1987); modification of a promoter sequence of the gene to alter its expression (Kaufmann et al., 2011); targeting induces local lesions in genomes (TILLING); conjugation, etc Another particular approach is gene inactivation by insertion of a foreign sequence, for example by transposon mutagenesis using mobile genetic elements, of natural or artificial origin or for example by insertion of an antibiotic cassette. According to another preferred embodiment, inhibition of gene expression is achieved by knockout techniques. Inhibition of gene expression can also be achieved by gene silencing using interfering, ribozyme or antisense RNAs (Daneholt, 2006. Nobel Prize in Physiology or Medicine). In the context of the present invention, the term "interfering RNA" or "RNAi" refers to any RNAi molecule (for example single-stranded RNA or double-stranded RNA) which can block the expression of a target gene and/or facilitate the degradation corresponding mRNAs. Gene inhibition can also be achieved by gene editing methods that allow direct genetic modifications to be made to a given genome, through the use of zinc finger nucleases (Kim et al., 1996), nucleases transcription activator type effectors, called “TALEN” (Ousterout et al. 2016), of a system combining Cas9-type nucleases with short grouped and regularly spaced palindromic repeats called “CRISPR” (Mali et al., 2013) , or meganucleases (Daboussi et al., 2012). The inhibition of the expression of the gene can also be obtained by inactivating the protein encoded by said gene. Inhibition of gene expression can also be obtained by deletion, mutation, insertion and/or substitution of one or more nucleotides in the promoter upstream of the gene considered. Inhibition of gene expression can also be obtained by deletion, mutation, insertion and/or substitution of one or more nucleotides in the ribosome binding site upstream of the gene considered.

By working on the modifications to be made to a bacterium which naturally oxidizes hydrogen genetically modified to enable it to produce lactate from CO ₂ , the inventors have demonstrated in these bacteria genes (lutA, lutB or lutC) coding for the components of an enzymatic complex using lactate, the inhibition of which makes it possible to substantially reduce or even completely eliminate the re-consumption of the lactate produced. These genes are united on an operon called lut.

The previously unknown lut operon in naturally hydrogen-oxidizing bacteria brings together genes that have less than 50% identity with the genes of the lut operon of B. subtilis (Chai et al ., 2009) known to allow the growth of this bacterium on L-lactate as a carbon source. For Cupriavidus necator , the genes joined in the lut operon have less than 44% identity with those of B. subtilis . For Rhodococcus opacus the genes united in the lut operon have less than 40% identity with those of B. subtilis. For Rhodococcus jostii the genes united in the lut operon have less than 40% identity with those of B. subtilis. For Helicobacter pylori , the genes joined in the lut operon have less than 37% identity with those of B. subtilis. For Bacillus tusciae , the genes joined in the lut operon have less than 55% identity with those of B. subtilis. For Pseudomonas putida, the genes united in the lut operon have less than 43% identity with those of B. subtilis. For Cupriavidus metallidurans , the genes joined in the lut operon have less than 42% identity with those of B. subtilis. For Paracoccus denitrificans , the genes joined in the lut operon have less than 42% identity with those of B. subtilis.

Table 3 below identifies the genes coding for lactate-using proteins found in several naturally occurring hydrogen-oxidizing bacteria.

Microorganism Embarrassed GenBank protein sequence Cupriavidus necator H16 lutA WP_011615044.1 SEQ ID No. 15 Cupriavidus necator H16 lutB WP_011615043.1 SEQ ID No. 16 Cupriavidus necator H16 lutC WP_011615045.1 SEQ ID No. 17 Cupriavidus necator H16 lutA WP_011616347.1 SEQ ID No. 18 Cupriavidus necator H16 lutB WP_010814062.1 SEQ ID No. 19 Cupriavidus necator H16 lutC WP_010814659.1 SEQ ID No. 20 Rhodococcus opacus lutA ANS31563.1 SEQ ID No. 21 Rhodococcus opacus lutB WP_105418126.1 SEQ ID No. 22 Rhodococcus opacus lutC RKM74017.1 SEQ ID No. 23 Rhodococcus jostii lutA WP_073359632.1 SEQ ID No. 24 Rhodococcus jostii lutB WP_073359631.1 SEQ ID No. 25 Rhodococcus jostii lutC WP_073359630.1 SEQ ID No. 26 Helicobacter pylori lutA WP_000867265.1 SEQ ID No. 27 Helicobacter pylori lutB WP_033777014.1 SEQ ID No. 28 Helicobacter pylori lutC WP_096411967.1 SEQ ID No. 29 Bacillus tusciae lutA WP_013076354.1 SEQ ID No. 30 Bacillus tusciae lutB WP_013076353.1 SEQ ID No. 31 Bacillus tusciae lutC WP_013076352.1 SEQ ID No. 32 Pseudomonas putida lutA WP_110966271.1 SEQ ID No. 33 Pseudomonas putida lutB WP_077182418.1 SEQ ID No. 34 Pseudomonas putida lutC WP_077182417.1 SEQ ID No. 35 Cupriavidus metallidurans lutA WP_029308115.1 SEQ ID No. 36 Cupriavidus metallidurans lutB WP_011518104. SEQ ID No. 37 Cupriavidus metallidurans lutC WP_035836231.1 SEQ ID No. 38 Paracoccus denitrificans lutA WP_011749200.1 SEQ ID No. 39 Paracoccus denitrificans lutB WP_011749199.1 SEQ ID No. 40 Paracoccus denitrificans lutC WP_011749198.1 SEQ ID No. 41

Those skilled in the art will be able to identify the genes encoding lactate-utilizing proteins united in lut operons in other species of naturally hydrogen-oxidizing bacteria from the information contained in the present application.

In the bacterium that naturally oxidizes hydrogen and is genetically modified to produce lactate from CO ₂ according to the invention, inhibiting the expression of the lut operon comprises inhibiting the expression of at least one of lutA, lutB or lutC genes, preferably at least two genes among the three, more preferably inhibition of the expression of the three lutA, lutB and lutC genes.

Preferably, the inhibition of the expression of a gene of the lut operon consists of the partial or total deletion of the sequence coding for said gene. The inhibition of the expression of the lut operon preferably consists of a deletion of said lut operon.

In some naturally hydrogen-oxidizing bacteria such as Cupriavidus necator, several lut operons are found. In Cupriavidus necator , the first lut operon bringing together the genes SEQ ID No. 15, SEQ ID No. 16 and SEQ ID No. 17, is named lut1. In Cupriavidus necator , the second lut operon bringing together the genes SEQ ID No. 18, SEQ ID No. 19 and SEQ ID No. 20, is named lut2. According to a first embodiment of the invention, in the bacteria which contain two or more lut operons, at least one of the lutA, lutB or lutC genes of one of the lut operons is inhibited. According to another embodiment of the invention, at least one of the lutA, lutB or lutC genes of at least two of the lut operons is inhibited. Advantageously, the expression of at least one of the lutA, lutB or lutC genes of each of the lut operons is inhibited.

According to another embodiment of the invention, the expression of at least one of the lut operons is inhibited. According to a preferred embodiment of the invention, the expression of at least two of the lut operons is inhibited. Advantageously, the expression of each of the lut operons is inhibited. This is in particular the case for Cupriavidus necator which comprises two lut1 and lut2 operons and where the expression of the two lut1 and lut2 operons is preferably inhibited.

In addition to the modifications described above, by working on the genetic modifications to be made to a bacterium which naturally oxidizes hydrogen to enable it to produce lactate from CO ₂ , the inventors have demonstrated that, in certain bacteria, it It is possible to at least partially inhibit a pyruvate degradation pathway competing with the lactate synthesis pathway, so as to promote the production of lactate from said pyruvate.

Indeed, certain bacteria which naturally oxidize hydrogen and capable of being genetically modified according to the invention, exhibit a pathway for the synthesis of polyhydroxybutyrate (PHB) from pyruvate. This is particularly the case for Cupriavidus necator (figure 2). Such a biosynthetic pathway can compete with the lactate synthesis pathway, forcing the consumption of pyruvate in this pathway. Also, in one embodiment, such a bacterium is genetically modified to at least partially inhibit the PHB synthesis pathway (FIG. 3). More particularly, it is possible to at least partially inhibit the expression of at least one gene chosen from the genes coding for Acetyl-CoA acetyltransferase (EC: 2.3.1.9), Acetoacetyl-CoA reductase (EC: 1.1.1.36) and poly(3-hydroxybutyrate) synthase (EC: 2.3.1.-), preferentially the expression of at least two of said genes, more preferentially the expression of said three genes.

In a particular embodiment, the genetically modified bacterium is Cupriavidus necator , in which the expression of at least one and preferentially of the three genes among the genes coding for a phaA (GenBank: CAJ91322.1), a phaB (GenBank: CAJ92574.1) and one phaC (GenBank: CAJ92572.1) is inhibited (Figure 2). The inventors have discovered that such a bacterium, also genetically modified to overexpress a lactate dehydrogenase, is capable of producing large quantities of lactate in the presence of CO ₂ as the sole carbon source, the pyruvate produced being little or not consumed by the pathway. production of PHB.

Alternatively or additionally, it is possible to genetically modify the bacterium so as to at least partially inhibit the expression of a gene coding for a phosphoenolpyruvate synthase (EC: 2.7.9.2), which converts pyruvate into phosphoenol pyruvate (PEP ), and/or a pyruvate carboxylase (EC: 6.4.1.1) and/or a pyruvate dehydrogenase complex (EC: 1.2.4.1) and/or a fumarate reductase (EC: 1.3.5.4).

In a particular embodiment, the genetically modified bacterium is Cupriavidus necator , in which the expression of at least one gene among the genes encoding a ppsa (GenBank: CAJ93138.1), a pyc (GenBank: CAJ92391.1) , pdhA (GenBank: CAJ92510.1) and sdhABCD (GenBank: CAJ93711.1, CAJ93712.1, CAJ93713.1, CAJ93714.1) is inhibited (Figure 2). The inventors have discovered that such a bacterium, also genetically modified to overexpress a lactate dehydrogenase, is capable of producing large quantities of lactate in the presence of CO ₂ as the sole carbon source, the pyruvate produced being little or not consumed by these pathways. competitors.

Alternatively or additionally, it is possible to genetically modify the bacterium so as to at least partially inhibit the conversion pathway from Acetyl-CoA to acetate and/or Acetaldehyde. For this, it is possible to at least partially inhibit the expression of at least one gene chosen from the genes encoding an acetyl-CoA hydrolase (EC: 3.1.2.1), a phosphate acetyltransferase (EC: 2.3.1.8) , an acetate kinase (EC: 2.7.2.1), a propionate CoA-transferase (EC: 2.8.3.1), a succinyl-CoA: acetate CoA-transferase (EC: 2.8.3.18) and an Acetaldehyde dehydrogenase (EC: 1.2. 1.10).

In a particular embodiment, the genetically modified bacterium is Cupriavidus necator , in which the expression of at least one gene among the genes encoding an Acetyl-CoA hydrolase (GenBank: CAJ96157.1), a Phosphate acetyltransferase (pta1, pta2) (GenBank: CAJ96416.1, GenBank: CAJ96653.1), an Acetate kinase (ackA, ackA2) (GenBank: CAJ91818.1, GenBank: CAJ96415.1), a Propionate CoA-transferase (GenBank: CAJ93797.1) , a Succinyl-CoA:acetate CoA-transferase (GenBank: CAJ92496.1) and an Acetaldehyde dehydrogenase (mhpf) (GenBank: CAJ92911.1) is inhibited (FIG. 2). The inventors have discovered that such a bacterium, also genetically modified to overexpress a lactate dehydrogenase and inhibit the expression of at least one gene of the lut operon, is capable of producing large quantities of lactate in the presence of CO ₂ as the sole source. of carbon, the pyruvate produced being little or not consumed by these competing pathways.

In some cases, bacteria that naturally oxidize hydrogen possess one or more genes coding for a Lactate ferricytochrome C reductase. This is particularly the case for Cupriavidus necator which has three genes lldD (EC: 1.1.2.3, GenBank: CAJ95257.1), lldA (EC: 1.1.2.3, GenBank: CAJ96599.1), dld (EC: 1.1.2.4 , GenBank: CAJ94166.1) encoding such Lactate ferricytochrome C reductase. Such an enzyme is also capable of reducing the level of lactate produced, by converting said lactate into pyruvate. Also, according to the invention, in the case where the bacterium has such an endogenous lactate ferricytochrome C reductase, the gene coding for said enzyme is advantageously at least partially inhibited.

The invention advantageously proposes using a genetically modified bacterium according to the invention to produce lactate from CO ₂ . Advantageously, a bacterium genetically modified to overexpress an L-lactate dehydrogenase, and if necessary inhibit a D-lactate dehydrogenase is used to produce L-lactate exclusively. Similarly, a bacterium genetically engineered to overexpress D-lactate dehydrogenase, and optionally inhibit L-lactate dehydrogenase is used to exclusively produce D-lactate.

The invention more particularly proposes a process for producing lactate from CO ₂ , according to which a genetically modified bacterium according to the invention is cultivated, for example in batch, fed-batch or in continuous culture, in the presence of CO ₂ and the lactate produced is recovered.

In one embodiment of the method, a stock solution is added during the fermentation to control the pH. Lactic acid is then found in the fermentation medium in the form of salt (sodium lactate, potassium lactate, calcium lactate or ammonium lactate, alone or in a mixture, depending on the base chosen to regulate the pH of the fermentation medium).

The fermentation broth containing the bacteria, the impurities of the fermentation medium (unconsumed proteins and inorganic salts of various kinds) and the lactate salt, is then treated in order to separate them. Such a separation step can in particular be implemented using a cell separator such as a centrifuge, a microfiltration or ultrafiltration device. After separation, a concentrated cell biomass is obtained on the one hand and a lactate salt solution on the other.

It is then possible to carry out a stage of conversion of the lactate salts into lactic acid in the free form. This step of recovering lactic acid from the fermentation medium can in particular be carried out via the extraction of lactic acid as such from the fermentation medium, or by acidification of the medium with sulfuric acid.

A purification step by extraction, esterification, distillation or hydrolysis can then be carried out, in order to obtain a high degree of purity lactic acid.

According to the invention, the source of CO ₂ can be pure CO ₂ or CO ₂ resulting from emissions from factories having industrial processes such as oil refineries, cement works, ammonia production, methanol production , etc. The CO ₂ can also be a fermentation product, a CO ₂ -enriched gas, an at least partially purified CO ₂ gas, a carbonate or bicarbonate solution, and/or formic acid. The CO ₂ content of the total gas can be from 10% to 100%. Such a gas containing CO ₂ can be injected directly or via an intermediate stage of CO ₂ capture or purification. Purification of the CO ₂ can be carried out by chemical treatment, for example in the presence of amines, or by enzymatic treatment using, for example, a carbonic anhydrase.

According to the invention, the hydrogen source can be a product of the steam reforming of methane, of the electrolysis of water or be a co-product (“fatal” hydrogen) of industrial processes such as the chlor-alkali process during the preparation of chlorine or the incineration of waste.

According to the invention, it is possible to optionally provide a stage of growth of the bacterium upstream, in the presence in particular of fermentable sugars, such as glucose, fructose or glycerol. It is also possible to cultivate the bacterium in the presence of CO ₂ and another carbon source during the lactate production step.

EXAMPLES.

Example 1 - CN0001 lactate-producing strain

Strain and genetic constructs. For the production of lactate, a strain Cupriavidus necator H16 PHB-4 (DSM no. 541) is used. This strain does not form poly-β-hydroxy-butyrate (PHB). The construction of the CN0001 lactate-producing strain is carried out according to the following protocol:

A plasmid carrying L-lactate dehydrogenase from Cupriavidus necator (ldh, EC:1.1.1.27) under an arabinose-inducible promoter is cloned in one step in vitro using the In-Fusion® assembly protocol (Clontech). Oligonucleotides are synthesized and purified (desalted) by Eurofins Genomics.

The ldh gene is amplified by PCR on the genomic DNA of the Cupriavidus necator H16 PHB-4 strain using oligonucleotides 1 (5' GGATCCAAAC TCGAGTAAGG ATCTCC 3') and 2 (5' ATGTATATCT CCTTCTTAAA AGATCTTTTG AATTCC 3') and Phusion High-Fidelity PCRMaster Mix with GC Buffer enzyme (New England Biolabs, Evry, France). The PCR product is purified on gel with the Nucleospin gel and PCR clean-up kit (Macherey-Nagel).

The backbone plasmid pJM3 (Müller et al. , 2013) derived from the plasmid pBBR1-MCS2 (Kovach et al, 1995), containing a pBAD promoter from Escherichia coli , is amplified using oligonucleotides 3 (5' GAAGGAGATA TACATATGAA GATCTCCCTC ACCAGCG 3') and 4 (5' CTCGAGTTTG GATCCTCAGG CCGTGGGGAC GGC 3') and the enzyme Phusion High-Fidelity PCRMaster Mix (New England Biolabs, Evry, France). The PCR product is digested with the enzyme DpnI (New England Biolabs, Evry, France): 1 μl of DpnI is added to 50 μL of PCR product, then incubated for 15-60 min at 37°C, the enzyme is then inactivated for 10 min at 80°C. The PCR product is purified on gel with the Nucleospin gel and PCR clean-up kit (Macherey-Nagel).

In vitro assembly by In-fusion is carried out with the two fragments in order to obtain the plasmid pJM3-ldh. 5 μl of the assembly product are transformed into chemo-competent Escherichia coli Stellar (Clontech) cells. The transformants are selected on an LB/Agar medium (Tryptone 10 g/L, yeast extract 5 g/L, NaCl 5 g/L, Sigma-Aldrich, agar 20 g/L) containing 25 μg/mL of kanamycin. The selection of positive clones is carried out by PCR on colonies using the enzyme DreamTaq Green PCR Master Mix (Thermo Scientific™). After extraction of the plasmids using the NucleoSpin® Plasmid kit (Macherey-Nagel), the correct insertion of the ldh gene is confirmed by sequencing (Eurofins genomic) with oligonucleotides 5 (5' GACGCTTTTT ATCGCAACTC TCTACTG 3') and 6 ( 5' CGAACGCCCT AGGTATAAAC GCAG 3').

The pBBAD-ldh plasmid is inserted into the Cupriavidus necator H16 PHB-4 strain by electroporation (protocol adapted from Taghavi et al. , 1994). Cupriavidus necator cells are cultured in TSB medium containing 27.5 g/L of tryptic soy broth (TSB, Becton Dickinson, Sparks, Maryland, USA, 3ml in 10ml tubes) and incubated overnight at 30°C with vigorous shaking . The following day, 1 ml of the culture is transferred into a 250 ml Erlenmeyer flask containing 25 ml of TSB medium and incubated at 30° C., 200 rpm until an OD _{600 nm} of 0.5-0.6 is reached. The cells are then harvested and washed twice with 25 mL of cold wash buffer (10% glycerol, 90% water [vol/vol]). The cells rendered electro-competent by this process are then concentrated in the washing solution in order to obtain an OD _{600 nm} of 50 and aliquoted with 150 μL.

An aliquot of competent cells is transformed with 2-5 μl of plasmid DNA (100-150 ng/μl). The DNA/cell mixture contained in a 1.5 mL tube is stirred by light tapping (4-5 times). The mixture is placed on ice for 5 min and transferred to a cold electroporation cuvette (0.2 cm, 10573463, Fisher scientific, Illkirch). Electroporation is performed with the following settings: voltage 2.5 kV, capacitance 25 µF, external resistance 200 Ω. Immediately after electroporation the cells are mixed with 1mL of SOC medium (2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl ₂ , 10 mM MgSO ₄ and 20 mM glucose ), incubated for 2 hours at 30°C then plated on Petri dishes maintained at 30°C containing TSB/Agar medium (20g/L) supplemented with 10 µg/mL of gentamycin and 200 µg/mL of kanamycin).

The Cupriavidus necator H16 PHB-4 pJM3-ldh strain is recovered and named CN0001. Genetic modifications are validated by sequencing.

Media For the precultures, the minimum medium A used contains: NaH2PO4, 4.0 g/L; Na ₂ HPO ₄ , 4.6 g/L; K ₂ SO ₄ , 0.45 g/L; MgSO ₄ 0.39 g/L, CaCl ₂ 0.062 g/L; NH ₄ Cl 0.05% (w/v), trace elements, 1ml/L (FeSO ₄ ·7H ₂ O, 15 g/L; MnSO ₄ ·H ₂ O, 2.4 g/L; ZnSO ₄ ·7H ₂ O, 2.4 g/L; CuSO ₄ 5H2O, 0.48 g/L in 0.1M HCl).

For the cultures in bioreactors, the minimum medium B used contains per liter: MgSO ₄ .7H ₂ O, 0.75 g; phosphate (Na ₂ HPO ₄ .12H ₂ O, 1.5 g; KH ₂ PO ₄ , 0.25 g); nitrilotriacetic acid, 0.285g; iron(III) ammonium citrate, 0.9 g; _CaCl2 , 0.015g; trace elements (H ₃ BO ₃ , 0.45 mg; CoCl ₂ .6H ₂ O, 0.3 mg; ZnSO ₄ .7H ₂ O, 0.15 mg; MnCl ₂ .4H ₂ O, 0.045 mg; Na ₂ MoO ₄ .2H ₂ O, 0.045 mg; NiCl ₂ .6H ₂ O, 0.03 mg; CuSO ₄ , 0.015 mg); kanamycin, 0.1 g.

Precultures An isolated colony of the CN0001 strain is used to inoculate the first culture which is cultured for 24 h with 10 mL of TSB containing 10 μg/mL of gentamycin and 200 μg/mL of kanamycin, in a 100 mL Erlenmeyer flask. Two further propagation steps are carried out for 12 h each (25 mL and 300 mL of minimum medium A, in 250 mL and 3 L Erlenmeyer flasks, respectively). Each culture is grown at 30°C and shaken at 100 rpm in an incubator. This preculture is used to inoculate the culture step in bioreactors.

Culture in bioreactors Culture of the CN0001 strain in fed-batch is carried out in three phases. The first phase consists of a controlled growth phase on fructose to reach 0.9 g/L of biomass at a specific growth rate of 0.16 h ^-1 . After the consumption of fructose, the second phase is carried out to allow the adaptation of cellular metabolism to gaseous substrates. It is started by a flow of 0.22 L/min of a commercial H ₂ /O ₂ /CO ₂ /N ₂ mixture (% molar: 60:2:10:28, Air Liquide, Paris, France). The third phase consists of nitrogen-limited growth on gaseous substrates coupled with the production of lactate. This phase is initiated by the addition of 1 g/L of L-arabinose to induce the expression of lactate dehydrogenase. Nitrogen is supplied from a 56 g/L solution of NH ₃ , to control a residual specific growth rate of 0.02 h ⁻¹ . This culture is carried out in 1.4 L BDCU B.Braun bioreactors. The temperature is regulated to 30°C and the pH to 7.0 by the addition of 2.5 M KOH solution.

Analysis of lactate and metabolites In order to determine the concentrations of organic acids, in particular that of lactate, the fermentation supernatant is analyzed by HPLC-UV-RI chromatography. The regularly sampled fermentation broth (1 mL) is first centrifuged for 10 min at 10,000 g. Then, it is filtered through 0.45 μm (Minicart RC4, Sartorius). The HPLC system used is a Thermo Scientific UltiMate 3000 HPLC, coupled to a refractometer and UV detector (210 nm). 10 μL of each sample is injected onto an Aminex HPX-87H H+ column, 300 mm×7.8 mm (Biorad). The eluent is an aqueous solution of 4 mM sulfuric acid. The flow rate is set at 0.5 ml/min. The oven temperature is 45°C. Isocratic elution is performed. The quantification is carried out using an appropriate standard range. If necessary, the samples are diluted.

Result The CN0001 strain produces under these culture conditions from 5 to 100 mg/L of lactate.

Example 2: Construction of a bacterium that naturally oxidizes hydrogen, Cupriavidus necator and genetically engineered to plasmidically overexpress an endogenous lactate dehydrogenase and to produce lactate from CO ₂ (CN0002).

Strain and Genetic Constructs For the production of lactate, a Cupriavidus necator H16 PHB-4 strain (DSM No. 541) is used. This strain does not form poly-β-hydroxy-butyrate (PHB).

The construction of the CN0002 lactate-producing strain is carried out according to the following protocol:

A plasmid carrying L-lactate dehydrogenase from Cupriavidus necator (ldh, EC:1.1.1.27) under an arabinose-inducible promoter is cloned in one step in vitro using the In-Fusion® assembly protocol (Clontech). Oligonucleotides are synthesized and purified (desalted) by Eurofins Genomics.

The ldh gene (GenBank: CAJ91814.1) is amplified by PCR on the genomic DNA of the Cupriavidus necator H16 PHB-4 strain using oligonucleotides oligonucleotides 7 (5' AAGGAGATAT ACATATGAAG ATCTCCCTCA CCAGCG 3') and 8 (5 'ACTCGAGTTT GGATCCTCAG GCCGTGGGGA CGGC 3') and the enzyme Phusion High-Fidelity PCRMaster Mix with GC Buffer (New England Biolabs, Evry, France). The PCR product is purified on gel with the Nucleospin gel and PCR clean-up kit (Macherey-Nagel).

The backbone plasmid pBADTrfp (Bi et al. , 2013) derived from the plasmid pBBR1-MCS (Kovach et al. , 1995), containing a pBAD promoter from Escherichia coli , is digested with the restriction enzymes BamHI-HF and NdeI (New England Biolabs, Evry, France) in order to eliminate the sequence coding for the RFP protein. The 5247 base pair DNA fragment containing the pBBR1 origin of replication, the selection gene (kan) and the pBAD promoter is purified on gel with the Nucleospin gel and PCR clean-up kit (Macherey-Nagel).

In vitro assembly by In-fusion is carried out with the two fragments in order to obtain the plasmid pBAD-ldh(cn). 5 μl of the assembly product are transformed into chemo-competent Escherichia coli Stellar™ (Clontech) cells. The transformants are selected on an LB/Agar medium (Tryptone 10 g/L, yeast extract 5 g/L, NaCl 5 g/L, Sigma-Aldrich, agar 20 g/L) containing 25 μg/mL of kanamycin (Gibco ™). The selection of positive clones is carried out by PCR on colonies using the enzyme DreamTaq Green PCR Master Mix (Thermo Scientific™) and oligonucleotides 9 (5' CGAAGGTGAG CCAGTGTGAC TC 3') and 10 (5' CCTGTCGATC CTGCCCAACT AC 3'). After extraction of the plasmids using the NucleoSpin® Plasmid kit (Macherey-Nagel), the correct insertion of the l.ldh gene is confirmed by sequencing (Eurofins Genomic) with oligonucleotides 9 and 11 (5' CATTGATTAT TTGCACGGCG TCAC 3' ).

The pBAD-1.ldh(cn) plasmid is inserted into an electrocompetent Escherichia coli S17-1 strain by electroporation using a Gene Pulser electroporator, BioRad (Datsenko and Wanner, 2000).

Positive clones are selected by colony PCR using the enzyme DreamTaq Green PCR Master Mix (Thermo Scientific™) and oligonucleotides 9 and 10. The clone of interest is isolated on LB/agar medium (Tryptone 10 g/L, yeast extract 5 g/L, NaCl 5 g/L, Sigma-Aldrich, agar 20 g/L) containing 25 µg/mL of kanamycin and incubated overnight at 37°C.

The conjugation between the Escherichia coli S17-1 cells and those of the Cupriavidus necator H16 PHB-4 strain (DSM no. 541) is carried out as follows:

The Cupriavidus necator cells are cultured in 3 mL of TSB medium containing 27.5 g/L of tryptic soy broth (TSB, Becton Dickinson, Sparks, Maryland, USA) and incubated for 20 h at 30° C. with vigorous stirring. The Escherichia coli cells are cultured in 3 mL of LB medium (Tryptone 10 g/L, yeast extract 5 g/L, NaCl 5 g/L, Sigma-Aldrich) containing 25 μg/mL of kanamycin and incubated overnight at 37°C with vigorous stirring. The next day, the optical density (OD _{600 nm} ) is measured for each culture and the equivalent of 1 OD of cells are transferred into a 1.5 ml tube and centrifuged (5 minutes, 3000 rpm). The supernatant is eliminated and the cells are washed in 1 ml of PBS (Sigma-Aldrich). Washing is repeated a second time. 50 μL of the Cupriavidus necator cell suspension and 50 μL of the Escherichia coli cell suspension are taken and mixed in a 1.5 mL tube. This mixture is deposited in the form of a drop on a non-selective LB/Agar medium containing 2% fructose and incubated for 6 hours at 30°C. The cells are then scraped and resuspended in 1 ml of PBS. This suspension is diluted to 1:1000th and 100 μl of this dilution are spread on selective LB/Agar medium containing 2% fructose, 300 μg/mL of kanamycin and 10 μg/mL of gentamycin (Sigma-Aldrich). The cells are incubated for 24 hours at 30°C. Isolated clones are taken, streaked on the same selective medium and incubated for 24 hours at 30°C. The selection of clones containing the pBAD-ldh(cn) plasmid is carried out by colony PCR using the enzyme DreamTaq Green PCR Master Mix (Thermo Scientific™) and oligonucleotides 9 and 10.

The Cupriavidus necator H16 PHB-4 pBAD-ldh(cn) strain is recovered and named CN0002. Genetic modifications are validated by sequencing.

Example 3: Production of lactate from Fructose by Erlenmeyer culture of a bacterium that naturally oxidizes hydrogen, Cupriavidus necator , genetically modified (CN0002)

Strain For the production of lactate, strain CN0002 ( Cupriavidus necator H16 PHB-4 pBAD-ldh) is used. In this strain, the LDH (lactate dehydrogenase) of C. necator is overexpressed on the plasmid via an arabinose-inducible promoter. This strain is naturally resistant to gentamicin and carries plasmid resistance to kanamycin.

Media The rich medium consisted of 27.5% (w / v) trypticase soy broth (TSB, Becton Dickinson, France). The minimum medium used for the cultures in Erlenmeyers (MMR medium) contained: 4.0 g/L of NaH ₂ PO ₄ 2H ₂ O; 4.6 g/L of Na ₂ HPO ₄ 12H ₂ O; 0.45 g/LK ₂ SO ₄ ; 0.39 g/L of MgSO ₄ 7H ₂ O; 0.062 g/L of CaCl ₂ 2H ₂ O and 1 ml/L of trace element solution. The trace element solution contained: 15 g/L of FeSO ₄ 7H ₂ O; 2.4 g/L MnSO ₄ H ₂ O; 2.4 g/L of ZnSO ₄ 7H ₂ O and 0.48 g/L of CuSO ₄ 5H ₂ O in 0.1 M HCl. Fructose (20 g/L) was used as the carbon source. NH ₄ Cl (0.5 g/L) was used as a nitrogen source to reach a biomass concentration of approximately 1 g/L. 0.04 g/L of NaOH was added.

Inoculation chain A clone of the CN0002 strain subcultured from a culture plate was first cultured for 8 h in 5 mL of rich medium with gentamycin (10 µg/mL) and kanamycin (200 µg /mL) (depending on the strain), in culture tubes, at 30°C with shaking (200 rpm). This first preculture was used to inoculate the second preculture at an initial OD ₆₀₀ of 0.05 in 25 mL of MMR medium in 250 mL baffled Erlenmeyer flasks which were incubated for 20 h at 30° C., 200 rpm. This second preculture was used to inoculate the culture in an Erlenmeyer flask in 50 mL of MMR medium in the presence of gentamicin (10 μg/mL) and kanamycin (200 μg/mL) (depending on the strain). The initial target OD ₆₀₀ was 0.05.

Culture in Erlenmeyer The heterotrophic culture was carried out in a volume of MMR of 50 mL in 500 mL baffled Erlenmeyer flasks, at a temperature of 30° C. and stirring at 200 rpm. A growth phase lasting 20 hours was carried out until a biomass of 1 g/L was obtained. After this first phase of growth, a modification of the aeration is made (2% air or 0.4% O ₂ ), induction of the LDH promoter is carried out by adding 1 g/L of arabinose (strain dependent) which leads to a lactate production phase lasting 120 h.

Sampling and analysis protocol Samples of 1mL were taken regularly during the culture. Growth was monitored by optical density (OD) measurement at 600 nm; converted to dry weight of bacterial cells (gCDW/L) according to a standard curve. Lactate production was analyzed by HPLC as described in example 1. The samples were centrifuged for 5 min at 13,000 rpm and the supernatants were filtered before being analyzed by HPLC. Calibration varied from 0.1 to 5 g/L in water.

Results The growth of the strain is represented in FIG. 5. During the exponential growth phase, the specific growth rate of CN0002 in heterotrophic condition reaches μ _max =0.14 h ⁻¹ . A maximum of lactate, that is to say 1.3 g/L, was reached after 139 h of culture as represented in FIG. 5. It should be noted that under the same culture conditions, the strain Cupriavidus necator H16 PHB-4 without overexpression of lactate dehydrogenase does not produce lactate.

Example 4: Production of lactate from CO ₂ by fermentation of a bacterium that naturally oxidizes hydrogen, Cupriavidus necator , genetically modified (CN0002).

Strain For the production of lactate, strain CN0002 ( Cupriavidus necator H16 PHB-4 pBAD-ldh) is used. In this strain, the LDH (lactate dehydrogenase) of C. necator is overexpressed on the plasmid via an arabinose-inducible promoter. This strain is naturally resistant to gentamicin and carries plasmid resistance to kanamycin.

EnvironmentsThe rich medium consisted of 27.5% (w/v) trypticase soy broth (TSB, Becton Dickinson, France). The minimum medium used for cultures in Erlenmeyer flasks (MMR medium) contained: 4.0 g/L of NaH₂PO₄2 hours₂O; 4.6 g/L Na₂HPO₄12 noon₂O; 0.45 g/L K₂N/A₄; 0.39 g/L of MgSO₄7 a.m.₂O; 0.062 g/L of CaCl₂2 hours₂O and 1 ml/L trace element solution. The trace element solution contained: 15 g/L of FeSO₄7 a.m.₂O; 2.4 g/L MnSO₄H₂O; 2.4 g/L of ZnSO₄7 a.m.₂O and 0.48 g/L of CuSO₄5H₂O in 0.1 M HCl. Fructose (20 g/L) was used as the carbon source. NH₄Cl (0.5 g/L) was used as a nitrogen source to achieve a biomass concentration of approximately 1 g/L. 0.04 g/L of NaOH was added.

The minimum medium used in the bioreactor for the gas fermentations (FAME medium) consisted of: 0.29 g/L of NitriloTriAcetic acid; 0.09 g/L of ferric ammonium citrate; 0.75 g/L of MgSO ₄ 7H ₂ O; 0.015 g/L of CaCl ₂ 2H ₂ O and 1.5 ml/L of trace element solution. The composition of the trace element solution was: 0.3 g/L of H ₃ BO ₃ ; 0.2 g/L of CoCl ₂ 6H ₂ O; 0.1 g/L of ZnSO ₄ 7H ₂ O; 0.03 g/L of MnCl ₂ 4H ₂ O; 0.03 g/L of Na ₂ MoO ₄ 2H ₂ O; 0.02 g/L of NiCl ₂ 6H ₂ O; 0.01 g/L of CuSO ₄ 5H ₂ O. (NH ₄ ) ₂ SO ₄ (1.6 g/L) was used as a nitrogen source to reach a biomass concentration of approximately 2.5 g/ I. The pH was adjusted to 7 with 2.5 M KOH. After autoclaving the medium, a sterile phosphate solution of Na ₂ HPO ₄ 12H ₂ O (final concentration of 1.6 g/L) and KH ₂ PO ₄ (final concentration of 2.9 g/L) was added under sterile conditions in the bioreactor (making it possible to produce approximately 10 g/L of biomass) as well as a sterile solution of FeSO ₄ 7H ₂ O (10.7 g/L; final concentration 0.032 g/L).

Chain of inoculation A clone of the CN0002 strain subcultured from a culture plate was first cultured for 24 h in 5 mL of TSB medium with gentamycin (10 mg/L) and kanamycin (50 mg /L) (depending on the strain), in 50 mL baffled Erlenmeyer flasks, at 30°C with stirring (110 rpm). The culture medium was then centrifuged for 10 min at 1900 g. The cells were resuspended in 5 mL of MMR medium, and were used to inoculate the 45 mL of MMR medium with 5.5 μg/mL gentamicin and 100 μg/mL kanamycin in 500 mL baffled Erlenmeyer flasks which were incubated for 20-24 h at 30°C, 110 rpm. The culture medium was centrifuged for 5 min at 4000 g and the cells were resuspended in 30 mL of FAME medium, and were used to inoculate the 300 mL of FAME medium with 100 mg/L of kanamycin in the gas bioreactor. The initial target OD ₆₀₀ was 0.2.

Gas bioreactor The autotrophic culture was carried out in a gas bioreactor with a working volume of 330 mL. The temperature was set at 30°C, the pH at 7. The pressure and stirring speed were set according to the needs of the culture. Gas flows were individually controlled (CO ₂ , H ₂ , air). A gas analyzer was used for the analysis of the exit gases (% O ₂ and CO ₂ ) and a volumeter to measure the total exit gas flow rates. After 53 hours of growth, approximately 3 g/L of biomass had been reached, the dissolved oxygen concentration had fallen to 0 and 160 mg/L of lactate had been produced.

Sampling and analysis protocol Samples (approximately 1 ml) were taken regularly (every 2-3 hours) by drawing through a septum with a syringe and a needle. Growth was monitored by optical density (OD) measurement at 600 nm; converted to bacterial cell dry weight (gCDW/L) according to a standard curve. Lactate production was analyzed by HPLC/HPAIC as described in Example 1. Samples were centrifuged for 3 min at 13,000 rpm and supernatants were analyzed. For the HPLC analysis, the sample supernatants were filtered before analysis. Calibration varied from 0.1 to 5 g/L in water.

Fermentation The production of lactate by strain CN0002 was characterized in a bioreactor under autotrophic conditions. The growth of the strain is represented in FIG. 6. During the exponential growth phase, the specific growth rate of CN0002 in autotrophic condition reaches μ _max =0.14 h ⁻¹ . A maximum of lactate, i.e. 160 mg/L, was reached after 53 h of culture as represented in figure 6.

Example 5: Construction and evaluation of a strain of Cupriavidus necator genetically modified in which a heterologous lactate dehydrogenase of Streptococcus bovis is overexpressed (CN0003).

Strain and genetic constructs. For the production of lactate, a strain Cupriavidus necator H16 PHB-4 (DSM no. 541) is used. This strain does not form poly-β-hydroxy-butyrate (PHB). The construction of the CN0003 lactate-producing strain is carried out according to the following protocol:

A plasmid carrying L-lactate dehydrogenase (ldh, EC:1.1.1.27) from Streptococcus bovis (ATCC 33317) under an arabinose-inducible promoter is cloned in one step in vitro using the In-Fusion® assembly protocol (Clontech ). Oligonucleotides are synthesized and purified (desalted) by Eurofins Genomics.

The ldh gene (GenBank: KFN85486.1) is recoded according to the codon usage bias described for Cupriavidus necator H16 and synthesized in vitro (GenScript®). It is amplified by PCR using oligonucleotides 12 (5' AAGGAGATAT ACATATGACC GCGACCAAGC AGCAC 3') and 13 (5' ACTCGAGTTT GGATCCTCAG TTCTTGCAGG CCGACGCGA 3') and the enzyme Phusion High-Fidelity PCRMaster Mix with GC Buffer (New England Biolabs, Evry, France). The PCR product is purified on gel with the Nucleospin gel and PCR clean-up kit (Macherey-Nagel).

The backbone plasmid pBADTrfp (Bi et al. , 2013) derived from the plasmid pBBR1-MCS (Kovach et al. , 1995), containing a pBAD promoter from Escherichia coli , is digested with the restriction enzymes BamHI-HF and NdeI (New England Biolabs, Evry, France) in order to eliminate the sequence coding for the RFP protein. The 5247 base pair DNA fragment containing the pBBR1 origin of replication, the selection gene (kan) and the pBAD promoter is purified on gel with the Nucleospin gel and PCR clean-up kit (Macherey-Nagel).

An in vitro assembly by In-fusion is carried out with the two fragments in order to obtain the plasmid pBAD-ldh(sb). 5 μl of the assembly product are transformed into chemo-competent Escherichia coli Stellar™ (Clontech) cells. The transformants are selected on an LB/Agar medium (10 g/L Tryptone, 5 g/L yeast extract, 5 g/L NaCl, Sigma-Aldrich, 20 g/L agar) containing 25 μg/mL of kanamycin (Gibco™) . The selection of the positive clones is carried out by colony PCR using the enzyme DreamTaq Green PCR Master Mix (Thermo Scientific™) and oligonucleotides 9 and 14 (5' GGAGCTGGGC ATCATCGAGA TC 3'). After extraction of the plasmids using the NucleoSpin® Plasmid kit (Macherey-Nagel), the correct insertion of the ldh gene is confirmed by sequencing (Eurofins genomic) with oligonucleotides 9 and 11.

The pBAD-ldh(sb) plasmid is inserted into an electrocompetent Escherichia coli S17-1 strain by electroporation using a Gene Pulser electroporator, BioRad (Datsenko and Wanner, 2000).

Positive clones are selected by PCR on colonies using the enzyme DreamTaq Green PCR Master Mix (Thermo Scientific™) and oligonucleotides 9 and 14. The clone of interest is isolated on LB/agar medium (Tryptone 10 g/L, yeast extract 5 g/L, NaCl 5 g/L, Sigma-Aldrich, agar 20 g/L) containing 25 µg/mL of kanamycin and incubated overnight at 37°C.

The conjugation between the Escherichia coli S17-1 cells and those of the Cupriavidus necator H16 PHB-4 strain (DSM no. 541) is carried out as follows:

The Cupriavidus necator cells are cultured in 3 mL of TSB medium containing 27.5 g/L of tryptic soy broth (TSB, Becton Dickinson, Sparks, Maryland, USA) and incubated for 20 h at 30° C. with vigorous stirring. The Escherichia coli cells are cultured in 3 mL of LB medium (Tryptone 10 g/L, yeast extract 5 g/L, NaCl 5 g/L, Sigma-Aldrich) containing 25 μg/mL of kanamycin and incubated overnight at 37°C with vigorous stirring. The next day, the optical density (OD _{600 nm} ) is measured for each culture and the equivalent of 1 OD of cells is transferred into a 1.5 ml tube and centrifuged (5 minutes, 3000 rpm). The supernatant is eliminated and the cells are washed in 1 ml of PBS (Sigma-Aldrich). Washing is repeated a second time. 50 μL of the Cupriavidus necator cell suspension and 50 μL of the Escherichia coli cell suspension are taken and mixed in a 1.5 ml tube. This mixture is deposited in the form of a drop on a non-selective LB/Agar medium containing 2% fructose and incubated for 6 hours at 30°C. The cells are then scraped and resuspended in 1 ml of PBS. This suspension is diluted to 1:1000th and 100 μl of this dilution are spread on selective LB/Agar medium containing 2% fructose, 300 μg/mL of kanamycin and 10 μg/mL of gentamycin (Sigma-Aldrich). The cells are incubated for 24 hours at 30°C. Isolated clones are taken, streaked on the same selective medium and incubated for 24 hours at 30°C. The selection of the clones containing the plasmid pBAD-ldh(sb) is carried out by colony PCR using the enzyme DreamTaq Green PCR Master Mix (Thermo Scientific™) and oligonucleotides 9 and 14.

The Cupriavidus necator H16 PHB-4 pBAD-ldh(sb) strain is recovered and named CN0003.

Evaluation of the Fructose Strain CN0003 The production of lactate on fructose of the strain Cupriavidus necator H16 PHB-4 pBAD-ldh(sb) (CN0003) was evaluated according to the method described in example 3. Under these culture conditions, strain CN0003 produces 1 g/L of lactate after 140 h of culture.

Extrapolation of lactate production by CN0003 from CO ₂ Lactate production of strain CN0003 was not achieved from CO₂. However, the fructose evaluation showed that the CN0003 strain produced 23% less lactate than the CN0002 strain under the same culture conditions. We can extrapolate that from CO₂as described in Example 4, strain CN0003 would produce 23% less lactate from CO₂than strain CN0002.

Example 6: Construction of a strain of Cupriavidus necator genetically modified in which a polyhydroxybutyrate (PHB) biosynthetic pathway is deleted and in which an endogenous lactate dehydrogenase is overexpressed (CN0004).

Strain For the production of lactate, the strain Cupriavidus necator H16 (ATCC 17699) is used. The deletion of the polyhydroxybutyrate (PHB) biosynthetic pathway operon is inhibited by insertion of an endogenous lactate dehydrogenase.

Genetic constructions The construction of the lactate-producing strain CN0004 is carried out according to the following protocol:

A plasmid carrying the L-lactate dehydrogenase of Cupriavidus necator (ldh, EC:1.1.1.27) under an inducible pLac promoter as well as the sequences of the homology zones upstream and downstream of the phaCAB operon is cloned in one step in vitro via the ^NEBuilder® HiFi DNA Assembly Cloning protocol (New England Biolabs, Evry, France). Oligonucleotides are synthesized and purified (desalted) by Eurofins Genomics.

The ldh gene is amplified by PCR on the genomic DNA of the Cupriavidus necator H16 strain using oligonucleotides 15 (5' AGACAATCAA ATC TTTACAC TTTATGCTTC CGGCTCGTAT GTTGTGTGGA ATGTGAGCG GATAACAATT TCACACAGGA AACAGCT ATG AAGATCTCCC TCACCAGCGC CC 3') presenting in 5' a 13 base pair overlap region with the 3' end of the upstream homology region of the phaCAB operon and the pLac promoter sequence from pJQ200mp18 (Quandt and Hynes, 1993) and 16 (5' CCAGGCCGGC AGGTCAGGCC GTGGGGACGG CCA 3') presenting in 5' a zone of overlap of 13 base pairs with the 5' end of the homology zone downstream of the phaCAB operon and of the enzyme KOD Hot Start DNA Polymerase (Novagen) with 5 % DMSO. The PCR product is purified on gel with the Nucleospin gel and PCR clean-up kit (Macherey-Nagel).

The sequence of the homology zone upstream of the phaCAB operon is amplified by PCR on the genomic DNA of the Cupriavidus necator H16 strain using oligonucleotides 17 (5' GCATGCCTGC AGGTCGACTC TAGAGGGTCG CTTCTACTCC TATCG 3') presenting at 5 ' a zone of overlap of 25 base pairs with the 3' end of the plasmid pJQ200mpTet and 18 (5' CATAAAGTGT AAAGATTTGA TTGTCTCTCT GCC 3') presenting in 5' a zone of overlap of 13 base pairs with the 5' end of the sequence of the pLac promoter and of the enzyme KOD Hot Start DNA Polymerase (Novagen) with 5% DMSO. The PCR product is purified on gel with the Nucleospin gel and PCR clean-up kit (Macherey-Nagel).

The sequence of the downstream homology zone of the phaCAB operon is amplified by PCR on the genomic DNA of the Cupriavidus necator H16 strain using oligonucleotides 19 (5' CCCCACGGCC TGACCTGCCG GCCTGGTTCA ACC 3') presenting at 5' an overlap zone of 13 base pairs with the 3' end of the sequence of the ldh gene of the Cupriavidus necator strain H16 and 20 (5' TACGAATTCG AGCTCGGTAC CCGGGTTCTG GATGTCGATG AAGGCCTG 3') presenting in 5' an overlap zone of 25 base pairs with the 5' end of the plasmid pJQ200mpTet and the enzyme KOD Hot Start DNA Polymerase (Novagen) with 5% DMSO. The PCR product is purified on gel with the Nucleospin gel and PCR clean-up kit (Macherey-Nagel).

The backbone plasmid pJQ200mpTet is a derivative of the plasmid pJQ200mp18 (Quandt and Hynes, 1993) where the gentamycin resistance gene has been replaced by the tetracycline resistance gene. The backbone plasmid pJQ200mpTet is cut with the restriction enzyme BamHI-HF (New England Biolabs, Evry, France). The digestion product is purified on gel with the Nucleospin gel and PCR clean-up kit (Macherey-Nagel).

In vitro assembly by ^NEBuilder® HiFi DNA Assembly Cloning (New England Biolabs, Evry, France), is carried out with the four fragments in order to obtain the plasmid pJQ200mp-ΔphaCABΩpLac L-ldh. 2 μl of the assembly product are transformed into chemo-competent Escherichia coli NEB 5-alpha competent cells (New England Biolabs, Evry, France). The transformants are selected on an LB/Agar medium (Tryptone 10 g/L, yeast extract 5 g/L, NaCl 5 g/L, Sigma-Aldrich, agar 20 g/L) containing 15 μg/mL of tetracycline. The selection of positive clones is carried out by colony PCR using the enzyme DreamTaq Green PCR Master Mix (Thermo Scientific™) and oligonucleotides 22 (5' CATGCAAAGT GCCGGCCAGG 3') and 23 (5' CTGCACGAAC ATGGTGCTGG CT 3 '). After extraction of the plasmids using the NucleoSpin® Plasmid kit (Macherey-Nagel), the correct insertion of the ldh gene, the sequence of the upstream homology zone and the sequence of the downstream homology zone is confirmed by sequencing (Eurofins genomic) with oligonucleotides 21 (5' TGCAAGGCGA TTAAGTTG 3'), 22, 23 and 24 (5' CTGGCACGAC AGGTTTCCCG A 3').

The pJQ200mp-ΔphaCABΩpLac L-ldh plasmid is inserted into an electrocompetent Escherichia coli S17-1 strain by electroporation using a Gene Pulser electroporator, BioRad (Datsenko and Wanner, 2000).

Positive clones are selected by PCR on colonies using the enzyme DreamTaq Green PCR Master Mix (Thermo Scientific™) and oligonucleotides 22 and 23. The clone of interest is isolated on LB/agar medium (Tryptone 10 g/L, yeast extract 5 g/L, NaCl 5 g/L, Sigma-Aldrich, agar 20 g/L) containing 15 µg/mL of tetracycline and incubated overnight at 37°C.

Conjugation between Escherichia coli S17-1 cells and those of the Cupriavidus necator H16 strain (ATCC 17699) was adapted from the protocol of Lenz et al. , 1994. The sucrose selection method (15% sucrose used) with SacB made it possible to precisely delete the phaCAB operon and insert pLac L-ldh into it. After the first homologous recombination, the clones are validated by colony PCR using the enzyme DreamTaq Green PCR Master Mix (Thermo Scientific™)™) and oligonucleotides 22 and 23. After the second homologous recombination, the selection positive clones among the clones sensitive to tetracycline is carried out by colony PCR using the enzyme Kod Xtreme™ Hot Start DNA Polymerase (Novagen) (Krauße et al. , 2009) and oligonucleotides 76 (5' CTGCATGTTC ATGAACTGCG AC 3') and 77 (5' CGACGCGTCA ACCATGTTCG 3').

The Cupriavidus necator H16 ΔphaCABΩpLac_L-ldh _C.necator strain is recovered and named CN0004. Genetic modifications are validated by sequencing using oligonucleotides 78 (5' GTACCTTGCC GACATCTATG C 3'), 79 (5' GCTGCTTGCA TCACGGACTT G 3'), 10, 77 and 76.

Evaluation of the Fructose Strain CN0004 The production of lactate on fructose of the strain Cupriavidus necator H16 ΔphaCABΩpLac_L-ldh C.necator (CN0004) was evaluated according to the method described in Example 3. Under these culture conditions, the strain CN0004 produces 1.5 g/L of lactate after 140 h of culture.

Extrapolation of CN0004 lactate production from CO ₂ Lactate production of strain CN0004 was not achieved from CO₂. However, the fructose evaluation showed that the CN0004 strain produced 25% more lactate than the CN0002 strain under the same culture conditions. We can extrapolate that from CO₂as described in Example 4, strain CN0004 would produce 25% more lactate from CO₂than strain CN0002.

Example 7: Construction of a strain of Cupriavidus necator genetically modified plant in which a polyhydroxybutyrate (PHB) biosynthetic pathway is deleted, an endogenous lactate dehydrogenase is overexpressed and in which a pyruvate carboxylase is deleted (CN0005)

Strain and genetic constructs The construction of the lactate-producing strain CN0005 is carried out according to the following protocol:

A plasmid carrying the sequences of the regions of homology upstream and downstream of the gene encoding the pyruvate carboxylase (pyc) of Cupriavidus necator is cloned in one step in vitro using the assembly protocol NEBuilder ^® HiFi DNA Assembly Cloning (New England Biolabs, Évry, France). Oligonucleotides are synthesized and purified (desalted) by Eurofins Genomics.

The sequence of the region of homology upstream of the gene coding for pyc is amplified by PCR on the genomic DNA of the Cupriavidus necator H16 strain using oligonucleotides 25 (5' AGATCCTTTA ATTCGAGCTC GGTACCGCAT GGCCAAGGTG GAAGAG 3') presenting at 5 'a zone of overlap of 25 base pairs with the 3' end of the plasmid pLO3 (Lenz and Friedrich, 1998) and 26 (5' TGCCGGCCAA CGTCACATGG GATGCAGGGA AGCGAAC 3') presenting in 5' a zone of overlap of 15 pairs of bases with the 5' end of the region of downstream homology of the gene coding for pyc and of the enzyme KOD Hot Start DNA Polymerase (Novagen) with 5% DMSO. The PCR product is purified on gel with the Nucleospin gel and PCR clean-up kit (Macherey-Nagel).

The sequence of the downstream homology zone of the gene coding for pyc is amplified by PCR on the genomic DNA of the Cupriavidus necator H16 strain using oligonucleotides 27 (5' TCCCTGCATC CCATGTGACG TTGGCCGGCA GGG 3') presenting at 5' a zone of overlap of 15 base pairs with the 3' end of the zone of homology upstream of the gene coding for pyc and 28 (5' ACTTAATTAA GGATCCGGCG CGCCCCCCGG GCTGATAGTT CTTCAACACC AGCAGTC 3') presenting in 5' an overlap zone of 31 base pairs with the 5' end of the pLO3 plasmid and the KOD Hot Start DNA Polymerase enzyme (Novagen) with 5% DMSO. The PCR product is purified on gel with the Nucleospin gel and PCR clean-up kit (Macherey-Nagel).

The backbone plasmid pLO3 (Lenz and Friedrich, 1998) is cut with the restriction enzyme XmaI-HF (New England Biolabs, Evry, France). The digestion product is purified on gel with the Nucleospin gel and PCR clean-up kit (Macherey-Nagel).

In vitro assembly by ^NEBuilder® HiFi DNA Assembly Cloning (New England Biolabs, Evry, France), is carried out with the three fragments in order to obtain the plasmid pLO3-Δpyc. 2 μl of the assembly product are transformed into chemo-competent Escherichia coli NEB 5-alpha competent cells (New England Biolabs, Evry, France). The transformants are selected on an LB/Agar medium (Tryptone 10 g/L, yeast extract 5 g/L, NaCl 5 g/L, Sigma-Aldrich, agar 20 g/L) containing 15 μg/mL of tetracycline. The selection of the positive clones is carried out by colony PCR using the DreamTaq Green PCR Master Mix enzyme (Thermo Scientific™) and oligonucleotides 51 and 80 (5' AATGGGACGT GCGAGTCCAT CC 3'). After extraction of the plasmids using the NucleoSpin® Plasmid kit (Macherey-Nagel), the deletion of the pyc gene is confirmed by sequencing (Eurofins genomic) with the oligonucleotides 29 (5' GCAAACAAAC CACCGCTGGT 3'), 30 (5' CGCCATATCG GATGCCGTTC 3') and 31 (5' TAGCAGCACG CCATAGTGAC TG 3').

The pLO3-Δpyc plasmid is inserted into an electrocompetent Escherichia coli S17-1 strain by electroporation using a Gene Pulser, BioRad electroporator (Datsenko and Wanner, 2000).

Positive clones are selected by colony PCR using the enzyme DreamTaq Green PCR Master Mix (Thermo Scientific™) and oligonucleotides 51 and 80. The clone of interest is isolated on LB/agar medium (Tryptone 10 g/L, yeast extract 5 g/L, NaCl 5 g/L, Sigma-Aldrich, agar 20 g/L) containing 15 µg/mL of tetracycline and incubated overnight at 37°C.

The conjugation between the Escherichia coli S17-1 cells and those of the CN0004 strain was adapted from the protocol of Lenz et al. , 1994. The sucrose selection method (15% sucrose used) with SacB made it possible to precisely delete the gene coding for pyc. After the first homologous recombination, the clones are validated by colony PCR using the enzyme DreamTaq Green PCR Master Mix (Thermo Scientific™) and oligonucleotides 51 and 80. After the second homologous recombination, the selection of clones positive among the tetracycline-sensitive clones is carried out by colony PCR using the enzyme Kod Xtreme™ Hot Start DNA Polymerase (Novagen) and oligonucleotides 81 (5' CCATTTGCCC AATACGACTA GC 3') and 30.

The Cupriavidus necator H16 Δpyc ΔphaCABΩpLac_L-ldh C.necator strain is recovered and named CN0005. Genetic modifications are validated by sequencing.

Evaluation of the Fructose Strain CN0005 The production of lactate on fructose of the strain Cupriavidus necator H16 Δpyc ΔphaCABΩpLac_L-ldh C.necator (CN0005) was evaluated according to the method described in Example 3. Under these culture conditions, the strain CN0005 produces 21% more lactate than strain CN0004 after 51 h of culture.

Extrapolation of CN0005 lactate production from CO ₂ Lactate production of strain CN0005 was not achieved from CO₂. However, the fructose evaluation showed that the CN0005 strain produced 21% more lactate than the CN0004 strain under the same culture conditions. We can extrapolate that from CO₂as described in Example 4, strain CN0005 would produce 25% more lactate from CO₂than strain CN0004.

Example 8: Construction of a strain of Cupriavidus necator genetically modified plant in which a polyhydroxybutyrate (PHB) biosynthetic pathway is deleted, an endogenous lactate dehydrogenase is overexpressed and in which a pyruvate dehydrogenase is deleted (CN0006)

Strain and genetic constructs The construction of the lactate-producing strain CN0006 is carried out according to the following protocol:

A plasmid carrying the sequences of the regions of homology upstream and downstream of the gene coding for the E1 component of the pyruvate dehydrogenase (pdhA2) of Cupriavidus necator is cloned in one step in vitro via the assembly protocol NEBuilder ^® HiFi DNA Assembly Cloning ( New England Biolabs, Évry, France). Oligonucleotides are synthesized and purified (desalted) by Eurofins Genomics.

The sequence of the region of homology upstream of the gene coding for pdhA2 is amplified by PCR on the genomic DNA of the Cupriavidus necator H16 strain using oligonucleotides 32 (5' TCCTTTAATT CGAGCTCGGT ACCCGGGTGC GTAATCCACT TCCAG 3') presenting at 5 'a zone of overlap of 22 base pairs with the 3' end of the plasmid pLO3 (Lenz and Friedrich, 1998) and 33 (5' CCCATCGTTC ACACGGCAAG TCTCCGTTAA GGAATTC 3') presenting in 5' a zone of overlap of 11 pairs of bases with the 5' end of the downstream homology zone of the gene coding for pdhA2 and of the enzyme KOD Hot Start DNA Polymerase (Novagen) with 5% DMSO. The PCR product is purified on gel with the Nucleospin gel and PCR clean-up kit (Macherey-Nagel).

The sequence of the downstream homology zone of the gene coding for pdhA2 is amplified by PCR on the genomic DNA of the strainCupriavidus necatorH16 using oligonucleotides 34 (5' GACTTGCCGT GTGAACGATG GGCCATCGGG CA 3') presenting in 5' a zone of overlap of 11 base pairs with the 3' end of the zone of homology upstream of the gene coding for pdhA2 and 35 (5' TAAGGATCCG GCGCGCCCCC GGGTTGAGCA GGATCACGTC GATCC 3') exhibiting at 5′ a zone of overlap of 22 base pairs with the 5′ end of the plasmid pLO3 and of the KOD Hot Start DNA Polymerase enzyme (Novagen) with 5% DMSO. The PCR product is purified on gel with the Nucleospin gel and PCR clean-up kit (Macherey-Nagel).

The backbone plasmid pLO3 (Lenz and Friedrich, 1998) is cut with the restriction enzyme XmaI-HF (New England Biolabs, Evry, France). The digestion product is purified on gel with the Nucleospin gel and PCR clean-up kit (Macherey-Nagel).

In vitro assembly by ^NEBuilder® HiFi DNA Assembly Cloning (New England Biolabs, Evry, France), is carried out with the three fragments in order to obtain the plasmid pLO3-ΔpdhA2. 2 μl of the assembly product are transformed into chemo-competent Escherichia coli NEB 5-alpha competent cells (New England Biolabs, Evry, France). The transformants are selected on an LB/Agar medium (Tryptone 10 g/L, yeast extract 5 g/L, NaCl 5 g/L, Sigma-Aldrich, agar 20 g/L) containing 15 μg/mL of tetracycline. The selection of the positive clones is carried out by PCR on colonies using the enzyme DreamTaq Green PCR Master Mix (Thermo Scientific™) and oligonucleotides 29 and 82 (5′ ATGTCCTGGA TTGCGCAGAT C 3′). After extraction of the plasmids using the NucleoSpin® Plasmid kit (Macherey-Nagel), the deletion of the pdhA2 gene is confirmed by sequencing (Eurofins genomic) with the oligonucleotides 36 (5' TAATCCACTT CCAGCGCGAT AAG 3'), 37 (5' CCTGAAGTCT CCGCGATAAC 3'), 38 (5' GTTCGAAGCC ACCGAGTATG AC 3') and 29.

The pLO3-ΔpdhA2 plasmid is inserted into an electrocompetent Escherichia coli S17-1 strain by electroporation using a Gene Pulser, BioRad electroporator (Datsenko and Wanner, 2000).

Positive clones are selected by colony PCR using the enzyme DreamTaq Green PCR Master Mix (Thermo Scientific™) and oligonucleotides 29 and 82. The clone of interest is isolated on LB/agar medium (Tryptone 10 g/L, yeast extract 5 g/L, NaCl 5 g/L, Sigma-Aldrich, agar 20 g/L) containing 15 µg/mL of tetracycline and incubated overnight at 37°C.

The conjugation between the Escherichia coli S17-1 cells and those of the CN0004 strain was adapted from the protocol of Lenz et al. , 1994. The sucrose selection method (15% sucrose used) with SacB made it possible to precisely delete the gene coding for pdhA2. After the first homologous recombination, the clones are validated by colony PCR using the enzyme DreamTaq Green PCR Master Mix (Thermo Scientific™) and oligonucleotides 29 and 82. After the second homologous recombination, the selection of clones positive among the tetracycline-sensitive clones is carried out by colony PCR using the enzyme Kod Xtreme™ Hot Start DNA Polymerase (Novagen).

The Cupriavidus necator H16 ΔpdhA2 ΔphaCABΩpLac_L-ldh _C.necator strain is recovered and named CN0006. Genetic modifications are validated by sequencing.

Evaluation of the Fructose Strain CN0006 The production of lactate on fructose of the strain Cupriavidus necator H16 ΔpdhA2 ΔphaCABΩpLac_L-ldh _C.necator (CN0006) was evaluated according to the method described in Example 3. Under these culture conditions, the strain CN0006 produces 13% more lactate than strain CN0004 after 51 h of culture.

Extrapolation of CN0006 lactate production from CO ₂ Lactate production of strain CN0006 was not achieved from CO₂. However, the fructose evaluation showed that the CN0006 strain produced 13% more lactate than the CN0004 strain under the same culture conditions. We can extrapolate that from CO₂as described in Example 4, strain CN0006 would produce 13% more lactate from CO₂than strain CN0004.

Example 9: Construction of a strain of Cupriavidus necator genetically modified plant in which a polyhydroxybutyrate (PHB) biosynthetic pathway is deleted, an endogenous lactate dehydrogenase is overexpressed and in which a phosphate acetyltransferase and an acetate kinase phosphoenolpyruvate synthase are deleted (CN0007)

Strain and Genetic Constructs

The construction of the CN0007 lactate-producing strain is carried out according to the following protocol:

A plasmid carrying the sequences of the regions of homology upstream and downstream of the operon coding for the acetate kinase (ackA) and phosphotransacetylase (pta1) genes of Cupriavidus necator is cloned in one step in vitro via the NEBuilder ^® HiFi assembly protocol DNA Assembly Cloning (New England Biolabs, Evry, France). Oligonucleotides are synthesized and purified (desalted) by Eurofins Genomics.

The sequence of the homology zone upstream of the gene coding for pta1 is amplified by PCR on the genomic DNA of the Cupriavidus necator H16 strain using oligonucleotides 39 (5' TCCTTTAATT CGAGCTCGGT ACGTGTCCAA TGAGATGACA GCACG 3') presenting at 5 ' a zone of overlap of 22 base pairs with the 3' end of the plasmid pLO3 (Lenz and Friedrich, 1998) and 40 (5' TGTAGCGGTG GTGCGTCAGG GTCGTCGGTG 3') presenting in 5' an overlap zone of 11 base pairs with the 5' end of the region of downstream homology of the gene coding for ackA and of the enzyme KOD Hot Start DNA Polymerase (Novagen) with 5% DMSO. The PCR product is purified on gel with the Nucleospin gel and PCR clean-up kit (Macherey-Nagel).

The sequence of the downstream homology zone of the gene coding for ackA is amplified by PCR on the genomic DNA of the Cupriavidus necator H16 strain using oligonucleotides 41 (5' ACCCTGACGC ACCACCGCTA CAGCCGACCA AG 3') presenting at 5' a zone of overlap of 11 base pairs with the 3' end of the zone of homology upstream of the gene coding for pta1 and 42 (5' TAAGGATCCG GCGCGCCCCC GGGCTGATAC GTTCACGCAT AGTGGTC 3') presenting in 5' an overlap zone of 22 base pairs with the 5' end of the pLO3 plasmid and the enzyme KOD Hot Start DNA Polymerase (Novagen) with 5% DMSO. The PCR product is purified on gel with the Nucleospin gel and PCR clean-up kit (Macherey-Nagel).

The backbone plasmid pLO3 (Lenz and Friedrich, 1998) is cut with the restriction enzyme XmaI-HF (New England Biolabs, Evry, France). The digestion product is purified on gel with the Nucleospin gel and PCR clean-up kit (Macherey-Nagel).

In vitro assembly by ^NEBuilder® HiFi DNA Assembly Cloning (New England Biolabs, Evry, France), is carried out with the three fragments in order to obtain the plasmid pLO3-Δpta1-ackA. 2 μl of the assembly product are transformed into chemo-competent Escherichia coli NEB 5-alpha competent cells (New England Biolabs, Evry, France). The transformants are selected on an LB/Agar medium (Tryptone 10 g/L, yeast extract 5 g/L, NaCl 5 g/L, Sigma-Aldrich, agar 20 g/L) containing 15 μg/mL of tetracycline. The selection of the positive clones is carried out by colony PCR using the enzyme DreamTaq Green PCR Master Mix (Thermo Scientific™) and oligonucleotides 29 and 43 (5' GACTTCCGGC AGGTCATGC 3'). After extraction of the plasmids using the NucleoSpin® Plasmid kit (Macherey-Nagel), the deletion of the ackA-pta1 operon is confirmed by sequencing (Eurofins genomic) with oligonucleotides 43, 44 (5' CAGTTGTTGC GCTGCAGTCA T 3' ), 45 (5' GCCAAGCCGG AACGCGTC 3') and 46 (5' GATGGTGGCA CGATGTTCAC 3').

The pLO3-Δpta1-ackA plasmid is inserted into an electrocompetent Escherichia coli S17-1 strain by electroporation using a Gene Pulser, BioRad electroporator (Datsenko and Wanner, 2000).

Positive clones are selected by PCR on colonies using the enzyme DreamTaq Green PCR Master Mix (Thermo Scientific™) and oligonucleotides 29 and 43. The clone of interest is isolated on LB/agar medium (Tryptone 10 g/L, yeast extract 5 g/L, NaCl 5 g/L, Sigma-Aldrich, agar 20 g/L) containing 15 µg/mL of tetracycline and incubated overnight at 37°C.

The conjugation between the Escherichia coli S17-1 cells and those of the CN0004 strain was adapted from the protocol of Lenz et al. , 1994. The sucrose selection method (15% sucrose used) with SacB allowed to precisely delete the pta1-ackA operon. After the first homologous recombination, the clones are validated by colony PCR using the enzyme DreamTaq Green PCR Master Mix (Thermo Scientific™) and oligonucleotides 29 and 43. After the second homologous recombination, the selection of clones positive among the tetracycline-sensitive clones is carried out by colony PCR using the enzyme Kod Xtreme™ Hot Start DNA Polymerase (Novagen) and oligonucleotides 44 and 83 (5' CAGAACTGGC TGTTCTCGGA C 3').

The Cupriavidus necator H16 Δpta1-ackA ΔphaCABΩpLac_L-ldh _C.necator strain is recovered and named CN0007. Genetic modifications are validated by sequencing.

Evaluation of the Fructose Strain CN0007 The production of lactate on fructose of the strain Cupriavidus necator H16 Δpta1-ackA ΔphaCABΩpLac_L-ldh C.necator (CN0007) was evaluated according to the method described in Example 3. Under these culture conditions, the CN0007 strain produces 11% more lactate than the CN0004 strain after 51 h of culture.

Extrapolation of CN0007 lactate production from CO ₂ Lactate production of strain CN0007 was not achieved from CO₂. However, the fructose evaluation showed that the CN0007 strain produced 11% more lactate than the CN0004 strain under the same culture conditions. We can extrapolate that from CO₂as described in Example 4, strain CN0007 would produce 11% more lactate from CO₂than strain CN0004.

Example 10: Construction of a strain of Cupriavidus necator genetically modified plant in which a polyhydroxybutyrate (PHB) biosynthetic pathway is deleted, an endogenous lactate dehydrogenase is overexpressed and in which a lactate ferricytochrome C reductase is deleted (CN0008)

Strain and genetic constructs. The construction of the CN0008 lactate-producing strain is carried out according to the following protocol:

A plasmid carrying the sequences of the regions of homology upstream and downstream of the active site of the L-Lactate cytochrome c reductase (lldD, 1.1.2.3) of Cupriavidus necator is cloned in one step in vitro via the NEBuilder ^® HiFi assembly protocol DNA Assembly Cloning (New England Biolabs, Evry, France). Oligonucleotides are synthesized and purified (desalted) by Eurofins Genomics.

The sequence of the homology zone upstream of the gene coding for lldD is amplified by PCR on the genomic DNA of the strainCupriavidus necatorH16 using oligonucleotides 47 (5' CCTGCAGGTC GACTCTAGAG AGCAATTGCT CCGCCATCAG C 3') presenting in 5' a zone of overlap of 20 base pairs with the 3' end of the plasmid pJQ200mpTet and 48 (5' AGTCGATGGC CACTTGGCGG CGCAAGGTAC 3') presenting in 5' a zone of overlap of 10 base pairs with the 5' end of the zone of homology downstream of the gene coding for lldD and of the enzyme KOD Hot Start DNA Polymerase (Novagen) with 5% DMSO. The PCR product is purified on gel with the Nucleospin gel and PCR clean-up kit (Macherey-Nagel).

The sequence of the region of homology downstream of the gene coding for lldD is amplified by PCR on the genomic DNA of the Cupriavidus necator H16 strain using oligonucleotides 49 (5' CCGCCAAGTG GCCATCGACT TGTTGCAGGC 3') presenting in 5' a overlapping zone of 10 base pairs with the 3' end of the zone of homology upstream of the gene coding for lldD and 50 (5' ATTCGAGCTC GGTACCCGGG CAAAGGCTGC GTCCAGCCAG 3') presenting in 5' an overlapping zone of 20 pairs of bases with the 5' end of the plasmid pJQ200mpTet and the enzyme KOD Hot Start DNA Polymerase (Novagen) with 5% DMSO. The PCR product is purified on gel with the Nucleospin gel and PCR clean-up kit (Macherey-Nagel).

The backbone plasmid pJQ200mpTet is a derivative of the plasmid pJQ200mp18 (Quandt and Hynes, 1993) where the gentamycin resistance gene has been replaced by the tetracycline resistance gene. The backbone plasmid pJQ200mpTet is cut with the restriction enzyme BamHI-HF (New England Biolabs, Evry, France). The digestion product is purified on gel with the Nucleospin gel and PCR clean-up kit (Macherey-Nagel).

In vitro assembly by ^NEBuilder® HiFi DNA Assembly Cloning (New England Biolabs, Evry, France), is carried out with the three fragments in order to obtain the plasmid pJQ200mpTet-ΔlldD. 2 μl of the assembly product are transformed into chemo-competent Escherichia coli NEB 5-alpha competent cells (New England Biolabs, Evry, France). The transformants are selected on an LB/Agar medium (Tryptone 10 g/L, yeast extract 5 g/L, NaCl 5 g/L, Sigma-Aldrich, agar 20 g/L) containing 15 μg/mL of tetracycline. The selection of positive clones is carried out by colony PCR using the enzyme DreamTaq Green PCR Master Mix (Thermo Scientific™) and oligonucleotides 24 and 86 (5' GACAACCTGA TGGTGCACAA GG 3'). After extraction of the plasmids using the NucleoSpin® Plasmid kit (Macherey-Nagel), the deletion of the lldD gene is confirmed by sequencing (Eurofins genomic) with the oligonucleotides 24, 51 (5' TGCAAGGCGA TTAAGTTGGG TAACG 3') and 52 ( 5' GAACAGCTGC ACGCCGAG 3').

The pJQ200mpTet -ΔlldD plasmid is inserted into an electrocompetent Escherichia coli S17-1 strain by electroporation using a Gene Pulser electroporator, BioRad (Datsenko and Wanner, 2000).

Positive clones are selected by colony PCR using the enzyme DreamTaq Green PCR Master Mix (Thermo Scientific™) and oligonucleotides 24 and 86. The clone of interest is isolated on LB/agar medium (Tryptone 10 g/L, yeast extract 5 g/L, NaCl 5 g/L, Sigma-Aldrich, agar 20 g/L) containing 15 µg/mL of tetracycline and incubated overnight at 37°C.

The conjugation between the Escherichia coli S17-1 cells and those of the CN0004 strain was adapted from the protocol of Lenz et al. , 1994. The sucrose selection method (15% sucrose used) with SacB made it possible to precisely delete the gene coding for lldD. After the first homologous recombination, the clones are validated by colony PCR using the enzyme DreamTaq Green PCR Master Mix (Thermo Scientific™) and oligonucleotides 24 and 86. After the second homologous recombination, the selection of clones positive among the tetracycline-sensitive clones is carried out by colony PCR using the enzyme Kod Xtreme™ Hot Start DNA Polymerase (Novagen)) and oligonucleotides 52 and 87 (5' CACATTGCGT CGCTGACATT G 3').

The Cupriavidus necator H16 ΔlldD ΔphaCABΩpLac_L-ldh _C.necator strain is recovered and named CN0008. Genetic modifications are validated by sequencing using oligonucleotides 88 (5' AGCTGTTGCA CACCAGGCGA C 3'), 89 (5' CATTGCCATA TCGCGCGAGA TC 3'), 90 (5' CTATCGATCATGTCAGCTCTTCATG 3') and 91 (5' CACTCCGTAC ACAATGACCA CG 3').

Evaluation of the Fructose Strain CN0008 The production of lactate on fructose of the strain Cupriavidus necator H16 ΔlldD ΔphaCABΩpLac_L-ldh C.necator (CN0008) was evaluated according to the method described in Example 3. Under these culture conditions, the strain CN0008 produces 1.4 g/L of lactate after 140 h of culture.

Extrapolation of CN0008 lactate production from CO ₂ Lactate production of strain CN0008 was not achieved from CO₂. However, the fructose evaluation showed that the CN0008 strain produced 7% less lactate than the CN0004 strain under the same culture conditions. We can extrapolate that from CO₂as described in Example 4, strain CN0008 would produce 7% less lactate from CO₂than strain CN0004.

Example 11: Construction of a strain of Cupriavidus necator genetically modified plant in which a polyhydroxybutyrate (PHB) biosynthetic pathway is deleted, an endogenous lactate dehydrogenase is overexpressed and in which two lactate ferricytochrome C reductases are deleted (CN0009)

Strain and genetic constructions The construction of the lactate-producing strain CN0009 is carried out according to the following protocol:

A plasmid carrying the sequences of the regions of homology upstream and downstream of the gene coding for L-Lactate cytochrome reductase (lldA) of Cupriavidus necator is cloned in one step in vitro using the NEBuilder ^® HiFi DNA Assembly Cloning protocol (New England Biolabs, Évry, France). Oligonucleotides are synthesized and purified (desalted) by Eurofins Genomics.

The sequence of the region of homology upstream of the gene coding for lldA is amplified by PCR on the genomic DNA of the Cupriavidus necator H16 strain using oligonucleotides 53 (5' CCTGCAGGTC GACTCTAGAG GATCCGCAAG ACGGTTTATC TCTCGGTC 3') presenting at 5 ' a zone of overlap of 25 base pairs with the 3' end of the plasmid pJQ200mpTet and 54 (5' GACGCTATCA CATGGGAACT CCCTTGAAAA AAACAAAAAG CTGC 3') presenting in 5' a zone of overlap of 10 base pairs with the end 5 'of the region of downstream homology of the gene coding for lldA and of the enzyme KOD Hot Start DNA Polymerase (Novagen) with 5% DMSO. The PCR product is purified on gel with the Nucleospin gel and PCR clean-up kit (Macherey-Nagel).

The sequence of the region of homology downstream of the gene coding for lldA is amplified by PCR on the genomic DNA of the Cupriavidus necator H16 strain using oligonucleotides 55 (5' AGTTCCCATG TGATAGCGTC TATGAGGCGT C 3') presenting at 5' an overlapping zone of 10 base pairs with the 3' end of the homology zone upstream of the gene coding for lldA and 56 (5' ATTCGAGCTC GGTACCCGGG GATCGAGGAA ATCGGCTGCG TAGG 3') presenting in 5' an overlapping zone of 24 base pairs with the 5' end of the plasmid pJQ200mpTet and the enzyme KOD Hot Start DNA Polymerase (Novagen) with 5% DMSO. The PCR product is purified on gel with the Nucleospin gel and PCR clean-up kit (Macherey-Nagel).

The backbone plasmid pJQ200mpTet is a derivative of the plasmid pJQ200mp18 (Quandt and Hynes, 1993) where the gentamycin resistance gene has been replaced by the tetracycline resistance gene. The backbone plasmid pJQ200mpTet is cut with the restriction enzyme BamHI-HF (New England Biolabs, Evry, France). The digestion product is purified on gel with the Nucleospin gel and PCR clean-up kit (Macherey-Nagel).

In vitro assembly by ^NEBuilder® HiFi DNA Assembly Cloning (New England Biolabs, Evry, France), is carried out with the three fragments in order to obtain the plasmid pJQ200mpTet-ΔlldA. 2 μl of the assembly product are transformed into competent Escherichia coli NEB 5-alpha cells (New England Biolabs, Evry, France), chemo-competent. The transformants are selected on an LB/Agar medium (Tryptone 10 g/L, yeast extract 5 g/L, NaCl 5 g/L, Sigma-Aldrich, agar 20 g/L) containing 15 μg/mL of tetracycline. Positive clones are selected by colony PCR using the enzyme DreamTaq Green PCR Master Mix (Thermo Scientific™) and oligonucleotides 92 (5' TGAGTCCAAC CCGGTAAGAC 3') and 57 (5' CCTCATAGAC GCTATCACAT GG 3 '). After extraction of the plasmids using the NucleoSpin® Plasmid kit (Macherey-Nagel), the deletion of the lldA gene is confirmed by sequencing (Eurofins genomic) with oligonucleotides 21, 57, 58 (5' CCATGTGATAGCGTCTATGAGG 3') and 24.

The pJQ200mpTet -ΔlldA plasmid is inserted into an electrocompetent Escherichia coli S17-1 strain by electroporation using a Gene Pulser electroporator, BioRad (Datsenko and Wanner, 2000).

Positive clones are selected by colony PCR using the enzyme DreamTaq Green PCR Master Mix (Thermo Scientific™) and oligonucleotides 92 and 57. The clone of interest is isolated on LB/agar medium (Tryptone 10 g/L, yeast extract 5 g/L, NaCl 5 g/L, Sigma-Aldrich, agar 20 g/L) containing 15 µg/mL of tetracycline and incubated overnight at 37°C.

The conjugation between the Escherichia coli S17-1 cells and those of the CN0008 strain was adapted from the protocol of Lenz et al. , 1994. The sucrose selection method (15% sucrose used) with SacB made it possible to precisely delete the gene coding for lldA. After the first homologous recombination, the clones are validated by colony PCR using the enzyme DreamTaq Green PCR Master Mix (Thermo Scientific™) and oligonucleotides 92 and 57. After the second homologous recombination, the selection of clones positive among the tetracycline-sensitive clones is carried out by colony PCR using the enzyme Kod Xtreme™ Hot Start DNA Polymerase (Novagen) and oligonucleotides 93 (5' CTGGCTGAAC GTGAAGAATG C 3') and 94 (5' GGAAGATAAA CCCGAGCAGA TC 3').

The Cupriavidus necator H16 ΔlldA ΔlldD ΔphaCABΩpLac_L-ldh _C.necator strain is recovered and named CN0009. The genetic modifications are validated by sequencing using oligonucleotides 57, 58, 95 (5' CAGGCTTATC GACAGAGAAA TTCG 3') and 96 (5' GTATTTCGCA TCGTGCCAGA C 3').

Evaluation of the Fructose Strain CN0009 The production of lactate on fructose of the strain Cupriavidus necator H16 ΔlldD ΔphaCABΩpLac_L-ldh C.necator (CN0009) was evaluated according to the method described in Example 3. Under these culture conditions, the strain CN0009 produces 1.4 g/L of lactate after 140 h of culture.

Extrapolation of CN0009 lactate production from CO ₂ Lactate production of strain CN0009 was not achieved from CO₂. However, the fructose evaluation showed that the CN0009 strain produced 7% less lactate than the CN0004 strain under the same culture conditions. We can extrapolate that from CO₂as described in Example 4, strain CN0009 would produce 7% less lactate from CO₂than strain CN0004.

Example 12: Construction of a strain of Cupriavidus necator genetically modified plant in which a polyhydroxybutyrate (PHB) biosynthetic pathway is deleted, an endogenous lactate dehydrogenase is overexpressed and in which a phosphoenolpyruvate synthase is deleted (CN0010)

Strain and genetic constructs. The construction of the CN0010 lactate-producing strain is carried out according to the following protocol:

A plasmid carrying the sequences of the regions of homology upstream and downstream of the gene coding for the phosphoenolpyruvate synthase (ppsA) of Cupriavidus necator is cloned in one step in vitro using the NEBuilder ^® HiFi DNA Assembly Cloning protocol (New England Biolabs, Évry, France). Oligonucleotides are synthesized and purified (desalted) by Eurofins Genomics.

The sequence of the homology zone upstream of the gene coding for ppsA is amplified by PCR on the genomic DNA of the Cupriavidus necator H16 strain using oligonucleotides 59 (5' AGATCCTTTA ATTCGAGCTC GGTACCCGAA GATCTTCGGC TTGAACG 3') presenting at 5 ' a zone of overlap of 25 base pairs with the 3' end of the plasmid pLO3 and 60 (5' ACGTCAAATG CTTCACATGT CCGGTATGTT CTTGGAGTTC 3') presenting in 5' a zone of overlap of 15 base pairs with the 5' end of the downstream homology zone of the gene coding for ppsA and of the enzyme KOD Hot Start DNA Polymerase (Novagen) with 5% DMSO. The PCR product is purified on gel with the Nucleospin gel and PCR clean-up kit (Macherey-Nagel).

The sequence of the downstream homology zone of the gene coding for ppsA is amplified by PCR on the genomic DNA of the Cupriavidus necator H16 strain using oligonucleotides 61 (5' AACATACCGG ACATGTGAAG CATTTGACGT CACAATAACG 3') presenting at 5' a zone of overlap of 15 base pairs with the 3' end of the zone of homology upstream of the gene coding for ppsA and 62 (5' ACTTAATTAA GGATCCGGCG CGCCCCTTGA GCACGTGCT TGTAGG 3') presenting in 5' an overlap zone of 25 base pairs with the 5' end of the pLO3 plasmid and the enzyme KOD Hot Start DNA Polymerase (Novagen) with 5% DMSO. The PCR product is purified on gel with the Nucleospin gel and PCR clean-up kit (Macherey-Nagel).

The backbone plasmid pLO3 (Lenz and Friedrich, 1998) is cut with the restriction enzyme XmaI-HF (New England Biolabs, Evry, France). The digestion product is purified on gel with the Nucleospin gel and PCR clean-up kit (Macherey-Nagel).

In vitro assembly by ^NEBuilder® HiFi DNA Assembly Cloning (New England Biolabs, Evry, France), is carried out with the three fragments in order to obtain the plasmid pLO3-ΔppsA. 2 μl of the assembly product are transformed into chemo-competent Escherichia coli NEB 5-alpha competent cells (New England Biolabs, Evry, France). The transformants are selected on an LB/Agar medium (Tryptone 10 g/L, yeast extract 5 g/L, NaCl 5 g/L, Sigma-Aldrich, agar 20 g/L) containing 15 μg/mL of tetracycline. The selection of positive clones is carried out by colony PCR using the enzyme DreamTaq Green PCR Master Mix (Thermo Scientific™). After extraction of the plasmids using the NucleoSpin® Plasmid kit (Macherey-Nagel), the deletion of the ppsA gene is confirmed by sequencing (Eurofins genomic) with the oligonucleotides 63 (5' ATGAACACCG GCACCTTCTA CC 3'), 64 (5' CAGGATGGAG TGGCTGAACG 3') and 29.

The pLO3-ΔppsA plasmid is inserted into an electrocompetent Escherichia coli S17-1 strain by electroporation using a Gene Pulser, BioRad electroporator (Datsenko and Wanner, 2000).

Positive clones are selected by colony PCR using the enzyme DreamTaq Green PCR Master Mix (Thermo Scientific™). The clone of interest is isolated on LB/agar medium (Tryptone 10 g/L, yeast extract 5 g/L, NaCl 5 g/L, Sigma-Aldrich, agar 20 g/L) containing 15 μg/mL of tetracycline and incubated overnight at 37°C.

The conjugation between the Escherichia coli S17-1 cells and those of the CN0004 strain was adapted from the protocol of Lenz et al. , 1994. The sucrose selection method (15% sucrose used) with SacB made it possible to precisely delete the gene coding for ppsA. After the first homologous recombination, the clones are validated by colony PCR using the enzyme DreamTaq Green PCR Master Mix (Thermo Scientific™). After the second homologous recombination, the selection of the positive clones among the tetracycline-sensitive clones is carried out by colony PCR using the enzyme Kod Xtreme™ Hot Start DNA Polymerase (Novagen).

The Cupriavidus necator H16 ΔppsA ΔphaCABΩpLac_L-ldh C.necator strain is recovered and named CN0010. Genetic modifications are validated by sequencing.

Example 13: Evaluation of lactate consumption by strains CN0004 and CN0009

Strains For the evaluation of lactate consumption, strains CN0004 ( Cupriavidus necator H16 ΔphaCABΩpLac_L-ldh _C.necator ) and CN0009 ( Cupriavidus necator H16 ΔlldA ΔlldD ΔphaCABΩpLac_L-ldh _C.necator ) are used.

Media The rich medium consisted of 27.5% (w/v) trypticase soy broth (TSB, Becton Dickinson, France). The minimum medium used for the precultures and the cultures in Erlenmeyers (MMR medium) contained: 4.0 g/L of NaH ₂ PO ₄ 2H ₂ O; 4.6 g/L of Na ₂ HPO ₄ 12H ₂ O; 0.45 g/LK ₂ SO ₄ ; 0.39 g/L of MgSO ₄ 7H ₂ O; 0.062 g/L of CaCl ₂ 2H ₂ O and 1 ml/L of trace element solution. The trace element solution contained: 15 g/L of FeSO ₄ 7H ₂ O; 2.4 g/L MnSO ₄ H ₂ O; 2.4 g/L of ZnSO ₄ 7H ₂ O and 0.48 g/L of CuSO ₄ 5H ₂ O in 0.1 M HCl. NH ₄ Cl (1 g/L) was used as a nitrogen source to reach a biomass concentration of approximately 2 g/L. 0.04 g/L of NaOH was added. For the precultures, fructose (20 g/L) was used as a carbon source. For cultures, lactate (6 g/L) was used as the carbon source.

Inoculation chain A clone of strain CN0004 and a clone of strain CN0009 transplanted from a culture plate were first cultured for 8 h in 5 mL of rich medium with gentamycin (10 μg/mL) , in culture tubes, at 30° C. with stirring (200 rpm). This first preculture was used to inoculate the second preculture at an initial OD ₆₀₀ of 0.05 in 25 mL of MMR medium + 20 g/L of fructose in 250 mL baffled Erlenmeyer flasks which were incubated for 20 h at 30° C, 200 rpm.

Culture in Erlenmeyer flask The second preculture was centrifuged, the cell pellet was washed twice with MMR without carbon source and used to inoculate the culture in a 500mL Erlenmeyer flask in 50 mL of MMR medium + 6g/L of lactate in the presence of gentamicin (10 µg/mL). The initial target OD ₆₀₀ was 0.2. Culture in the presence of lactate as the sole carbon source was carried out at a temperature of 30° C. and stirring at 200 rpm. A growth phase lasting 8 hours was carried out until the total consumption of lactate.

Sampling and analysis protocol Samples of 1mL were taken regularly during the culture. Growth was monitored by optical density (OD) measurement at 600 nm. Lactate consumption was analyzed by HPLC as described in example 1. The samples were centrifuged for 5 min at 13,000 rpm and the supernatants were filtered before being analyzed by HPLC. Calibration varied from 0.1 to 5 g/L in water.

ResultsThe growth of strains CN0004 and CN0009 with lactate as the sole carbon source is shown in Figure 7. During the exponential growth phase, the lactate consumption rate for strain CN0004 is 0.89 g/L/h and for strain CN0009 of 0.88 g/L/h. The deletion of the two lactate ferricytochrome C reductases leads to less than 1% reduction in lactate consumption byCupriavidus necator . These results demonstrate that the deletion of the lldD and lldA genes usually described is not sufficient to substantially inhibit the consumption of lactate byCupriavidus necator.

Example 14: Construction of a strain of Cupriavidus necator genetically modified plant in which a polyhydroxybutyrate (PHB) biosynthetic pathway is deleted, an endogenous lactate dehydrogenase is overexpressed, two lactate ferricytochrome C reductases are deleted and in which I' lut1 operon carrying genes coding for For the components of an enzyme complex using lactate is deleted (CN0011).

Strain and genetic constructs:

The construction of the CN0011 lactate-producing strain is carried out according to the following protocol:

A plasmid carrying the sequences of the regions of homology upstream and downstream of the operon comprising the A1390, A1391 and A1392 genes of the AM260479 chromosome of Cupriavidus necator is cloned in one step in vitro using the NEBuilder ^® HiFi DNA Assembly Cloning protocol (New England Biolabs, Évry, France). Oligonucleotides are synthesized and purified (desalted) by Eurofins Genomics.

The sequence of the homology zone upstream of the A1390 gene is amplified by PCR on the genomic DNA of the Cupriavidus necator H16 strain using oligonucleotides 65 (5' AGATCCTTTA ATTCGAGCTC GGTACTCATA GATGCCGGCG GCAATGC 3') presenting in 5' a overlapping zone of 25 base pairs with the 3' end of the plasmid pLO3 and 66 (5' TTTACTGAAG CCTCACATTG TCGACTTCTC CAGACCCG 3') presenting at 5' an overlapping zone of 15 base pairs with the 5' end of the zone of downstream homology of the A1392 gene and the enzyme KOD Hot Start DNA Polymerase (Novagen) with 5% DMSO. The PCR product is purified on gel with the Nucleospin gel and PCR clean-up kit (Macherey-Nagel).

The sequence of the downstream homology zone of the A1392 gene is amplified by PCR on the genomic DNA of the Cupriavidus necator H16 strain using oligonucleotides 67 (5' GAGAAGTCGA CAATGTGAGG CTTCAGTAAA GAACCAC 3') presenting in 5' a zone of overlap of 15 base pairs with the 3' end of the zone of homology upstream of the A1390 gene and 68 (5' ACTTAATTAA GGATCCGGCG CGCCCCCAAG CTGCTGTACG TGTC 3') presenting in 5' a zone of overlap of 25 base pairs with the 5' end of the pLO3 plasmid and of the KOD Hot Start DNA Polymerase enzyme (Novagen) with 5% DMSO. The PCR product is purified on gel with the Nucleospin gel and PCR clean-up kit (Macherey-Nagel).

The backbone plasmid pLO3 (Lenz and Friedrich, 1998) is cut with the restriction enzyme XmaI-HF (New England Biolabs, Evry, France). The digestion product is purified on gel with the Nucleospin gel and PCR clean-up kit (Macherey-Nagel).

In vitro assembly by ^NEBuilder® HiFi DNA Assembly Cloning (New England Biolabs, Evry, France), is carried out with the three fragments in order to obtain the plasmid pLO3-Δlut1. 2 μl of the assembly product are transformed into chemo-competent Escherichia coli NEB 5-alpha competent cells (New England Biolabs, Evry, France). The transformants are selected on an LB/Agar medium (Tryptone 10 g/L, yeast extract 5 g/L, NaCl 5 g/L, Sigma-Aldrich, agar 20 g/L) containing 15 μg/mL of tetracycline. Positive clones are selected by PCR on colonies using the enzyme DreamTaq Green PCR Master Mix (Thermo Scientific™) using oligonucleotides 97 (5' AGGCATTGAC CTTGGTGCGT ATC 3') and 31. After extraction plasmids using the NucleoSpin® Plasmid kit (Macherey-Nagel), the deletion of the lut1 operon is confirmed by sequencing (Eurofins genomic) with oligonucleotides 69 (5' GTGACAGCTC TTTCAGGCTG AC 3'), 31 and 29.

The pLO3-Δlut1 plasmid is inserted into an electrocompetent Escherichia coli S17-1 strain by electroporation using a Gene Pulser, BioRad electroporator (Datsenko and Wanner, 2000).

Positive clones are selected by PCR on colonies using the enzyme DreamTaq Green PCR Master Mix (Thermo Scientific™) and oligonucleotides 97 and 31. The clone of interest is isolated on LB/agar medium (Tryptone 10g/L, yeast extract 5g/L, NaCl 5g/L, Sigma-Aldrich, agar 20g/L) containing 15µg/mL of tetracycline and incubated overnight at 37°C.

The conjugation between the Escherichia coli S17-1 cells and those of the CN0009 strain was adapted from the protocol of Lenz et al. , 1994. The sucrose selection method (15% sucrose used) with SacB allowed to precisely delete the lut1 operon. After the first homologous recombination, the clones are validated by colony PCR using the enzyme DreamTaq Green PCR Master Mix (Thermo Scientific™). After the second homologous recombination, the selection of the positive clones among the clones sensitive to tetracycline is carried out by PCR on colonies using the enzyme Kod Xtreme™ Hot Start DNA Polymerase (Novagen) and oligonucleotides 98 (5' GGTCGGAGAA GACCTGATCG 3') and 69.

The Cupriavidus necator H16 Δlut1 ΔlldA ΔlldD ΔphaCABΩpLac_L-ldh _{C.necator strain} is recovered and named CN0011. Genetic modifications are validated by sequencing using oligonucleotides 99 (5' GCTTCGGTCA CGTAGCCCAT G 3'), 100 (5' CTGGAATTCG TGGATACGCT GAC 3'), 101 (5' TGTCCGCCAG CCTTAACATC AG 3'), and 102 (5 'CTCACCGGAA GTGATGTGCA TTG 3').

Example 15: Construction of a strain of Cupriavidus necator genetically modified plant in which a polyhydroxybutyrate (PHB) biosynthetic pathway is deleted, an endogenous lactate dehydrogenase is overexpressed, two lactate ferricytochrome C reductases are deleted and in which I' lut2 operon carrying genes coding for For the components of an enzyme complex using lactate is deleted (CN0012).

Strain and genetic constructs:

The construction of the CN0012 lactate-producing strain is carried out according to the following protocol:

A plasmid carrying the sequences of the regions of homology upstream and downstream of the lactate utilization operon comprising the genes B0091, B0092, B0093 of the AM260480 chromosome of Cupriavidus necator is cloned in one step in vitro using the NEBuilder ^® HiFi DNA assembly protocol Assembly Cloning (New England Biolabs, Evry, France). Oligonucleotides are synthesized and purified (desalted) by Eurofins Genomics.

The sequence of the region of homology upstream of the B0093 gene is amplified by PCR on the genomic DNA of the Cupriavidus necator H16 strain using oligonucleotides 70 (5' AGATCCTTTA ATTCGAGCTC GGTACGGCTG CGCACGAAGT CGATATG 3') presenting in 5' a overlapping zone of 25 base pairs with the 3' end of the plasmid pLO3 and 71 (5' CGTGATGACGGATTATCATGTCTCGCTCCGGACGTG 3') presenting at 5' an overlapping zone of 15 base pairs with the 5' end of the downstream homology of B0091 gene and KOD Hot Start DNA Polymerase enzyme (Novagen) with 5% DMSO. The PCR product is purified on gel with the Nucleospin gel and PCR clean-up kit (Macherey-Nagel).

The sequence of the downstream homology zone of the B0091 gene is amplified by PCR on the genomic DNA of the Cupriavidus necator H16 strain using oligonucleotides 72 (5' CGGAGCGAGA CATGATAATC CGTCATCACG GGCGCGAG 3') presenting in 5' a zone of overlap of 15 base pairs with the 3' end of the zone of homology upstream of the B0093 and 73 gene (5' ACTTAATTAA GGATCCGGCG CGCCCGCGGC TGCCATACCT TCAGGAAC 3') presenting in 5' a zone of overlap of 25 base pairs with the 5' end of the pLO3 plasmid and of the KOD Hot Start DNA Polymerase enzyme (Novagen) with 5% DMSO. The PCR product is purified on gel with the Nucleospin gel and PCR clean-up kit (Macherey-Nagel).

The backbone plasmid pLO3 (Lenz and Friedrich, 1998) is cut with the restriction enzyme XmaI-HF (New England Biolabs, Evry, France). The digestion product is purified on gel with the Nucleospin gel and PCR clean-up kit (Macherey-Nagel).

In vitro assembly by ^NEBuilder® HiFi DNA Assembly Cloning (New England Biolabs, Evry, France), is carried out with the three fragments in order to obtain the plasmid pLO3-Δlut2. 2 μl of the assembly product are transformed into chemo-competent Escherichia coli NEB 5-alpha competent cells (New England Biolabs, Evry, France). The transformants are selected on an LB/Agar medium (Tryptone 10 g/L, yeast extract 5 g/L, NaCl 5 g/L, Sigma-Aldrich, agar 20 g/L) containing 15 μg/mL of tetracycline. The selection of positive clones is carried out by PCR on colonies using the enzyme DreamTaq Green PCR Master Mix (Thermo Scientific™) and oligonucleotides 31 and 74 (5' GTGGCCGATT GCATGGATCA TC 3'). After extraction of the plasmids using the NucleoSpin® Plasmid kit (Macherey-Nagel), the deletion of the lut2 operon is confirmed by sequencing (Eurofins genomic) with oligonucleotides 29, 74, 75 (5' TGTTGCGCGC CACCTCGTAT TGC 3') and 31.

The pLO3-Δlut2 plasmid is inserted into an electrocompetent Escherichia coli S17-1 strain by electroporation using a Gene Pulser, BioRad electroporator (Datsenko and Wanner, 2000).

Positive clones are selected by PCR on colonies using the enzyme DreamTaq Green PCR Master Mix (Thermo Scientific™) and oligonucleotides 31 and 74. The clone of interest is isolated on LB/agar medium (Tryptone 10 g/L, yeast extract 5 g/L, NaCl 5 g/L, Sigma-Aldrich, agar 20 g/L) containing 15 µg/mL of tetracycline and incubated overnight at 37°C.

The conjugation between the Escherichia coli S17-1 cells and those of the CN0009 strain was adapted from the protocol of Lenz et al., 1994. The sucrose selection method (15% sucrose used) with SacB made it possible to precisely delete the operon lut2. After the first homologous recombination, the clones are validated by colony PCR using the enzyme DreamTaq Green PCR Master Mix (Thermo Scientific™) and oligonucleotides 31 and 74. After the second homologous recombination, the selection of clones positive among the tetracycline-sensitive clones is carried out by PCR on colonies using the enzyme Kod Xtreme™ Hot Start DNA Polymerase (Novagen) and oligonucleotides 103 (5' CGCGGAAGCG GGAATTATGC 3') and 104 (5' TCCTGCTTCG GTGGTGTTTC G 3').

The Cupriavidus necator H16 Δlut2 ΔlldA ΔlldD ΔphaCABΩpLac_L-ldh C.necator strain is recovered and named CN0012. The genetic modifications are validated by sequencing using oligonucleotides 103, 104, 105 (5' CCTATCTTCT ATGCCTACCT GAAG 3') and 106 (5' TGTAGGGCGA AGCCTTGC 3').

Example 16: Construction of a strain of Cupriavidus necator genetically modified in which a polyhydroxybutyrate (PHB) biosynthetic pathway is deleted, an endogenous lactate dehydrogenase is overexpressed, two lactate ferricytochrome C reductases are deleted and in which the two lut1 and lut2 operons carrying the genes coding for the components of an enzyme complex using lactate are deleted (CN0013).

Strain and genetic constructs.

The construction of the CN0013 lactate-producing strain is carried out according to the following protocol:

A plasmid carrying the sequences of the regions of homology upstream and downstream of the lactate utilization operon comprising the genes B0091, B0092, B0093 of the AM260480 chromosome of Cupriavidus necator is cloned in one step in vitro using the NEBuilder ^® HiFi DNA assembly protocol Assembly Cloning (New England Biolabs, Evry, France). Oligonucleotides are synthesized and purified (desalted) by Eurofins Genomics.

The sequence of the region of homology upstream of the B0093 gene is amplified by PCR on the genomic DNA of the Cupriavidus necator H16 strain using oligonucleotides 70 presenting in 5' an overlapping region of 25 base pairs with the 3' end of the plasmid pLO3 and 71 presenting in 5' a zone of overlap of 15 base pairs with the 5' end of the homology zone downstream of the B0091 gene and of the enzyme KOD Hot Start DNA Polymerase (Novagen) with 5% DMSO. The PCR product is purified on gel with the Nucleospin gel and PCR clean-up kit (Macherey-Nagel).

The sequence of the downstream homology zone of the B0091 gene is amplified by PCR on the genomic DNA of the Cupriavidus necator H16 strain using oligonucleotides 72 presenting in 5' a zone of overlap of 15 base pairs with the 3' end of the zone of homology upstream of the B0093 and 73 gene presenting in 5' a zone of overlap of 25 base pairs with the 5' end of the plasmid pLO3 and the enzyme KOD Hot Start DNA Polymerase (Novagen) with 5% DMSO. The PCR product is purified on gel with the Nucleospin gel and PCR clean-up kit (Macherey-Nagel).

The backbone plasmid pLO3 (Lenz and Friedrich, 1998) is cut with the restriction enzyme XmaI-HF (New England Biolabs, Evry, France). The digestion product is purified on gel with the Nucleospin gel and PCR clean-up kit (Macherey-Nagel).

In vitro assembly by ^NEBuilder® HiFi DNA Assembly Cloning (New England Biolabs, Evry, France), is carried out with the three fragments in order to obtain the plasmid pLO3-Δlut2. 2 μl of the assembly product are transformed into chemo-competent Escherichia coli NEB 5-alpha competent cells (New England Biolabs, Evry, France). The transformants are selected on an LB/Agar medium (Tryptone 10 g/L, yeast extract 5 g/L, NaCl 5 g/L, Sigma-Aldrich, agar 20 g/L) containing 15 μg/mL of tetracycline. Positive clones are selected by PCR on colonies using the enzyme DreamTaq Green PCR Master Mix (Thermo Scientific™) and oligonucleotides 31 and 74. After extraction of the plasmids using the NucleoSpin® Plasmid kit ( Macherey-Nagel), the deletion of the lut2 operon is confirmed by sequencing (Eurofins genomic) with oligonucleotides 29, 74, 75 and 31.

The pLO3-Δlut2 plasmid is inserted into an electrocompetent Escherichia coli S17-1 strain by electroporation using a Gene Pulser, BioRad electroporator (Datsenko and Wanner, 2000).

Positive clones are selected by PCR on colonies using the enzyme DreamTaq Green PCR Master Mix (Thermo Scientific™) and oligonucleotides 31 and 74. The clone of interest is isolated on LB/agar medium (Tryptone 10g/L, yeast extract 5g/L, NaCl 5g/L, Sigma-Aldrich, agar 20g/L) containing 15 µg/mL of tetracycline and incubated overnight at 37°C.

The conjugation between the Escherichia coli S17-1 cells and those of the CN0011 strain was adapted from the protocol of Lenz et al., 1994. The sucrose selection method (15% sucrose used) with SacB made it possible to precisely delete the operon lut2. After the first homologous recombination, the clones are validated by colony PCR using the enzyme DreamTaq Green PCR Master Mix (Thermo Scientific™) and oligonucleotides 31 and 74. After the second homologous recombination, the selection of clones positive among the tetracycline-sensitive clones is carried out by PCR on colonies using the enzyme Kod Xtreme™ Hot Start DNA Polymerase (Novagen) and oligonucleotides 103 (5' CGCGGAAGCG GGAATTATGC 3') and 104 (5' TCCTGCTTCG GTGGTGTTTC G 3').

The Cupriavidus necator H16 Δlut1 Δlut2 ΔlldA ΔlldD ΔphaCABΩpLac_L-ldh _{C.necator strain} is recovered and named CN0013. The genetic modifications are validated by sequencing using oligonucleotides 103, 104, 105 (5' CCTATCTTCT ATGCCTACCT GAAG 3') and 106 (5' TGTAGGGCGA AGCCTTGC 3').

Example 17: Evaluation of lactate consumption by strains CN0009, CN0011, CN0012 and CN0013

Strains: For lactate consumption assessment, strains CN0009 ( Cupriavidus necator H16 ΔlldA ΔlldD ΔphaCABΩpLac_L-ldh _C.necator ), CN0011 ( Cupriavidus necator H16 Δlut1 ΔlldA ΔlldD ΔphaCABΩpLac_L-ldh _C.necator (), CN1vidus necator H161 Δlut2 ΔlldA ΔlldD ΔphaCABΩpLac_L-ldh _C.necator ), CN0013 ( Cupriavidus necator H16 Δlut1 Δlut2 ΔlldA ΔlldD ΔphaCABΩpLac_L-ldh _C.necator ) are used.

Media, Culture Conditions and Sampling and Analysis Protocol The media, culture conditions and sampling and analysis protocol used are the same as those described in Example 13.

Results The growth of strains CN0009, CN0011, CN0012 and CN0013 with lactate as the sole carbon source is shown in Figure 8. During the growth phase, the rate of consumption of lactate for strain CN0009 is 0.89 g/ L/h, for strain CN0011 0.72 g/L/h, for strain CN0012 0.05 g/L/h and for strain CN0013 0 g/L/h. The deletion of the lut1 operon therefore leads to a reduction in the rate of consumption of lactate by Cupriavidus necator by 19% . The deletion of the lut2 operon therefore leads to a reduction in the rate of consumption of lactate by Cupriavidus necator by 94% . The concomitant deletion of the lut1 and lut2 operons therefore leads to a total reduction in the consumption of lactate by Cupriavidus necator.

Abbreviations Name F6P Fructose 6 phosphate F16BP Fructose 1,6 bisphosphate G3P Glyceraldehyde 3 Phosphate 13BPG 1.3a Phospho Glycerate 3PG 3 PhosphoGlycerate 2PG 2 PhosphoGlycerate PEP Phospho Enol Pyruvate R-HB-CoA 3 hydroxybutyrate-CoA PHB PolyHydroxyButyrate Quote Citrate Isocit Isocitrate AKG 2 oxoglutarate SucCoA Succinyl-CoA Success Succinate Smoke fumarate Evil malate OAA OxaloAcetate DHAP Dihydroxyacetone Phosphate E4P Erythrosis 4 phosphate X5P Xylulose 5 phosphate R5P Ribose 5 Phosphate Ru5P Ribulose 5 Phosphate R15BP Ribose 1.5 Bis Phosphate G6P Glucose 6 phosphate 15PGL 1.5 Phospho-glucono-lactone 6PGA 6 Phosphogluconate S7P Sedoheptulose 7 phosphate S17BP Sedoheptulose 1,7 biphosphate fbp Fructose 1,6 BisPhosphatase cbbF2, cbbFp Fructose 1,6 BisPhosphatase, Sedoheptulose 1,7 bisphosphatase fba, cbbA2, cbbAp Fructose-bisphosphate aldolase class II fbaB Fructose-bisphosphate aldolase class I tpiA triose phosphate isomerase gapA, cbbG2, cbbGp Glyceraldehyde 3-phosphate dehydrogenase pgk, cbbK2, cbbKp Phosphoglycerate kinase cbbS2, cbbL2, cbbSp, cbbLp rbcL Ribulose bisphosphate carboxylase small chain
Ribulose-1,5-bisphosphate carboxylase/large subunit oxygenase
Ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit
Ribulose-1,5-bisphosphate carboxylase/large subunit oxygenase cbbP2, cbbPp Phosphoribulokinase tktA, cbbT2 Transketolase rpe, cbbE2, cbbEp Ribulose-phosphate 3-epimerase pgam1, pgam2 phosphoglycerate mutase 1, phosphoglycerate mutase 2 eno enolase pyk1, pyk2, pyk2 pyruvate kinase pdhA1, pdhA2, ace, bkdA1, bkdA2 Pyruvate dehydrogenase phaA Acetyl-CoA acetyltransferase phaB Acetoacetyl-CoA reductase phaC Poly(3-hydroxybutyrate) synthase ppsA Phosphoenolpyruvate synthase ldh L-lactate dehydrogenase ldhA1, ldhA2 D-lactate dehydrogenase lldD, lldA L-lactate dehydrogenase (cytochrome) dld D-lactate dehydrogenase (cytochrome) pyc Pyruvate carboxylase mhpf Acetaldehyde dehydrogenase pta Phosphate acetyltransferase ackA acetate kinase cisY Citrate synthase acnA, acnB Aconitate hydratase icd1, icd2, icd3 Isocitrate dehydrogenase odhA, odhB 2-Oxoglutarate dehydrogenase sucC, sucD Succinyl-CoA synthetase sdhA,B,C,D Succinate dehydrogenase fumA, fumC Fumarate hydratase mdh1 Malate dehydrogenase lutA, lutB, lutC Enzyme complex using lactate

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Claims (12)

Bacterium which naturally oxidizes hydrogen and genetically modified to produce lactate from CO ₂ , said bacterium being genetically modified to overexpress at least one gene encoding a lactate dehydrogenase and to inhibit the expression of a lut operon. Bacterium according to Claim 1, characterized in that the inhibition of the expression of a lut operon comprises the inhibition of the expression of at least one lutA, lutB or lutC gene of the said lut operon. Bacterium according to one of Claims 1 or 2, characterized in that the inhibition of the expression of the lut operon consists of a deletion of the said lut operon. Bacterium according to one of Claims 1 to 3, characterized in that when the bacterium comprises several lut operons, the expression of at least one operon is inhibited. Bacterium according to one of Claims 1 to 4, characterized in that it is genetically modified to overexpress at least one gene encoding an endogenous lactate dehydrogenase. Bacterium according to Claim 1 to 5, characterized in that it is genetically modified to overexpress at least one gene encoding an exogenous lactate dehydrogenase, preferentially derived from a bacterium, a fungus, a yeast or a mammal , more preferably derived from a bacterium. Bacterium according to one of Claims 1 to 6, characterized in that it is also genetically modified to at least partially inhibit at least one pyruvate degradation pathway competing with the lactate synthesis pathway. Bacterium according to one of Claims 1 to 7, characterized in that the expression of at least one gene encoding a lactate ferricytochrome C reductase is at least partially inhibited. Bacterium according to one of Claims 1 to 8, characterized in that it is chosen from Cupriavidus sp . , such as Cupriavidus necator , Hydrogenobacter sp . , such as Hydrogenobacter thermophilus , Rhodococcus sp., such as Rhodococcus opacus , and Pseudomonas sp., such as Pseudomonas hydrogenothermophila . Bacterium according to one of Claims 1 to 9, characterized in that it is Cupriavidus necator . Use of a bacterium defined according to one of Claims 1 to 10, for the production of lactate from CO ₂ . Process for the production of lactate from CO ₂ , comprising the steps consisting in cultivating the bacterium according to one of Claims 1 to 10 in the presence of CO ₂ as the sole carbon source, then in recovering the lactate.