CN110857449A - Improved method for producing polyhydroxyalkanoate - Google Patents

Abstract

The present invention provides an improved method for microbial production of polyhydroxyalkanoates (PHA), the method comprising adding acetic acid, acetate, acetate or other basal medium to a basal medium comprising a carbon source for microbial synthesis of PHA acetic acid derivatives; preferably acetic acid or an acetate salt is added. This method can be used to increase the yield of microbially synthesized PHA polymers and/or to control the monomer ratio in the PHA copolymer product.

Description

An improved method for producing polyhydroxyalkanoates

technical field

The present invention relates to the field of biotechnology, in particular, the present invention relates to an improved method for producing polyhydroxyalkanoates (PHA). More specifically, the present invention relates to a method for increasing the yield of microbially synthesized PHA and/or controlling the ratio of monomers in the PHA copolymer product by adding a certain amount of acetic acid to the culture medium.

Background technique

At present, the large-scale production of biopolymers in industry usually uses glucose or sodium gluconate as a carbon source, wherein glucose or sodium gluconate first generates pyruvate through the glycolysis pathway, and then starts from pyruvate to generate acetyl-CoA, and then enters Synthetic pathways for the synthesis of various polymer monomers starting from acetyl-CoA. Glycolysis will produce a large amount of NADH and ATP, and the process of generating acetyl-CoA from pyruvate will also produce NADH. Under high-density cell culture, the lack of oxygen will lead to the accumulation of NADH, which will affect product synthesis and waste resources. . Therefore, there is a need in the art for a method that can alleviate or solve this problem.

According to the composition of monomers, polymers can be divided into copolymers and homopolymers. Homopolymers are composed of one kind of monomers, while copolymers are composed of two or more kinds of monomers. The material properties of homopolymers are single and not adjustable, while the material properties of copolymers can be adjusted by changing the proportion of monomers in them. Copolymers have higher added value due to better material properties, in which the proportion of monomers largely determines their material properties.

In addition, many microorganisms are known to be able to produce PHA copolymers through metabolic engineering, but it is difficult to control the ratio of monomers. The current methods for controlling the proportion of monomers are mainly divided into two categories, one of which is to genetically engineer the metabolic pathways of microorganisms, optimize the expression of specific genes to increase the proportion of specific monomers, or reduce the expression of specific genes to reduce specific monomers proportion. The other type is to change the proportion of the corresponding monomers in the polymer by directly controlling the amount of specific related precursors added, but the related precursors of most monomers are expensive, and some precursors are even toxic to cells, resulting in Such a method is difficult to apply to industrial production on a large scale. Therefore, finding a low-cost method to adjust the ratio of monomers in copolymers is of great significance in industrial production.

SUMMARY OF THE INVENTION

The present invention has been made in order to solve the aforementioned problems of the prior art.

Regarding the glycolytic pathway of microbial synthesis of PHA polymers, the inventors of the present invention have repeatedly conducted experimental research and analysis and found that the synthesis of polymer products is inhibited under high cell density fermentation because the accumulation of NADH leads to the formation of acetyl-CoA from pyruvate. The pathway is inhibited, which in turn leads to the inhibition of the synthetic pathway for the synthesis of various PHA polymer monomers from acetyl-CoA, which ultimately results in limited polymer product synthesis. In view of this, finding an inexpensive method to reduce or relieve the inhibition of pyruvate metabolism is of great significance for the industrial production of polymers.

In order to reduce or release the inhibition of pyruvate metabolism, the inventors have found through research that adding acetic acid, acetate, acetate or other acetic acid derivatives to the microbial fermentation medium can reduce the accumulation of NADH on pyruvate to generate acetyl coenzyme. A Inhibition of this pathway reduces pyruvate accumulation and increases product yield. Furthermore, the present inventors unexpectedly found that the addition of acetic acid, acetate salt, acetate ester or other acetic acid derivatives can even have the effect of adjusting the proportion of monomers in the copolymer.

Based on the above findings, the present invention relates to the following aspects.

(one)

It is an object of the present invention to increase the yield of polymers obtained in processes for microbial synthesis of polymers such as polyhydroxyalkanoates (PHA). The inventors of the present invention found that the purpose can be achieved by adding acetic acid, acetate, acetate or other acetic acid derivatives to the basal medium, and the production process of the present invention is simple, low in cost, and has wide application prospect.

Accordingly, in one aspect, the present invention provides an improved method for the production of polyhydroxyalkanoates (PHA) comprising adding acetic acid, acetic acid to a basal medium comprising a carbon source for culturing microorganisms to produce polymers Salts, acetates or other acetic acid derivatives, preferably acetic acid or acetates are added. Through such a method, the inhibition of pyruvic acid metabolism produced in the process of microbial production of polymers can be alleviated, the utilization rate of carbon sources can be improved, and finally the purpose of increasing the polymer content and final yield in microbial cells can be achieved.

The microorganisms involved in the method of the present invention are Gram-positive or negative bacteria capable of synthesizing biopolymers. The bacteria include, but are not limited to, Escherichia, Halophile, Halomonas, Pseudomonas, or Bacillus bacteria of the same genus, or a combination thereof. Preferably the microorganism is a bacterium of the genus Halomonas. More specifically, the microorganism can be, for example, Escherichia coli (Escherichia coli), Pseudomonas putida (Pseudomonas putida), Halomonas campaniensis, or Halomonas bluephagenesis, etc. or a combination thereof, preferably Halomonascampaniensis or Halomonas bluephagenesis or a combination thereof .

The above-mentioned microorganisms involved in the method of the present invention can be wild-type (that is, in their natural form, capable of synthesizing polymers, such as PHA, using suitable substrates under suitable culture conditions), or they can be artificially engineered recombinant types, including but not limited to: It is not limited to bacteria obtained by mutagenesis, genetic engineering, or the like. For example, in the case of a recombinant type, the above-mentioned microorganisms may be artificially modified (such as introducing genes related to PHA synthesis) in microorganisms that are not capable of synthesizing PHA by themselves, so that they can utilize suitable A recombinant microorganism for synthesizing a polymer as a substrate, for example, a recombinant microorganism obtained by introducing the phaC gene, a synthetic gene of polyhydroxybutyrate, into Escherichia coli. It can also be obtained by disrupting/knockout etf gene on the basis of wild-type microorganism capable of synthesizing PHA (such as Halomonas bluephagenesis TD01), for example, by disrupting/knockout etf-alpha or etf-beta gene, more specifically Ground, for example, bacteria obtained by knocking out etf-alpha or etf-beta genes on the basis of Halomonas bluephagenesis, Halomonas campaniensis and Pseudomonas putida. For example, the microorganism involved in the method of the present invention may be Halomonas bluephagenesis TD01, or an engineered bacteria obtained by mutagenesis, genetic engineering and other methods on the basis of Halomonas bluephagenesis TD01.

More preferably, the microorganism can be selected from one or more of the following:

Escherichia coli JM109-pBHR68;

Escherichia coli JM109SG-p68orfZ+pMCSH5;

Halomonas bluephagenesis TD01;

Halomonas bluephagenesis TD08AB;

Halomonas bluephagenesis TD△gabD2-D2;

Halomonas bluephagenesis TDΔβ;

Halomonas bluephagenesis TDΔα.

The microorganisms involved in the present invention can be cultured under appropriate culture conditions (temperature, rotational speed, dissolved oxygen, pH, etc.) as long as the culture enables them to synthesize the desired PHA polymer. For example, the temperature and rotational speed during the cultivation process can be appropriately set by those skilled in the art according to the characteristics of microorganisms, or selected by routine optimization experiments.

In the above method, the medium for fermentation culture can be obtained by adding substrates (also called carbon sources) related to product synthesis and regulating substances (eg, acetic acid) on the basis of basal medium. For example, the final medium may contain glucose, gluconic acid, gluconate, gluconate, or a combination thereof as a carbon source for microbial culture. For the sake of distinction, the carbon sources described herein do not encompass acetic acid. Preferably, the medium contains glucose as carbon source. Optionally, the medium may or may not also contain carbon sources other than glucose.

The gluconate mentioned above may be any one or more gluconates as long as it can be used as a carbon source for the microorganism involved in the present invention for polymer production, for example, sodium gluconate, potassium gluconate, Calcium gluconate, etc. The concentration of glucose, gluconic acid, gluconate or gluconate as a carbon source can be appropriately adjusted by those skilled in the art according to the culture conditions and microorganisms used, and can be, for example, about 1-100 g/L, about 1- 90g/L, about 1-80g/L, about 1-70g/L, or about 1-60g/L; preferably, the concentration may be about 3-60g/L, about 3-50g/L or In the range of about 3-40 g/L; more preferably in the range of about 5-60 g/L, about 10-60 g/L, about 20-40 g/L, including, for example, about 25.5-34.5 g/L. It should be understood that the above concentration ranges are not an exhaustive enumeration, but can be appropriately adjusted by those skilled in the art through experiments according to the conditions of the fermentation system, and these concentration ranges are all covered within the scope of the present invention, as long as they will not adversely affect The purpose of the present invention is sufficient.

In the above method, acetic acid, acetate salts, acetate esters or other acetic acid derivatives may be added to the basal medium prior to the initiation of microbial culture. Alternatively, acetic acid, acetate salts, acetate esters, or other acetic acid derivatives can also be added to the medium once, in multiple batches, or as a stream during microbial cultivation. Still alternatively, acetic acid, acetate salts, acetate esters or other acetic acid derivatives may be added to the medium at the same time as the microorganisms are inoculated into the medium. Acetic acid, acetate salts, acetate esters or other acetic acid derivatives can be added at concentrations, for example, at about 1-12 g/L, about 1-11 g/L, about 1-10 g/L, about 1-9 g/L or in the range of about 1-8 g/L; preferably about 1-8 g/L, for example at about 2-8 g/L, about 2-7 g/L, about 2-6 g/L, about 2-5 g/L, about In the range of 2-4 g/L, or 2-3 g/L; more preferably 2-6 g/L, for example in the range of about 3-6 g/L or about 3-5 g/L. These concentrations can be appropriately adjusted on the basis of the present invention according to factors such as the composition of the medium and the culturing conditions, as long as they do not affect the effects of the present invention.

The acetate salts mentioned above may be any one or more of the acetate salts, as long as those skilled in the art can reasonably expect that their addition will not adversely affect the purpose of the present invention, for example, the acetate salts may be, But not limited to: sodium acetate, potassium acetate, calcium acetate, etc. Likewise, the above-mentioned acetates may be any one or more acetates, as long as those skilled in the art can reasonably expect their addition to not adversely affect the purposes of the present invention, for example, methyl acetate , ethyl acetate, propyl acetate, etc.

The basal medium mentioned above refers to a medium containing nutrients that can be used to support the growth of the microorganisms of the present invention. The above-mentioned basal medium can be the medium conventionally used in the art for microbial culture, such as mineral medium, LB medium, MM medium or beef extract peptone, etc., or can be based on these mediums according to the desired purpose. modified medium. That is, those skilled in the art can routinely select a suitable basal medium as long as it can allow the growth of microorganisms.

In the method of the present invention, the synthesized polymer may be polyhydroxyalkanoate (PHA), but is not limited thereto. Specifically, the PHA can be a copolymer or a homopolymer, or a combination thereof. In the case of homopolymers, it may include, but is not limited to, polyhydroxypropionate, polyhydroxybutyrate, polyhydroxyvalerate, and the like, eg, poly-3-hydroxybutyrate (P3HB), poly- 4-hydroxybutyrate (P4HB), poly-3-hydroxypropionate (P3HP) or poly-3-hydroxyvalerate (P3HV), etc. In the case of a copolymer, the copolymer may be a dimer, a trimer, but not limited thereto, for example, the copolymer may be a copolymer of hydroxypropionate and hydroxybutyrate; hydroxypropionate and hydroxybutyrate Copolymers of hydroxyvalerate; copolymers of hydroxybutyrate and hydroxyvalerate; copolymers of hydroxypropionate, hydroxybutyrate, hydroxyvalerate, etc. More specifically, it may be poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P3HB3HV) , poly(3-hydroxybutyrate-co-4-hydroxybutyrate-co-3-hydroxyvalerate) (P3HB4HB3HV) and the like.

According to the technical solution of the present invention, by adding an appropriate amount of acetic acid to a basal medium containing a carbon source for culturing microorganisms, the metabolic inhibition of pyruvate is alleviated or eliminated, the ability of microorganisms to synthesize polymers is improved, and cell stemness is improved. The weight also increases, as does the final polymer yield.

Based on the foregoing, the present invention also provides a method for increasing the yield of polyhydroxyalkanoates in the microbial synthesis of polyhydroxyalkanoates, comprising adding a carbon source to a basal medium for culturing microorganisms to produce polymers Add acetic acid, acetate, acetate or other acetic acid derivatives, preferably acetic acid or acetate. Definitions regarding the specific microorganism, carbon source, culture medium, acetic acid (or acetate, acetate or other acetic acid derivative) involved in the method are as previously described.

(two)

Another object of the present invention is to adjust the monomer ratio in the copolymer product produced in the microbial production of the copolymer.

Accordingly, in a second aspect, the present invention provides an improved method for the production of polyhydroxyalkanoates (PHA), said method comprising adding a carbon source to a basal medium for growth and cultivation of said microorganisms Add acetic acid, acetate, acetate or other acetic acid derivatives, and the polymer is a copolymerized polyhydroxyalkanoate. Through such an approach, it is possible to change the shunting of microbial metabolic flow, change the internal environment of the microorganism, and affect the metabolic flow of acetyl-CoA into different monomer synthesis pathways, thereby changing the ratio of various monomers in the cell-accumulated biopolymer. The production process of the invention is simple, the cost is low, and the application prospect is broad.

The microorganisms involved in the method of the present invention are Gram-positive or negative bacteria capable of synthesizing biopolymers. The bacteria include, but are not limited to, Escherichia, Halophile, Halomonas, Pseudomonas, or Bacillus bacteria of the same genus, or a combination thereof. Preferably the microorganism is a bacterium of the genus Halomonas. More specifically, the microorganism can be, for example, Escherichia coli (Escherichia coli), Pseudomonas putida (Pseudomonas putida), Halomonas campaniensis, or Halomonas bluephagenesis, etc. or a combination thereof, preferably Halomonascampaniensis or Halomonas bluephagenesis or a combination thereof .

The microorganisms involved in the method of the present invention may be wild-type (ie, in their natural form, capable of synthesizing polymers, such as PHA, using suitable substrates under suitable culture conditions), or they may be artificially engineered recombinant types. In the case of recombinant type, it can be artificially engineered (such as introducing genes related to PHA synthesis) in microorganisms that are not capable of synthesizing PHA themselves, so that they can be synthesized using appropriate substrates under appropriate culture conditions A recombinant microorganism of a polymer, for example, a recombinant microorganism obtained by introducing a polyhydroxybutyrate synthesis gene phaC gene into Escherichia coli. It can also be obtained by disrupting/knockout etf gene on the basis of wild-type microorganism capable of synthesizing PHA (such as Halomonas bluephagenesis TD01), for example, by disrupting/knockout etf-alpha or etf-beta gene, more specifically Ground, for example, bacteria obtained by knocking out the etf-alpha or etf-beta gene on the basis of Halomonas bluephagenesis, Halomonas campaniensis, Pseudomonas putida. For example, the microorganism involved in the method of the present invention can be Halomonas bluephagenesis TD01, or an engineered bacteria obtained by mutagenesis, genetic engineering and other methods on the basis of Halomonas bluephagenesis TD01.

More preferably, the microorganism can be selected from one or more of the following:

Escherichia coli JM109-pBHR68;

Escherichia coli JM109SG-p68orfZ+pMCSH5;

Halomonas bluephagenesis TD01;

Halomonas bluephagenesis TD08AB;

Halomonas bluephagenesis TD△gabD2-D2;

Halomonas bluephagenesis TDΔβ;

Halomonas bluephagenesis TDΔα.

The microorganisms involved in the present invention can be cultured under appropriate culture conditions (temperature, rotational speed, dissolved oxygen, pH, etc.) as long as the culture enables them to synthesize a desired polymer. For example, the temperature and rotational speed during the cultivation process can be appropriately set by those skilled in the art according to the characteristics of microorganisms, or selected by routine optimization experiments.

In the above method, the medium for fermentation culture can be obtained by adding substrates (also called carbon sources) related to product synthesis and regulating substances (eg, acetic acid) on the basis of basal medium. For example, the final medium can be glucose, gluconic acid, gluconate, gluconate, or a combination thereof as a carbon source for microbial culture. Preferably, the medium contains glucose as carbon source. For the sake of distinction, the carbon sources described herein do not encompass acetic acid. Optionally, the medium may or may not also contain carbon sources other than glucose.

The gluconate mentioned above may be any one or more gluconates as long as it can be used as a carbon source for the microorganism involved in the present invention for polymer production, for example, sodium gluconate, potassium gluconate, Calcium gluconate, etc. The concentration of glucose, gluconic acid, gluconate or gluconate as a carbon source can be appropriately adjusted by those skilled in the art according to the culture conditions and microorganisms used, and can be, for example, about 1-100 g/L, about 1- 90g/L, about 1-80g/L, about 1-70g/L, or about 1-60g/L; preferably, the concentration may be about 3-60g/L, about 3-50g/L or In the range of about 3-40 g/L; more preferably in the range of about 5-60 g/L, about 10-60 g/L, about 20-40 g/L, including, for example, about 25.5-34.5 g/L. It should be understood that the above concentration ranges are not an exhaustive enumeration, but can be appropriately adjusted by those skilled in the art through experiments according to the conditions of the fermentation system, and these concentration ranges are all covered within the scope of the present invention, as long as they will not adversely affect The purpose of the present invention is sufficient.

In the above method, acetic acid, acetate salts, acetate esters or other acetic acid derivatives may be added to the basal medium prior to the initiation of microbial culture. Alternatively, acetic acid, acetate salts, acetate esters, or other acetic acid derivatives can also be added to the medium once, in multiple batches, or as a stream during microbial cultivation. Still alternatively, acetic acid, acetate salts, acetate esters or other acetic acid derivatives may be added to the medium at the same time as the microorganisms are inoculated into the medium. Acetic acid, acetate salts, acetate esters or other acetic acid derivatives can be added at concentrations, for example, at about 1-12 g/L, about 1-11 g/L, about 1-10 g/L, about 1-9 g/L or in the range of about 1-8 g/L; preferably about 1-8 g/L, for example at about 2-8 g/L, about 2-7 g/L, about 2-6 g/L, about 2-5 g/L, about In the range of 2-4 g/L, or 2-3 g/L; more preferably 2-6 g/L, for example in the range of about 3-6 g/L or about 3-5 g/L. These concentrations can be appropriately adjusted on the basis of the present invention according to factors such as the composition of the medium and the culturing conditions, as long as they do not affect the effects of the present invention.

The acetate salts mentioned above may be any one or more of the acetate salts, as long as those skilled in the art can reasonably expect that their addition will not adversely affect the purpose of the present invention, for example, the acetate salts may be, But not limited to: sodium acetate, potassium acetate, calcium acetate, etc. Likewise, the above-mentioned acetates may be any one or more acetates, as long as those skilled in the art can reasonably expect their addition to not adversely affect the purposes of the present invention, for example, methyl acetate , ethyl acetate, propyl acetate, etc.

The basal medium mentioned above refers to a medium containing nutrients that can be used to support the growth of the microorganisms of the present invention. The above-mentioned basal medium can be the medium conventionally used in the art for microbial culture, such as mineral medium, LB medium, MM medium or beef extract peptone, etc., or can be based on these mediums according to the desired purpose. modified medium. That is, those skilled in the art can routinely select a suitable basal medium as long as it can allow the growth of microorganisms.

In the method of the present invention, the synthesized polymer is a copolymer, which can be a dimer, a trimer, but not limited thereto, for example, the copolymer can be a copolymer of hydroxypropionate and hydroxybutyrate ; copolymers of hydroxypropionate and hydroxyvalerate; copolymers of hydroxybutyrate and hydroxyvalerate; copolymers of hydroxypropionate, hydroxybutyrate, hydroxyvalerate, etc. More specifically, it may be poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P3HB3HV) , poly(3-hydroxybutyrate-co-4-hydroxybutyrate-co-3-hydroxyvalerate) (P3HB4HB3HV) and the like.

Based on the foregoing, the present invention also provides a method for adjusting the ratio of monomers in the copolymer in the microbial synthesis of polyhydroxyalkanoate copolymers, which comprises adding a carbon source to a basis for culturing microorganisms to produce polymers comprising a carbon source Acetic acid, acetate salt, acetate ester or other acetic acid derivatives are added to the medium, preferably acetic acid or acetate salt is added. Definitions regarding the specific microorganism, carbon source, culture medium, acetic acid (or acetate, acetate or other acetic acid derivative) involved in the method are as previously described.

(three)

In a third aspect, the present invention provides a method of reducing pyruvic acid accumulated during the production of polyhydroxyalkanoates (PHA) by microorganisms, the method comprising adding acetic acid to a basal medium comprising a carbon source for culturing the microorganisms , acetate, acetate or other acetic acid derivatives in the concentrations described in the first to second aspects above. In addition, the basal medium, carbon source, etc. in the method are also as described in the aforementioned first to second aspects.

(Four)

In a fourth aspect, the present invention provides a recombinant bacteria obtained by knocking out/inactivating any one or all of etf-α and etf-β subunits of etf gene on the basis of bacteria of the genus Halomonas, The bacteria belonging to the genus Halomonas are preferably Halomonas bluephagenesis, more preferably Halomonas bluephagenesis TD01 with the deposit number of CGMCC No.4353.

The present invention also provides a method for producing poly(3-hydroxybutyrate-co-4-hydroxybutyrate), the method comprising using the aforementioned etf gene knocked out/inactivated recombinant bacteria in a carbon source containing Fermentation is carried out in a basal medium under conditions suitable for culturing the recombinant bacteria to produce poly(3-hydroxybutyrate-co-4-hydroxybutyrate). Preferably, acetic acid, acetate salts, acetate esters or other acetic acid derivatives may be added to the basal medium at concentrations as described in the first to second aspects above. In addition, the basal medium, carbon source, etc. in the method are also as described in the aforementioned first to second aspects.

Description of drawings

FIG. 1 is a schematic diagram of the agarose gel result of knocking out etf-β subunit in Example 2. FIG.

Figure 2 is a schematic diagram of the results of transmission electron microscopy of the accumulation of P3HB by Halomonas bluephagenesis TD01 and TDΔβ in Example 2. The left picture is the picture obtained by TD01 under the condition of adding 6g/L acetic acid; the right picture is the picture obtained by adding TDΔβ under the condition of adding 3g/L acetic acid. obtained graph.

Detailed ways

The dry cell weight (CDW, g/L) mentioned herein is the ratio of the mass of ice-dried cells to the volume of the fermentation broth.

The PHA referred to herein refers to polyhydroxyalkanoate, which can be divided into homopolymers and copolymers according to the monomer composition. Depending on the number of carbon atoms in the monomer, the PHA of the present invention can be a short-chain PHA (ie, a hydroxy fatty acid whose monomer is C3-C5) or a medium-to-long-chain PHA (ie, a hydroxy fatty acid whose monomer is C6-C16), but Not limited to this. In some embodiments of the invention, the PHA may be a homopolymer including, but not limited to, polyhydroxypropionate, polyhydroxybutyrate, polyhydroxyvalerate, and the like, eg, poly-3-hydroxybutyrate (P3HB), poly-4-hydroxybutyrate (P4HB), poly-3-hydroxypropionate (P3HP) or poly-3-hydroxyvalerate (P3HV), etc. In some embodiments of the present invention, PHA may be a copolymer such as dimer, trimer, etc., but not limited thereto, for example, the copolymer may be a copolymer of hydroxypropionate and hydroxybutyrate; hydroxypropionate Copolymers of acid esters and hydroxyvalerates; copolymers of hydroxybutyrate and hydroxyvalerate; copolymers of hydroxypropionate, hydroxybutyrate, hydroxyvalerate, etc. More specifically, in some embodiments of the present invention, the PHA may be poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB), poly(3-hydroxybutyrate-co-3 -hydroxyvalerate) (P3HB3HV) or poly(3-hydroxybutyrate-co-4-hydroxybutyrate-co-3-hydroxyvalerate) (P3HB4HB3HV), or a combination thereof, and the like. In some embodiments of the invention, the PHA may be P3HB (ie, poly-beta-hydroxybutyrate or poly-3-hydroxybutyrate). In other embodiments of the invention, the PHA may be P3HB3HV (ie, 3-hydroxybutyric acid 3-hydroxyvaleric acid copolymer).

The P3HB3HV content (wt%) mentioned in this paper is the percentage of the mass of P3HB3HV in the ice-dried cells and the mass of the ice-dried cells involved in esterification, wherein the mass of P3HB3HV is the mass of 3HV and 3HB obtained after esterification. In the Examples, unless otherwise stated, "%" means "wt%".

The P3HB content (wt%) mentioned in this paper is the percentage of the mass of P3HB to the mass of the ice-dried cells involved in esterification, wherein the mass of P3HB is the total mass of 3HB obtained after esterification. In the Examples, unless otherwise stated, "%" means "wt%".

The 3HV (mol%) mentioned in Example 3 herein is the percentage of the moles of 3HV monomers to the total moles of P3HB3HV monomers, wherein the total moles of P3HB3HV monomers are the moles of 3HV monomers+the moles of 3HB monomers number, where 3HV represents 3-hydroxyvaleric acid (ester) and 3HB represents 3-hydroxybutyric acid (ester). Similarly, the 4HB (mol%) mentioned in Example 3 herein represents the percentage of the moles of 4HB monomers to the total moles of P3HB4HB monomers.

The "basal medium" referred to herein refers to a medium suitable for culturing microorganisms and suitable for the microorganisms to synthesize polyhydroxyalkanoates using a carbon source added to the medium, such as MM medium, LB culture medium base, mineral medium, etc., but not limited thereto. The formulations of these media are routinely known to those skilled in the art, and those skilled in the art can routinely adjust their components or component concentrations appropriately. Herein, unless otherwise specified, the medium or basal medium used for culturing microorganisms to synthesize target products refers to liquid medium.

The general formula of LB liquid medium is: 4-6g/L yeast extract, 8-12g/L peptone, 8-12g/L NaCl, the rest are distilled water (pH adjusted to 7.0-7.2); preferably: 5g/L Yeast extract, 10g/L peptone, 10g/L NaCl, the rest are distilled water (pH adjusted to 7.0-7.2).

The general formula of MM liquid medium is: 0.1‰-2‰(NH ₄ ) ₂ SO ₄ or urea, 0.1‰-1‰ MgSO ₄ , 5‰-10‰ Na ₂ HPO ₄ ·12H ₂ O, 0.5‰-2 ‰KH ₂ PO ₄ , other trace elements not exceeding 0.1% (Fe(III)-NH ₄ -Citrate, CaCl ₂ 2H ₂ O, ZnSO ₄ 7H ₂ O, MnCl ₂ 4H ₂ O, H ₃ BO ₃ , CoCl ₂ ·6H ₂ O, CuSO ₄ ·5H ₂ O, NiCl ₂ ·6H ₂ O, NaMoO ₄ ·2H ₂ O trace) (pH adjusted to about 9.0). Preferably: 0.1% (NH ₄ ) ₂ SO ₄ or 0.2% urea, 0.02% MgSO ₄ , 1.0% Na ₂ HPO ₄ ·12H ₂ O, 0.15% KH ₂ PO ₄ , no more than 0.1% of other trace elements (Fe ( _III ) _{-NH4 - Citrate , CaCl2.2H2O} , _ZnSO4.7H2O , _{MnCl2.4H2O , H3BO3} , _CoCl2.6H2O , _CuSO4.5H2O , _{NiCl 2.6H2O} , _{NaMoO4.2H2O ) (} pH adjusted to about 9.0).

A "carbon source" is a nutrient that provides carbon for the growth and reproduction of microorganisms. In the present invention, "carbon source" is a source of substrates used by the microorganisms of the present invention to synthesize PHA, excluding acetic acid. Thus, "carbon source" may be used interchangeably with "substrate" herein. The carbon source can be: glucose, gluconic acid, gluconate, gluconate, starch, sucrose, etc., or a combination thereof, but is not limited thereto. Glucose is preferably used as the carbon source in the present invention.

The etf gene referred to herein is the gene encoding the electron transfer flavoprotein (ETF), which is known to exist in, for example, Halomonas and Pseudomonas, which comprises two subunits etf -α and etf-β, the NCBI numbers in Halomonas bluephagenesis TD01 are: WP_009724031.1, WP_009724032.1, respectively.

Example

The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

Unless otherwise specified, the enzyme reagents used were purchased from Thermo Scientific Fermentas Company, the kit used for plasmid extraction was purchased from Beijing Bomed Technology Development Company, and the kit used for DNA fragment recovery was purchased from U.S. omega Company. The corresponding operation steps were as follows: All media were prepared with deionized water unless otherwise specified; PCR-related reagents and enzymes were sourced from NEB Company (USA).

Experimental Materials:

LB medium contains: 5g/L yeast extract (purchased from British OXID company, product catalog number LP0021), 10g/L peptone (purchased from British OXID company, product catalog number LP0042), 10g/L NaCl, and the rest are distilled water; Adjust pH to 7.0-7.2; then autoclave.

LB60 medium contains: 5g/L yeast extract (purchased from British OXID company, product catalog number LP0021), 10g/L peptone (purchased from British OXID company, product catalog number LP0042), 60g/L NaCl, and the rest are distilled water; Adjust pH to 7.0-7.2; then autoclave.

MM60 medium configuration method: prepare a NaCl solution of yeast extract (purchased from OXID, UK, product catalog number LP0021), the concentration of yeast extract is 1g/L, and the concentration of NaCl is 60g/L; autoclave after dissolving; after cooling Add 1 ml of component I (to 10 g (NH ₄ ) ₂ SO ₄ and 2 g of MgSO ₄ to 200 ml with distilled water, then autoclave) and 1 mL of component II (to 96.5 g of Na ₂ HPO ₄ ·12H) per 50 ml of solution ₂ O and 15g KH ₂ PO ₄ were added to distilled water to make up to 200ml, and then autoclaved); finally, the pH value of the system was adjusted to about 9.0 with 5M aqueous NaOH solution.

(NH ₄ ) ₂ SO ₄ , NaCl, MgSO ₄ , Na ₂ HPO ₄ ·12H ₂ O, KH ₂ PO ₄ used in the above preparation process were purchased from Sinopharm Chemical Reagent Co., Ltd., catalog numbers are 10002992, 10019308, 10034998 , 10020392, 1017628; glucose was purchased from Sinopharm Chemical Reagent Co., Ltd., catalog number 63005518; sodium gluconate was purchased from SIGMA-ALDRICH company in the United States, catalog number S2054-1KG.

During the culturing process, a certain concentration of antibiotics, such as 100 μg/mL ampicillin, was added to the medium according to actual needs to maintain the stability of the plasmid. Acetic acid and glucose were added after the medium was sterilized, and the pH was adjusted to an appropriate value.

The following strains were used in the examples:

E.coli S17-1: described in Simon R,Priefer U,Pühler A.A broad host range mobilization system for in vivo genetic engineering:transposon mutagenesis ingram negative bacteria[J].Nature biotechnology,1983,1(9):784. By Switzerland Gift from Prof. Uwe Sauer of ETH Zurich; public can obtain the bacteria from Tsinghua University.

Halomonas bluephagenesis TD01: This strain was deposited in the General Microbiology Center of the China Microbial Culture Collection Management Committee on November 19, 2010, and the deposit registration number is CGMCCNo.4353, and the classification name is Halomonas sp.TD01 (also known as Halomonas sp. bluephagenesis TD01, see XB Chen et al. (2017), Construction of Halomonas bluephagenesis capable of high cell density growth for efficient PHA production. Appl Microbiol Biotechnol. Volume 244, Part 1, pages 534-541); it is described in Patent Application Publication No. CN102120973A; The public can obtain the bacteria from Tsinghua University.

Escherichia coli JM109-pBHR68: prepared by transforming the pBHR68 plasmid into Escherichia coli JM109 (purchased from Beijing Bomed Gene Technology Co., Ltd.), wherein pBHR68 is a plasmid containing a gene related to the synthesis of polyhydroxybutyrate, which is described in the following In the literature: Spiekermann P, Rehm BHA, Kalscheuer R, Baumeister D, Steinbüchel A (1999) A sensitive, viaable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other storage com-pounds. Arch Microbiol 171:73 -80, the plasmid contains the polyhydroxybutyrate synthesis gene phaC gene, β-ketothiolase phaA and NADPH-dependent acetoacetyl-CoA reductase phaB derived from Ralstonia eutropha. The public can download it from Tsinghua University The bacteria are obtained from universities, and those skilled in the art can also obtain Escherichia coli JM109-pBHR68 by conventional molecular biological means according to the above disclosure.

Escherichia coli JM109SG-p68orfZ+pMCSH5: This strain was described in Li Z J, Shi Z Y, Jian J, et al.Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate)fromunrelated carbon sources by metabolically engineered Escherichia coli[J]. Metabolic engineering, 2010, 12(4): 352-359. It can produce P3HB4HB using glucose as a single carbon source, which is preserved in Tsinghua University and publicly available from Tsinghua University.

Halomonas bluephagenesis TD08AB: This strain was described in Yin J, Wang H, Fu X Z, et al. Effects of chromosomal gene copy number and locations on polyhydroxyalkanoate synthesis by Escherichia coli and Halomonas sp[J]. Applied Microbiology and Biotechnology, 2015, 99(13) : 5523-5534., can utilize glucose as a single carbon source to produce P3HB3HV, which is preserved in Tsinghua University and the bacteria can be obtained from Tsinghua University by the public.

Halomonas bluephagenesis TD△gabD2-D2: This strain was documented in Ye J, Hu D, Che X, et al. Engineering of Halomonas bluephagenesis for low cost production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) from glucose[J]. In Metabolic Engineering, 2018, 47:143-152., the bacteria can use glucose as a single carbon source to produce P3HB4HB, which is preserved in Tsinghua University and the bacteria can be obtained from Tsinghua University by the public.

Halomonas bluephagenesis TDΔβ: obtained by taking Halomonas bluephagenesis TD01 as the starting strain and knocking out etf-β subunit, this strain was constructed in the experiments of this application and stored in Tsinghua University. The specific construction process is shown in Example 2. Those skilled in the art can prepare the bacteria according to the disclosure of the present application, and the public can obtain the bacteria from Tsinghua University.

Halomonas bluephagenesis TDΔα: obtained by taking Halomonas bluephagenesis TD01 as the starting strain and knocking out etf-α subunit, this strain was constructed in the experiments of this application and stored in Tsinghua University. The specific construction process is shown in Example 2. Those skilled in the art can prepare the bacteria according to the disclosure of the present application, and the public can obtain the bacteria from Tsinghua University.

Method for detecting the content of polyhydroxyalkanoates (PHA) by gas chromatography:

Set the furnace temperature to 80°C, the injector temperature to 200°C, the detector temperature to 220°C, the column head pressure to be 0.25Mpa, and the temperature programmed conditions to be: dwell at 80°C for 1.5 minutes, and increase the temperature to 140°C at a rate of 30°C/min. °C, followed by raising the temperature to 220 °C at a rate of 40 °C/min and holding at this temperature for 0.5 minutes. The injection volume of the sample was 1 μl, and a microinjector produced by Agilent was used.

Gas-phase sample preparation: take 40-60 mg of stem cells of the sample to be tested (take the bacterial solution and centrifuge it at room temperature for 10 minutes at 10,000 rpm, and wash the obtained cell pellet once with water, then freeze-dried to obtain stem cells, and homopolymers are produced in the cells), add 2 ml of chloroform, 2ml of esterification solution (containing 3% (v/v) concentrated sulfuric acid and 2g/L benzoic acid in pure methanol as internal standard) was placed in an esterification tube, sealed with a cap, and heated at 100° C. for 4 hours. After cooling, 1 ml of distilled water was added, fully shaken and left to stand. After the chloroform phase and the aqueous phase were completely separated, 1 μl of the lower chloroform phase was taken and injected into a gas chromatograph (Hewlett Packard 6890 from HP) for chromatographic analysis. The gas chromatograph was operated according to the instructions of the Hewlett Packard 6890 gas chromatograph from Hewlett-Packard Company.

Standard sample preparation: Take 10-20 mg of standard sample into an esterification tube, add 2 ml of chloroform and 2 ml of esterification solution, cover and seal, and then carry out esterification at 100 °C.

Analysis of the results: Taking the standard sample as a control, if the esterification sample (sample to be tested) of the cells to be tested has obvious peaks at the standard sample, the mass of each monomer can be calculated according to the peak area, and then the mass of each monomer can be calculated according to the peak area. The molar ratio is calculated from the mass fraction; the specific gravity (wt%) of the polymer contained in the dry weight of the cells can be calculated according to the amount of the added sample.

Embodiment 1, by adding acetic acid in basal medium, improve the output of microbial fermentation to synthesize P3HB

1. Optimization of culture medium and culture conditions

(1) Optimization of glucose concentration

Using Halomonas bluephagenesis TD01 as the P3HB producing strain, the experiment was carried out to determine the optimum glucose concentration for producing P3HB. First pick a single colony into 20 mL of LB60 (LB containing 60 g/L NaCl) medium at 37°C, 200 rpm overnight culture; then according to 5% inoculum (v/v), inoculate to 50 mL of 50 mL containing a series of different concentrations In the MM60 medium (pH 8.5-9.0, NaCl concentration 60g/L) of glucose (see Table 1), shake the flask at 37°C and 200rpm for 48 hours to obtain bacterial liquid, and measure the sample after centrifugation, water washing and ice drying dry cell weight (CDW; unit g/L), and then the content percentage (%) of P3HB in dry cell weight was detected by gas chromatography. Three sets of parallels were set for each condition. Cell dry weight and P3HB content are shown in Table 1.

Table 1. Results of shake flask experiments for the production of P3HB by Halomonas TD01 with different glucose concentrations

From the results in Table 1, it can be seen that 30 g/L of glucose is the optimal concentration for TD01 to accumulate P3HB, therefore, this concentration was selected for subsequent experiments.

(2) Optimization of the rotation speed of shake flask culture

The culture speed of the shake flask has a direct impact on the oxygen environment for the growth of microorganisms, and the external oxygen environment directly affects the internal environment of the microorganism. Therefore, we control the speed of the shake flask to find the best environment for microorganisms to accumulate P3HB. Taking Halomonas bluephagenesis TD01 as the P3HB producing bacteria, first pick a single colony in 20 mL of LB60 (LB containing 60 g/L NaCl) medium, and cultivate at 37° C. at 200 rpm overnight; then press 5% of the inoculum (v /v), inoculated into 50mL of MM60 medium containing 30g/L glucose (pH 8.5-9.0, NaCl concentration 60g/L), respectively, set the rotational speed to three rotational speed gradients of 100rpm, 200rpm and 300rpm, at 37°C Shake flasks were cultured for 48 hours to obtain bacterial liquid. After centrifugation, water washing and ice drying, the dry cell weight of the sample was obtained, and then the content percentage (%) of P3HB in the dry cell weight was detected by gas chromatography. Three sets of parallels were set for each condition. Cell dry weight and P3HB content are shown in Table 2.

Table 2. Shake flask experiment results of P3HB production by Halomonas TD01 under different rotational speed conditions

From Table 2, we can see that the optimal rotation speed of Halomonas TD01 to produce P3HB is 200 rpm, so the rotation speed of 200 rpm was selected for subsequent experiments.

2. Adding acetic acid to the medium supplemented with carbon source to test its effect on the synthesis of Halomonas bluephagenesisTD01 and P3HB

It is known that acetic acid is a carbon source with lower reducibility than glucose, and many literatures have reported that acetic acid has an inhibitory effect on the growth of microorganisms. In this application, the inventors found that by adding acetic acid and controlling the appropriate amount of addition, not only did not observe the inhibitory effect of acetic acid on cell growth and accumulation of P3HB, but also found that it can increase the accumulation of P3HB, which may be by regulating It is realized by the microenvironment inside the microorganism, eliminating the metabolic inhibition of pyruvic acid, and saving carbon sources.

Taking the production of P3HB by Halomonas bluephagenesis TD01 as an example, the experiment was carried out. First, pick a single colony in 20 mL of LB60 (LB containing 60 g/L NaCl) medium and cultivate at 37 °C overnight at a speed of 200 rpm; then press 5 The % inoculum volume (v/v) was inoculated into 50 mL of MM60 medium (pH 8.5-9.0, NaCl concentration 60 g/L) supplemented with glucose and acetic acid, wherein the glucose concentration was 30 g/L, and the acetic acid was set between 0-12 g/L. During this time, a concentration gradient experiment was performed (see Table 3). The culture temperature was set to 37°C, the rotational speed was set to 200 rpm, and the flask was cultured for 48 hours to obtain bacterial liquid. After centrifugation, washing, and ice drying, the dry cell weight of the sample was obtained, and then the content of P3HB in the dry cell weight was detected by gas chromatography. percentage(%). Three sets of parallels were set for each condition. Cell dry weight and P3HB content are shown in Table 3.

Table 3. Effects of adding different concentrations of acetic acid on the production of P3HB by Halomonas TD01

It can be seen from the data in Table 3 that acetic acid supplementation can increase the ability of microbial cells to accumulate P3HB per unit weight, and can also increase the dry cell weight of microorganisms within a certain range. After calculation, supplementing 1-8g/L acetic acid, especially 2-8g/L acetic acid can improve the total output (g/L) of P3HB product, and the output obtained in the range of adding 2-8g/L acetic acid is different from that without adding acetic acid. increased by at least about 12% compared to the control group. Among them, under the condition of acetic acid addition below 6g/L, as the acetic acid concentration increases, the growth of cells and the accumulation of P3HB are promoted more greatly, but when the acetic acid concentration rises above 6g/L, as the acetic acid concentration increases, The dry cell weight and the percentage of P3HB did not increase further and even started to decrease.

We also detected the contents of various substances in the fermentation broth after fermentation, including glucose, acetate and pyruvate (Table 4).

Table 4. Substance analysis and detection in fermentation broth after fermentation

According to the results of this example, compared with the case of not adding acetic acid, adding acetic acid can indeed alleviate the inhibition of pyruvate metabolism by NADH in the fermentation system, and reduce the residual pyruvate, thereby improving the utilization rate of glucose.

Combined with the P3HB accumulation and cell dry weight data in Table 3, we can find that adding 1-8 g/L acetic acid, especially 2-8 g/L acetic acid can significantly improve cell dry weight and carbon source utilization, and effectively improve P3HB synthesis At the same time, the residual glucose and pyruvate are reduced, thereby alleviating the metabolic inhibition of glucose and pyruvate; and the residual proportion of acetic acid is low, so that the entire fermentation system is optimized.

3. Comparison of adding acetic acid and other carbon sources

In order to verify that the addition of acetic acid has a relieving effect on the metabolic inhibition of pyruvate and glucose rather than the effect of additional carbon sources, we set up a control experiment with additional addition of pyruvate and glucose. In this experiment, the strains, culture medium and culture conditions used are the same as those in the second part of Example 1, the difference is that acetic acid, pyruvate or glucose are additionally added to the basal medium, and the amount of addition is shown in Table 5. Their additions would theoretically translate into the same amount of acetyl-CoA (Table 5).

Table 5. Effects of addition of acetic acid, pyruvate and glucose on P3HB synthesis

From Table 5 we can conclude that the addition of additional carbon sources such as glucose or pyruvate did not increase dry cell weight or P3HB accumulation, and even addition of pyruvate decreased dry cell weight and P3HB content, confirming that pyruvate accumulation does indeed It will have a certain inhibitory effect on the synthesis of P3HB; this experiment further proves that adding acetic acid reduces the residual pyruvate, which in turn can promote the growth of microbial cells and the synthesis of P3HB.

4. The effect of adding acetate on the synthesis of P3HB

Given that acetic acid exists in ionic form in the medium solution, the inventors expected that the addition of acetate would achieve substantially the same effect as the addition of acetic acid, and performed the same experiments as in the second part of Example 1, except that the acetic acid was replaced by acetic acid Salts (sodium acetate, potassium acetate, calcium acetate, etc.).

The results showed that the addition of acetic acid and sodium acetate had essentially the same effect on dry cell weight and percentage of P3HB synthesis.

Example 2. Acetic acid was added to medium supplemented with carbon source to test its effect on Halomonas bluephagenesis TDΔβ and P3HB synthesis.

1. Construction of engineering bacteria TDΔβ:

1), the construction of knockout plasmid pKObet23 homology arm

The genome of Halomonas bluephagenesis TD01 was extracted and used as a template for PCR amplification with pfu enzyme (NEB, USA), with beta-H1-F and beta-H1-R, beta-H2-F and beta, respectively -H2-R is a primer to amplify the left homology arm H1 and the right homology arm H2 of etf-β (nucleotide sequence is shown in SEQ ID NO: 1; amino acid sequence is shown in SEQ ID NO: 2); Using plasmid pSEVA241 as a template (this plasmid is recorded in the literature: Silva-Rocha, Rafael, et al. "The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes." Nucleic Acids Research 41.D1 ( 2012): D666-D675.), using primers donor-H-F and donor-H-R as primers for amplification to obtain the plasmid template Donor-1.

Nucleotide sequence of etf-beta (SEQ ID NO: 1):

ATGCGGCCTGAACAACAGCAAAAACAACAAAAGCCGGGTATCGACGTGGCGGTGCTGGTCTCCATAGGCCGTCACCCCACAACCGGTCGTGCGAGGCGCGCCGAGCAGGATGCACGGGGTCTGGAACTTGCTCTAGCCATGGAAGCAGAACTGCCGGGTAGCCGGATTGACGTACTGCACGCTGGTTCTCAGGATGTCGACAGCGAGGCAGCTCTGCGCAGTTACTTGGGTATGGCCACAGGCGTTGGTATGGGGCTTGAGTCTATGACCTTGCTGGAGCAGCCCGATGGCAGCGATGCCATTCTACCGCTGGTGGAACATTTAGCCGCTACCTCGCCCCAACTAGTGATCACCGGTGCCCGGGCCGAGCGCGGAGAAGGTTCTGGGCTTTTGCCCTATGCGTTGGCTGAGCATCTCGGTTGGCCGTTAGTGAATAGCTTGGCGTCGATAGAGACAGTGGAGAATGGTGTGATAACGCTGCTTCAAGCGTTGCCACGGGGCCAGCGTCGCCGTCTCAAGGTGCGCTTGCCCGCCATCATCAGCGTGGATGAAGCAGCGCCGGCGGCGCGTCAGAGCGCCTTCGGCCCGGCTCGCCGAGCCAGCTTCTCGGTGGCGCCCACTACCTCTGAAGCCGACAGTGAGTTGGCCCAGTGGCACTTGAGCCCCGCACGTAAACGGCCTAAGCGTCTGAAAATCATTAAAGCCGCCTCAGCCAGAGATCGTTTCAAAGCGGCGGCTTCCAAAGCCGAGGGCAAGGGCGGGCAAGTGCTCACCGACGTCACCCCCGAAGAGGGCGCCGAGGCGATCTACAAGCTACTCAAGGAAGAGGACGTGCTGCGCTGA

Amino acid sequence of etf-beta (SEQ ID NO: 2):

MRPEQQQKQQKPGIDVAVLVSIGRHPTTGRARRAEQDARGLELALAMEAELPGSRIDVLHAGSQDVDSEAALRSYLGMATGVGMGLESMTLLEQPDGSDAILPLVEHLAATSPQLVITGARAERGEGSGLLPYALAEHLGWPLVNSLASIETVENGVITLLQALPRGQRRRLKVRLPAIISVDEAAPAARQSAFGPARRASFSVAPTTSEADSELAQWHLSPARKRPKRLKIIKAASARDRFKAAASKAEGKGGQVLTDVTPEEGAEAIYKLLKEEDVLR*

The primers for H1 are:

beta-H1-F: 5' CGAGGCGATCTACAAGCTACT 3' (SEQ ID NO: 3)

beta-H1-R: 5'

AGGGTTTTCCCAGTCACGACGCCGCTATCCAGCCATTGAC 3' (SEQ ID NO: 4)

The primers for H2 are:

beta-H2-F: 5'

AGTCGGTGCTTTTTTTGAACCCTGACCTTGGCAGCGTCCATT 3' (SEQ ID NO: 5)

beta-H2-R: 5'

AGTAGCTTGTAGATCGCCTCGTATGGAGACCAGCACCGCC 3' (SEQ ID NO:6)

The primers for Donor-1 are:

Donor-H-F: 5' GTCGTGACTGGGAAAACCCT 3' (SEQ ID NO: 7)

Donor-H-R: 5'GGGTTCAAAAAAAGCACCGACT 3' (SEQ ID NO:8)

PCR reaction conditions:

First, pre-denaturation at 95°C for 5 minutes; then denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 30 seconds, 30 cycles; and post-extension at 72°C for 10 minutes.

PCR reaction system (50 μL system):

When the PCR amplification system is prepared, the DNA polymerase is added last.

The 504bp PCR product obtained is the homology arm H1 of the target gene etf-β; the 565bp product is the homology arm H2 of the target gene etf-β; the 5291bp product is the plasmid backbone Donor-1 derived from pSEVA241. Then, the three amplified products were ligated with Gibson kit (purchased from NEB Company, USA, catalog number E2611L).

Gibson ligation reaction conditions:

The target fragments were first mixed, filled up to 10 μl with water, and then treated at 50° C. for 1 hour according to the instructions of the Gibson kit. The Gibson ligation system (10 μL system) is as follows:

The ligation product was obtained, transformed into Escherichia coli JM109, and the transformant was obtained and sequenced. The correct sequence was selected and stored, and the plasmid was extracted after overnight culture in LB medium. The plasmid was named pSEVA241-HR.

2), knock out the insertion of sgRNA in pKObet23 plasmid

The pSEVA241-HR plasmid inserted into the homology arms H1 and H2 in the above step was used as a template, and donor-G-F and donor-G-R were used as primers, and pfu enzyme was used for PCR amplification to obtain the plasmid backbone derived from pSEVA241-HR; The primers sgRNA-F and sgRNA-R are annealed and connected to obtain a DNA double-strand with sticky ends; the above plasmid backbone and the DNA double-strand with sticky ends are digested and connected with BsaI to obtain the target gene etf - β-knockout plasmid pKObet23.

Amplify using pSEVA241-HR with homology arms H1 and H2 as template:

donor-G-F: 5'

CTAGGGTCTCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGT 3' (SEQ ID NO: 9)

donor-G-R: 5'

CTAGGGTCTCAACTAGTATTATACCTAGGACTGAGCTAGCTGT 3’

(SEQ ID NO: 10)

A PCR product of 6300 bp was obtained, the obtained plasmid backbone was digested with the restriction enzyme BsaI, and the digested fragment was recovered.

Annealing conditions for sgRNA-F and sgRNA-R primers:

First, pre-denature at 95°C for 5 minutes; decrease by 10°C every 3 minutes, and hold at 15°C for 5 minutes at the end.

The primer sequences are:

sgRNA-F: 5' TAGTTGGGCTTTTGCCCTATGCGT 3' (SEQ ID NO: 11)

sgRNA-R: 5'AAACACGCATAGGGCAAAAGCCCA 3' (SEQ ID NO: 12)

Annealing system (10 μL):

sgRNA-F 5μL

sgRNA-R 5μL

The final primer concentration was 10 pM.

The obtained plasmid backbone after digestion and the annealed DNA double-strand were connected with T4 DNA ligase, transformed into Escherichia coli JM109, the grown recombinant colony was sequenced, the sequenced correct was stored, and the plasmid was extracted, which is The knockout plasmid pKObet23 was used to knock out the target gene etf-β.

3) Knockout of etf-β gene in Halomonas TD01

First, the plasmid pQ08 expressing Cas9 protein was transferred into Halomonas TD01 (for the transfer process, see: Qin, Qin, et al. "CRISPR/Cas9 editing genome of extremophile Halomonas spp." Metabolic Engineering (2018): 219-229.) , the plasmid contains the Cas9 protein derived from Streptococcus pyogenes, which is preserved in Tsinghua University.

The above-mentioned Halomonas TD01 containing the Cas9 protein was used as the recipient bacteria, and E.coli S17-1 carrying the plasmid pKObet23 was used as the donor bacteria for conjugation transformation. Single clones were screened on a plate of LB60 solid medium (add 15 g/L agar powder to the formula of LB60 liquid medium, autoclave at 121°C for 20 min, and pour it onto the plate after cooling). Using the monoclonal genome as a template and Test-beta-F and Test-beta-R as primers, colony PCR amplification was performed with tagase.

The primers are:

Test-beta-F: 5' GGGTGTGGCAGATTGAAGCT 3' (SEQ ID NO: 13)

Test-beta-R: 5' GTGCCGCTAAGAACGTTGTT 3' (SEQ ID NO: 14)

Colony PCR reaction conditions:

First, pre-denaturation at 95°C for 5 minutes; then denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 72°C for 30 seconds, 30 cycles; and post-extension at 72°C for 10 minutes.

PCR reaction system (50 μL system):

After the PCR amplification system was prepared and packaged, single colonies were picked with a toothpick. The colony with the amplified band size of 1338 bp was the knockout type, and the colony with the amplified band size of 2069 bp was the wild type (see Figure 1), and finally the etf-β gene knockout type of TD01 was obtained, which was named Halomonas bluephagenesis TDΔβ.

2. The effect of adding acetic acid on the synthesis of P3HB by recombinant bacteria TDΔβ

In order to test the ability of TDΔβ to synthesize P3HB, we performed shake flask experiments with TD01 and TDΔβ; first, single colonies were picked into 20 mL of LB60 medium, and cultured overnight at 37°C and 200 rpm; v), were inoculated into 50mL MM60 medium (pH8.5-9.0, NaCl concentration 60g/L), glucose concentration was set to 30g/L, and acetic acid concentration gradient was set: 0g/L, 1g/L, 2g/L, 3g /L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L, 12g/L. The culture temperature is 37°C, the rotation speed is set to 200rpm, and the shake flask is cultured for 48 hours to obtain bacterial liquid. After centrifugation, water washing, and ice drying, the dry cell weight of the sample is obtained, and then the content percentage of P3HB in the dry cell weight is detected by gas chromatography. (%). Three sets of parallels were set for each condition. Cell dry weight and P3HB content are shown in Table 6.

Table 6. Effects of acetic acid addition on Halomonas TD01 and TDΔβ

From the data in Table 6, we can draw the following conclusions: TDΔβ is obtained after knocking out etf-β from Halomonas TD01. Under the same conditions, Halomonas TDΔβ can synthesize significantly higher content of P3HB than TD01. Moreover, similar to the results obtained by adding acetic acid to the TD01 culture, adding acetic acid within a certain concentration range to the medium of Halomonas TDΔβ can also increase the dry cell weight of the microorganism and the content of P3HB accumulated in the cells. After adding 3 g/L acetic acid, the P3HB content of TDΔβ accounted for 94% of the dry cell weight, which was the highest content reported in the world so far.

The results of this experiment show that by deleting microbial etf-β, combined with adding acetic acid, the proportion of P3HB in cells can be increased to as high as 94%, and the cell volume is also increased (see Figure 2).

3. Construct the engineered strain TDΔα and test the effect of acetic acid on the synthesis of P3HB by the recombinant strain

Referring to the first part of Example 2, the etf-α subunit of the etf gene in Halomonas bluephagenesis TD01 was knocked out, and the engineering bacteria Halomonas bluephagenesis TDΔα was constructed, and the experiment and analysis were carried out as the second part of Example 2 to determine the acetate. Effects of additions on P3HB synthesis.

The nucleotide sequence of the etf-alpha subunit is (SEQ ID NO: 15):

ATGAGTGAAATTATTCGCCGCGACCCACGTCGTGAGTGGATCGCCCGAAACCGTCTGCACCCCGACAACGCCGCGGTGCTGGCCGCTCTTGGTGTAAACAGGGGCGGCGGAGCGGTCAGCGAATGGGTGGGGCCCAACGGCGTGGTGCGTAAGAATCCTCGCGCTATTGGCTTTATCGGCCCCAATGGCGTTAAACGAATTGATCGTAGCGGCCTCCAGCAAGGAGGGCATTCCAGCGCGGCAACCGCCGTGGCTAGCGATAGCCGCCGTACAGTGACTATTGATCAGCCCGCTTTTCTAGTGGCCGGGGTACCTGACCTGATCGGTGGACGCCTTTCTAGCCATGATAAGGATCTGCTGGGACTAGCCAGACGCGTGGCGGATGCCGACGCCGAACAGCCAGGCGCCGTGGTGGCGATCCTGTTTGGTGAGCATAAAGGCGAGTGGGGCGGCGAGCTTAAGAAGCAAGCGCTGGGCGAAGCTGGGATTGATCGCGTCGTACATCTTGATGATGAGCTTTACGCTGGGTTTGCTCCCGAGGCACGGTTAGCGGTGTTAAGTGCCGTCGAACGGGAGATGACGCCGCGTTTCTGGCTGTTGCCGGATTCACCCCTAGGCGGAGCTGATCTGGGGCGGCGGCTTGCCTTACGCCTGGGTGAACGCGCTGCTACAGGGGTGTGGCAGATTGAAGCTGACGATGAGGCACCTCTGGGCTGGCAGTGCACTGCCCGTGGCGCCGCTGGAAGTTTGGATATCCAGCGCCCTCTGCCACGGGTTGCCCTGGCGTTGGCCGAGTGCGCCGAACCGGTGGACGAGACCCGACATGCGGCCGAACACCTGACCTTGGCAGCGTCCATTCCCACCACTCTATCGCGCATTGAAGATCTGGGTCAGGTGGCGGTGGATCCCGCTGGCGTGGCGTTGGCCGAGGCTGAGTTCATCCTCTCTGGTGGCAATGGTGTTAAACAGTGGGACGCCTTCCATCATGCCGCGAAAGTAC TGGGCGCTACCGAAGGGGCCTCGCGTGTTGCGGTAGACGACGGCTTTATGGCCCGCGACCGGCAGGTAGGCGCGACCGGTACCTGGGTAACCGCCCGAGTCTATATGGCGGTGGGGATTTCAGGCGCTATCCAGCACCTGCAGGGCATTCAGCGCTGCGACAAGGTGGTGGCCATCAATCTCGATCCGGGGTGCGACATGATCgAACGCGCAGACCTGGCGGTGATAGGCGACAGCACGCAAATTCTTGCTGCGTTAGTGGCGATGGTGGAACAGCAGCGAGGAGGGCAGCGCGATGCGGCCTGA

The amino acid sequence of the etf-alpha subunit is (SEQ ID NO: 16):

MSEIIRRDPRREWIARNRLHPDNAAVLAALGVNRGGGAVSEWVGPNGVVRKNPRAIGFIGPNGVKRIDRSGLQQGGHSSAATAVASDSRRTVTIDQPAFLVAGVPDLIGGRLSSHDKDLLGLARRVADADAEQPGAVVAILFGEHKGEWGGELKKQALGEAGIDRVVHLDDELYAGFAPEARLAVLSAVEREMTPRFWLLPDSPLGGADLGRRLALRLGERAATGVWQIEADDEAPLGWQCTARGAAGSLDIQRPLPRVALALAECAEPVDETRHAAEHLTLAASIPTTLSRIEDLGQVAVDPAGVALAEAEFILSGGNGVKQWDAFHHAAKVLGATEGASRVAVDDGFMARDRQVGATGTWVTARVYMAVGISGAIQHLQGIQRCDKVVAINLDPGCDMIERADLAVIGDSTQILAALVAMVEQQRGGQRDAA*

The results showed that compared with the original strain Halomonas bluephagenesis TD01, under the same culture conditions, Halomonas bluephagenesis TDΔα also showed significantly increased P3HB content, presumably because of the knockout of etf-α subunit or the knockout of etf-β subunit. Both lead to the inactivation of etf gene, so the effects on the P3HB synthesis pathway are basically the same.

In addition, adding acetic acid within a certain concentration range can also increase the cell dry weight and P3HB content of the knockout strain, and the experimental results and trends are basically consistent with Halomonas bluephagenesis TDΔβ.

Example 3. Controlling the ratio of monomers in the copolymer by adding acetic acid to the basal medium

The material properties of copolymers P3HB3HV and P3HB4HB depend to a large extent on the monomer ratios, especially for P3HB3HV. At present, the biggest problem in the production of copolymers by Halomonas bluephagenesis TD is that the proportion of 3HB monomer is high, while the proportion of 3HV and 4HB is low. Acetic acid is a carbon source with lower reducibility than glucose, and the inventors considered using acetate and glucose together to regulate the internal redox environment of microorganisms, thereby controlling the metabolic flow path of acetyl-CoA. Specifically, the copolymers P3HB3HV and P3HB4HB are both dimers, the former is composed of 3-hydroxybutyric acid (3HB) and 3-hydroxyvaleric acid (3HV), and the latter is composed of 3-hydroxybutyric acid (3HB) and 4-Hydroxybutyric acid (4HB) copolymerization. 3HB is directly synthesized from acetyl-CoA by anaerobic fermentation, while 3HV and 4HB are both derived from precursors that enter the TCA cycle from acetyl-CoA. Therefore, the key to changing the monomer ratio in P3HB3HV and P3HB4HB is to control The metabolic pathway of acetyl-CoA. This experiment proves that the ratio of monomers in the copolymers P3HB3HV and P3HB4HB can also be adjusted by changing the amount of acetic acid added in an appropriate range.

1. Adjust the content of 3HV in P3HB3HV by changing the concentration of added acetic acid

Halomonas bluephagenesis TD08AB was used as the synthetic strain of P3HB3HV, which was recorded in the following documents: Yin J, Wang H, Fu X Z, et al. Effects of chromosomal gene copy number and locations on polyhydroxyalkanoate synthesis by Escherichia coliand Halomonas sp[J ]. Applied microbiology and biotechnology, 2015, 99(13): 5523-5534., the bacteria can use glucose as a single carbon source to produce P3HB3HV.

First, pick a single colony in 20 mL of LB60 (LB containing 60 g/L NaCl) medium, and cultivate it overnight at 37°C and 200 rpm; 8.5-9.0, NaCl concentration 60g/L), glucose concentration is set to 30g/L, acetic acid is set to 8 concentration gradients: 0g/L, 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L. The culture temperature is 37°C, the rotation speed is set to 200rpm, and the shake flask is cultured for 48 hours to obtain bacterial liquid. After centrifugation, washing, and ice drying, the dry cell weight of the sample is obtained, and then the polymer content in the dry cell weight is detected by gas chromatography. percentage(%). Three sets of parallels were set for each condition. Cell dry weight, P3HB3HV content and 3HV ratio are shown in Table 7.

Table 7. Halomonas bluephagenesis TD08AB synthesis of P3HB3HV at different acetic acid concentrations

From the data in Table 7, we can draw the following conclusions: The copolymer can be changed by adding 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L acetic acid The monomer ratio of P3HB3HV, especially after adding 1g/L and 2g/L acetic acid, the monomer ratio of 3HV was nearly doubled compared with the result without adding acetic acid, which greatly promoted the improvement of material properties. effect. In addition, the results also showed that the addition of 1-8 g/L of acetic acid increased the dry cell weight, resulting in a significant increase in the final P3HB3HV production compared to no addition (data not shown).

2. Control the content of 4HB in P3HB4HB by changing the concentration of added acetic acid

Halomonas bluephagenesis TD△gabD2-D2 was used as P3HB4HB synthetic bacteria, and the strain was recorded in the following literature: Ye J, Hu D, Che X, et al. Engineering of Halomonas bluephagenesis for low cost production of poly(3-hydroxybutyrate -co-4-hydroxybutyrate) from glucose [J]. Metabolic engineering, 2018, 47: 143-152., the strain can use glucose as a single carbon source to produce P3HB4HB, the strain is preserved in Tsinghua University, and the public can obtain from Tsinghua University .

First, pick a single colony in 20 mL of LB60 (LB containing 60 g/L NaCl) medium, and cultivate it overnight at 37°C and 200 rpm; 8.5-9.0, NaCl concentration 60g/L), glucose concentration fixed at 30g/L, 9 concentration gradients for acetic acid: 0g/L, 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L. The culture temperature is 37°C, the rotation speed is set to 200rpm, and the shake flask is cultured for 48 hours to obtain bacterial liquid. After centrifugation, washing, and ice drying, the dry cell weight of the sample is obtained, and then the polymer content in the dry cell weight is detected by gas chromatography. percentage(%). Three sets of parallels were set for each condition. Cell dry weight, P3HB4HB content and 4HB ratio are shown in Table 8.

Table 8. Halomonas bluephagenesis TDΔgabD2-D2 synthesis of P3HB4HB at different acetic acid concentrations

The experimental results show that adding acetic acid can also increase the cell content and yield of the copolymer P3HB4HB, compared with the control group without adding acetic acid (cell dry weight: 8.79±0.07g/L; P3HB4HB content: 66.89±0.37%), adding 1g In the range of /L-8g/L acetic acid, the dry cell weight and the content of P3HB4HB were increased, and the yield improvement effect was the most significant under the condition of adding 6g/L acetic acid, and the dry cell weight was measured to be 9.11±0.28g/L. L, P3HB4HB content was measured to be 69.91±1.93%. In addition, from the data in Table 8, we can also draw the following conclusions: the monomer ratio of the copolymer P3HB4HB can be changed by adding acetic acid, especially after adding 4g/L acetic acid, the 3HV monomer ratio is increased by more than 50%, which is a The improvement of material properties will have a very significant promotion effect.

The above two sets of experiments prove that the present invention can indeed adjust the internal environment of microbial cells by adding an appropriate amount of acetic acid to the basal medium, and control the metabolic flow of acetyl-CoA into 3HV and 4HB, thereby changing each monomer in the polymer synthesized by the microorganism proportion. In a specific embodiment, the present invention can significantly increase the proportion of 3HV monomer in P3HB3HV polymer (from 4% to 8%) by adjusting the concentration of added acetic acid; and can also significantly increase the proportion of 4HB monomer in P3HB4HB polymer (from 7.8% to 12%).

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Claims (11)

1. An improved microbial method of producing Polyhydroxyalkanoates (PHAs), the method comprising adding acetic acid, acetate salt, acetate ester or other acetic acid derivative to a basal medium comprising a source of carbon for microbial synthesis of PHAs.

2. The method of claim 1, wherein the microorganism is a bacterium of the genus Escherichia (Escherichia), halophilic (Halophile), Halomonas (Halomonas), Pseudomonas (Pseudomonas), or Bacillus (Bacillus), or a combination thereof; for example, the microorganism is Escherichia coli (Escherichia coli), Pseudomonas putida (Pseudomonas putida), Halomonas campestris or Halomonas bluephasegenes or a combination thereof, preferably Halomonas campestris or Halomonas bluephasegenes; in particular, the microorganism is selected from one or more of the following:

Escherichia coli JM109-pBHR68；

Escherichia coli JM109SG-p68orfZ+pMCSH5；

Halomonas bluephagenesis TD01；

Halomonas bluephagenesis TD08AB；

Halomonas bluephagenesis TD△gabD2-D2；

halomonas bluephagene TD delta β, and

Halomonas bluephagenesis TDΔα。

3. the method of claim 1, wherein the microorganism is a recombinant bacterium obtained after knocking out or inactivating the etf gene in halomonas, preferably, by knocking out either or both of etf- α subunit and etf- β subunit of etf gene.

4. The method of claim 1, wherein the carbon source is glucose, gluconic acid, gluconate ester or a combination thereof, the carbon source being added at a concentration of 1-100 g/L.

5. The method of claim 1, wherein the PHA is a homopolymeric PHA or a copolymeric PHA.

6. The process according to any of the preceding claims, wherein the concentration of acetic acid, acetate salt, acetate ester or other acetic acid derivative added is 1-12 g/L.

7. The process according to claim 5, wherein the concentration of acetic acid, acetate or other acetic acid derivative added is 1-12g/L, the microorganism is Halomonas bluePhagenesis TD01, Halomonas bluephagenesis TD Δ α or Halomonas bluephagenesis TD Δ β, and the PHA is poly-3-hydroxybutyrate (P3 HB).

8. The method of claim 5, wherein the concentration of acetic acid, acetate or other acetic acid derivative added is 1-8g/L, wherein the microorganism is Halomonas bluephagene TD08AB and the PHA is poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (P3HB3HV), or the microorganism is Halomonas bluephagene TD △ gabD2-D2 and the PHA is poly (3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4 HB).

9. A method of reducing pyruvate accumulated during microbial production of Polyhydroxyalkanoate (PHA), the method comprising adding acetic acid, acetate salt, acetate ester or other acetic acid derivative to a basal medium comprising a source of carbon for microbial synthesis of PHA.

10. A recombinant bacterium obtained by knocking out/inactivating etf gene on the basis of a bacterium of the genus Halomonas, preferably Halomonas bluephagesis, more preferably Halomonas bluephagesis TD01 with the accession number cgmccno. 4353.

11. A method of producing poly 3-hydroxybutyrate, comprising fermenting using the recombinant bacterium of claim 10 in a basal medium comprising a carbon source under conditions suitable for culturing the recombinant bacterium to produce poly 3-hydroxybutyrate.

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