CN116904384A - Recombinant microorganism and application thereof in production of polyhydroxyalkanoate - Google Patents

Abstract

The invention belongs to the technical field of microorganism culture, and particularly relates to a recombinant microorganism and application thereof in production of polyhydroxyalkanoates. The invention provides a culture medium of halophilic microorganisms, which takes amino acid or amino acid salt as a nitrogen source, and also provides a recombinant halophilic microorganism which knocks out a coding gene of a glutamic acid synthesis related protein in the recombinant halophilic microorganism, and the amino acid or amino acid salt (especially sodium glutamate) is used as a unique nitrogen source for fermentation culture, so that the carbon source conversion rate in the fermentation process can be remarkably improved, and the PHA yield is further improved; when fermentation is performed using the mixed amino acid as a nitrogen source, the dry cell weight, the PHA yield, the PHA percentage content and the carbon source conversion rate can be simultaneously improved.

Description

A recombinant microorganism and its application in the production of polyhydroxyalkanoate

Technical field

The invention belongs to the field of microbial culture technology, and specifically relates to a recombinant microorganism and its application in the production of polyhydroxyalkanoate.

Background technique

Microbial fermentation refers to the process of using microorganisms to convert raw materials into products needed by humans through specific metabolic pathways under appropriate conditions. Microbial fermentation is widely used in food, medicine, energy, environmental protection and other fields. Polyhydroxyalkanoate (PHA) produced using microorganisms is a biosynthetic degradable polymer material with good biocompatibility and thermal processing properties and can be used in the production of biomedical materials and degradable plastics (Ye J W , Lin Y N, YiX Q, et al. Synthetic biology of extremophiles: a new wave of biomanufacturing[J]. Trends in Biotechnology, 2022.).

At present, the main method for producing PHA using microorganisms is microbial fermentation. This method uses microorganisms to synthesize PHA using carbon sources such as sugars, fatty acids or glycerol in a fermentation tank. The traditional microbial fermentation medium uses urea or ammonium salt as a nitrogen source to perform two-stage fermentation in a fermentation tank. The first stage is the growth stage, which provides sufficient carbon sources and other nutrients to promote the growth of the strain; the second stage is the synthesis stage, which limits the nitrogen source, maintains the supply of carbon sources, and promotes the accumulation of PHA in the cell (Chen GQ, Zhang X, Liu X, et al. Halomonas spp., as chassis for low-costproduction of chemicals[J]. Applied Microbiology and Biotechnology, 2022, 106(21): 6977-6992.). The use of high concentrations of inorganic nitrogen during the fermentation process will lead to a significant decrease in the conversion rate of carbon sources to synthesize PHA, leading to a decrease in PHA production (Liu X, Li D, Yan X, et al. Rapid quantification ofpolyhydroxyalkanoates accumulated in living cells based on green fluorescence protein-labeled phasins: the qPHA method[J]. Biomacromolecules, 2022, 23(10):4153-4166.).

Therefore, developing a fermentation method that can increase the conversion rate of carbon sources and increase the accumulation of PHA is of great significance for industrial microbial production and manufacturing of PHA products.

Contents of the invention

In order to solve the above problems, the present invention provides a recombinant microorganism in which nitrogen source assimilation-related proteins (such as glutamate synthesis-related proteins) are inactivated. The present invention provides a culture medium using which Base fermentation culture microorganisms can improve the carbon source conversion rate. Using the medium fermentation to produce the recombinant microorganisms can increase the cell dry weight, PHA yield, PHA percentage content and carbon source conversion rate in fermentation production (i.e., increase conversion rate from substrate to product).

A first aspect of the present invention provides a recombinant microorganism in which glutamate synthesis-related proteins are inactivated.

The glutamate synthesis-related protein includes one of glutamate dehydrogenase (GdhA), glutamate synthase (GltBD), glutamine synthetase (GlnA) or glutamine synthetase (GlnN) Or two or more.

Preferably, the inactivation can be carried out by any method in the prior art.

Further preferably, the deactivation includes:

A) Knock out all or part of the gene encoding glutamate synthesis-related proteins, or knock down the gene encoding glutamate synthesis-related proteins;

B) Mutation of part of the bases of the gene encoding glutamate synthesis-related proteins makes the gene unable to express the protein normally, or the activity of the expressed protein is weakened or inactive.

In a specific embodiment of the present invention, the inactivation is knocking out the gene encoding glutamine synthetase.

Preferably, the knockout can be carried out using any method in the prior art.

It is further preferred to use methods including but not limited to DNA homologous recombination, RNA interference, zinc finger nucleases, TALENs, CRISPR/Cas, CRISPR/AID and other methods.

In a specific embodiment of the present invention, CRISPR/AID is used to knock out the gene encoding glutamine synthetase (GlnA or GlnN).

Preferably, the CRISPR/AID includes the use of sgRNA, and the sgRNA sequence includes the nucleotide sequence shown in any one of SEQ ID NO: 1 and 4-8.

Preferably, the recombinant microorganism also expresses exogenous PHA synthesis-related proteins.

Preferably, an expression vector containing a gene encoding an exogenous PHA synthesis-related protein can be introduced into the recombinant microorganism.

Preferably, the PHA synthesis-related protein includes one or more than two of PhaA, PhaB, PhaC, SucD, 4hbD, OrfZ, AldD or DhaT proteins.

Preferably, the growth of the recombinant microorganism requires the medium to contain inorganic salts with a certain salt concentration.

It is further preferred that the salt concentration in the culture medium is any value from 2.5 to 100g/L, and further preferably 40 to 60g/L, such as 2.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100g/L. Preferably, the recombinant microorganism is selected from eukaryotic microorganisms or prokaryotic microorganisms.

Further preferably, the recombinant microorganism is selected from prokaryotic microorganisms.

More preferably, the recombinant microorganism is selected from the genus Escherichia , Pseudomonas , Ralstonia , Aeromonas , Corynebacterium ) or Halomonas .

Preferably, the Escherichia genus includes but is not limited to Escherichia coli and its derivatives.

Preferably, the Pseudomonas genus includes but is not limited to Pseudomonas putida and its derivatives.

Preferably, the Ralstonia genus includes but is not limited to Ralstonia eutropha and its derivatives.

Preferably, the Aeromonas genus includes but is not limited to Aeromonas hydrophila and its derivatives.

Preferably, the Halomonas genus ( Halomonas ) includes but is not limited to Halomonas bluephagenesis TD01 (CGMCC No. 4353), Halomonas campaniensis LS21 (CGMCC No. 6593), Halomonas aydingkolgenesis M1 (CGMCC No. 19880), Halomonas bluephagenesis WZY254 and/or Halomonas bluephagenesis WZY278, as well as their derivative strains or combinations thereof.

The recombinant microorganism can produce PHA.

A second aspect of this aspect provides a method for preparing the recombinant microorganism described in the first aspect, which method includes inactivating glutamate synthesis-related proteins in the starting strain.

Preferably, the preparation method further includes introducing a gene encoding a protein related to PHA synthesis.

Relevant limitations on recombinant microorganisms, glutamate synthesis-related proteins, inactivation, and PHA synthesis-related proteins are the same as in the first aspect of the present invention.

A third aspect of the present invention provides a culture medium for microorganisms. The culture medium includes a nitrogen source. The nitrogen source includes amino acids or amino acid salts. The adding method of the amino acids includes directly adding one or two amino acids. More than one type of amino acid, or a substance that produces amino acids after hydrolysis is added.

Preferably, the substances that produce amino acids after hydrolysis include but are not limited to one or more of hair hydrolyzate, algae powder hydrolyzate or casein hydrolyzate.

Preferably, the amino acid is selected from but not limited to glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, One or more of cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine or histidine .

Preferably, the amino acid salt includes one or more of potassium salt, sodium salt, calcium salt, magnesium salt or zinc salt of amino acid.

In a specific embodiment of the present invention, the amino acid salt is sodium glutamate.

Preferably, the hair hydrolyzate is selected from hydrolysates of human hair or animal hair.

Further preferably, the hair hydrolyzate includes amino acids or salts thereof, and the amino acids or salts thereof include but are not limited to glycine, alanine, valine, leucine, isoleucine, and methionine. One of acid, proline, serine, tyrosine, cystine, phenylalanine, threonine, aspartic acid, glutamic acid, lysine, arginine or histidine or Various amino acids or their salts.

In a specific embodiment of the present invention, the hair hydrolyzate is wool hydrolyzate.

Preferably, in the culture medium, the concentration of amino acids or amino acid salts is any value in 0.01-2 mol/L, preferably any value in 0.01-1 mol/L, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.1, 0.2, 0.25, 0.3, 0.33, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2mol/L.

Depending on the needs of the specific implementation, the nitrogen source may include inorganic nitrogen and/or organic nitrogen. The organic nitrogen includes the above-mentioned amino acids or amino acid salts, and may also include but is not limited to peanut cake powder, soybean cake powder, cottonseed cake powder, corn steep liquor, yeast powder, fish meal, silkworm pupa powder, peptone, bran, and waste bacteria. Silk body and so on. The inorganic nitrogen includes but is not limited to ammonium salts (such as ammonium sulfate), nitrates or ammonia water, etc.

Preferably, the culture medium contains a carbon source.

Preferably, in the culture medium, the concentration of the carbon source can be any value from 1 to 300 g/L, preferably any value from 10 to 80 g/L, such as 1, 5, 10, 20, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 g/L.

Preferably, the carbon source is selected from one or more of glucose, gluconate, gluconate, sucrose, fructose or organic acids.

Further preferably, the organic acid is selected from but not limited to acetic acid, propionic acid, butyric acid and/or medium and long chain fatty acids, etc. (such as lauric acid, palm oil, oleic acid).

Further preferably, the gluconate is selected from, but is not limited to, one or more of calcium gluconate, magnesium gluconate, zinc gluconate, sodium gluconate or potassium gluconate.

In a specific embodiment of the present invention, the carbon source is glucose, fructose and/or organic acid (such as lauric acid).

Preferably, the culture medium also includes other substances used for growth, screening, metabolism, etc. of the microorganism.

Further preferably, the other substances include but are not limited to one or more of antibiotics, additional carbon sources, vitamins or growth factors used to maintain plasmid stability.

Preferably, the culture medium contains inorganic salts, and the salt concentration in the culture medium is any value from 2.5 to 100 g/L, more preferably 40 to 60 g/L, such as 2.5, 5, 10, 15 , 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100g/L.

Preferably, the pH value of the culture medium is any value from 6 to 10, such as 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10.

The culture medium includes but is not limited to replacing the inorganic nitrogen source of MM culture medium, LB culture medium, TB culture medium or SB culture medium with amino acids or amino acid salts.

Under normal conditions, the MM culture medium contains MgSO ₄ , KH ₂ PO ₄ , 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. Preferably glucose and/or NaCl are also included.

Under general conditions, the LB medium contains tryptone, yeast extract and NaCl.

The TB culture medium is a culture medium that contains more tryptone and yeast extract than the LB culture medium and provides glycerol as an additional carbon source; the SB culture medium is a culture medium that contains more tryptone and yeast extract than the LB culture medium. content of the culture medium.

According to the needs of the specific implementation, any value from 1 to 300 g/L (preferably any value from 10 to 80 g/L, such as 1, 5, 10, 20, 30, 35) can also be added to the culture medium. ,40,50,60,70,80,90,100,110,120,130,140,150,160,170,180,190,200,210,220,230,240,250,260,270,280 , 290 or 300 g/L, further preferably 35g/L) glucose, and/or add any value from 2.5-100g/L (preferably 40-60g/L, such as 2.5, 5, 10, 15, 20 , 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100g/L) of inorganic salts (such as NaCl, KCL) to maintain osmotic pressure and salt concentration.

Preferably, the microorganism is selected from the genus Escherichia , Pseudomonas , Ralstonia , Aeromonas , Corynebacterium or Halomonas . Further definitions for each genera are the same as in the first aspect of the invention.

Preferably, the microorganism expresses exogenous PHA synthesis-related proteins.

Further preferably, the glutamate synthesis-related protein of the microorganism is inactivated.

Preferably, the glutamate synthesis-related protein includes one, two or three of glutamate dehydrogenase, glutamate synthase or glutamine synthetase.

The microorganisms described can produce PHA.

The relevant limitations on inactivation and PHA synthesis-related proteins are the same as in the first aspect of the present invention.

A fourth aspect of the present invention provides a fermentation method, which method includes using the culture medium described in the above third aspect.

A fifth aspect of the present invention provides a method for cultivating microorganisms. The method includes culturing microorganisms using the medium described in the third aspect.

Preferably, the microorganism includes the recombinant microorganism of the first aspect.

A sixth aspect of the present invention provides a method for improving substrate conversion rate during fermentation, which method includes using the culture medium described in the third aspect.

Preferably, the substrate is a carbon source.

Preferably, the fermentation includes fermenting the recombinant microorganism of the first aspect.

The limitations on the culture medium and carbon source are the same as in the first aspect of the present invention.

A seventh aspect of the present invention provides a use of the medium described in the third aspect in cultivating microorganisms.

Preferably, the microorganism includes the recombinant microorganism described in the first aspect.

An eighth aspect of the present invention provides a method for producing PHA, which method includes culturing microorganisms using the medium described in the third aspect.

Preferably, the microorganism includes the recombinant microorganism of the first aspect.

Preferably, the PHA includes but is not limited to short-chain PHA (SCL-PHA) with a monomer chain length of 3-5 carbon atoms and/or medium-long chain PHA (MCL) with a monomer chain length of 6-14 carbon atoms. -PHA).

Preferably, the PHA contains a homopolymer or copolymer of the monomers constituting the PHA.

Further preferably, the monomers constituting PHA include but are not limited to 2-hydroxypropionic acid, 3-hydroxybutyric acid, 4-hydroxybutyric acid, 3-hydroxypentanoic acid, 3-hydroxypropionic acid, 5-hydroxypentanoic acid. Acid, 3-hydroxycaproic acid, 3-hydroxyheptanoic acid, 6-hydroxycaproic acid, 3-hydroxyoctanoic acid, 3-hydroxynonanoic acid, 3-hydroxydecanoic acid, 3-hydroxyundecanoic acid or 3-hydroxydodecanoic acid One, two or more of them.

More preferably, the PHA includes but is not limited to one or more of P3HP, PHB, P(HB-LA), PHV, P34HB, PHBV, PHBHHx, PHBHHp, PHO, PHN, PHD, P3HB4HB3HV or P3HB4HB5HV.

The term "and/or" as used herein includes all combinations of the items connected by this term, and each combination should be deemed to have been separately listed herein. For example, "A and/or B" includes "A", "A and B" and "B". For another example, "A, B and/or C" includes "A", "B", "C", "A and B", "A and C", "B and C" and "A and B and C" ".

The word "comprising" or "includes" used in the present invention is an open-ended method. When used to describe the sequence of a protein or nucleic acid, the protein or nucleic acid may be composed of the sequence, or may be at one end of the protein or nucleic acid. Or it can have additional amino acids or nucleotides at both ends, but still have the same or similar activity as the original sequence.

Description of the drawings

Figure 1: Sequencing results of the glnN gene in the Halomonas aydingkolgenesis M1 strain;

Figure 2: Component detection results of wool hydrolyzate.

Detailed ways

The present invention will be further explained below through specific examples. It should be noted that these embodiment illustrations are only for better describing and helping to understand the present invention, but do not limit the present invention to these examples. For example, the example uses Halomonas as an example, but it does not mean that the culture medium, culture method and genetic engineering method are not applicable to other microorganisms.

Furthermore, those skilled in the art may join and combine the different embodiments or examples described in this specification. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

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

The strains used in the examples are Halomonas bluephagenesis TD01, Halomonas campaniensis LS21, Halomonas aydingkolgenesis M1, Pseudomonas putida (Wang H, Zhou , 89:1497-1507.), Escherichia coli (Meng DC, Shi ZY, Wu LP, et al. Production and characterization of poly (3-hydroxypropionate-co-4-hydroxybutyrate) with fully controllable structures by recombinant Escherichia coli containing anengineered pathway[ J]. Metabolic Engineering, 2012, 14(4): 317-324.), Ralstonia eutropha (Zheng Z, Li M, Xue XJ, et al. Mutation on N-terminus ofpolyhydroxybutyrate synthase of Ralstonia eutropha enhanced PHB accumulation[J] . Applied microbiology and biotechnology, 2006, 72: 896-905.) and Aeromonas hydrophila (Zhao YH, Li HM, Qin LF, et al. Disruption of thepolyhydroxyalkanoate synthase gene in Aeromonas hydrophila reduces its survival ability under stress conditions[J]. FEMS microbiology letters, 2007,276(1): 34-41.).

Halomonas bluephagenesis TD01 strain was deposited in the General Microbiology Center of China Microbial Culture Collection Committee on November 19, 2010. The deposit registration number is CGMCC No.4353, and the classification name is Halomonas sp. TD01 (also known as Halomonas bluephagenesis). TD01; it is recorded in patent application publication number CN102120973A; the public can obtain this bacterium from Tsinghua University.

Halomonas campaniensis LS21 strain is a Gram-negative halophilic bacterium screened in our laboratory. It has very good prospects for industrial production and application. The preservation registration number is CGMCC No. 6593, which is recorded in the patent application publication number CN102925382A, and "Jiang X, Yao Z, Chen G Q. Controlling cell volume for efficient PHB production by Halomonas [J]. Metabolic Engineering, 2017, 44:30-37", the public can obtain the bacteria from Tsinghua University.

The deposit registration number of the Halomonas aydingkolgenesis M1 strain is CGMCC No. 19880, which is recorded in the patent application publication number CN111593006A; the public can obtain the strain from Tsinghua University.

The composition of MM medium used to culture microorganisms is as follows:

MgSO ₄ 0.2g/L; KH ₂ PO ₄ 1.5g/L; and a total of <0.1 g/L 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.

The 60MM medium is to add 35 g/L glucose as a carbon source to the above MM medium, and add 60 g/L NaCl to maintain osmotic pressure; use NaOH solution to adjust the pH between 6.5-9.0. The nitrogen source of the culture medium is added as needed, which can be different concentrations of sodium glutamate or wool hydrolyzate (mixed amino acids).

The composition of LB medium used to cultivate microorganisms is as follows:

Tryptone 10g/L Yeast extract 5g/L Sodium chloride (NaCl) 10g/L

The sodium chloride concentration in the 60LB medium composition is 60g/L.

Calculation method of cell dry weight in the examples:

Cell Dry Weight (CDW) refers to the weight of bacteria after losing all water, and its unit is g/L. The method to detect the dry weight of cells is to weigh the mass of the empty centrifuge tube as a, add 35 mL of bacterial liquid sample into the 50 mL centrifuge tube with a pipette, centrifuge to remove the supernatant, wash and shake the precipitate with distilled water to remove residual culture medium components. After freeze-drying for 18-24 hours, the weight measured at this time is b.

Cell dry weight = (b-a) ÷ 0.035; the weight unit of the centrifuge tube is g; 0.035 represents 0.035L.

Calculation method of carbon source conversion rate in the examples:

Carbon source conversion rate = amount of PHA produced (g) ÷ amount of glucose utilized (g).

Detection of polyhydroxyalkanoate (PHA) by gas chromatography:

Take about 30 mg of the dry product and add it to the esterification tube. Take about 16 mg, 18 mg, 20 mg and 22 mg of pure P3HB standard samples, and add 4 ml of the esterification mixture (mix the esterification liquid and trichloride). Mix methane at a volume ratio of 1:1), seal and place in a 100°C metal bath for heating and esterification for 4 hours. Take out the esterification reaction bottle from the esterifier and place it in a fume hood to cool to room temperature for 10-15 minutes. After the esterification is completed, cool to room temperature, add 1 ml of ultrapure water to the esterification tube, cap the bottle tightly, shake with a multi-tube vortex at 2500 rpm for 5 minutes to extract, wait for 1 hour after shaking, wait for the organic phase and water Phase separation, use a syringe to extract 1ml of the lower organic phase in the fume hood, and inject it into the injection bottle for gas chromatography detection.

The gas chromatograph is Shimadzu GC-2014 gas chromatograph, and the chromatographic column is HP-5 column. Set the temperature rise program for GC analysis as follows: the inlet temperature is 240°C, the detector temperature is 250°C, the starting temperature is 80°C and maintained for 1.5 minutes, and then enters the first heating stage with a heating rate of 30°C/min. The heating stage is 40°C/min. When the temperature rises to 240°C, it is maintained for 2 minutes.

The data processing method is: obtain the internal standard peak area measured by gas chromatography, the PHA monomer methyl ester peak area of the standard sample, the internal standard peak area of the sample, and the PHA monomer methyl ester peak area of the sample to calculate the 3HB monomer ratio. Make a standard curve based on the PHA monomer methyl ester peak area of the standard product/the peak area of the internal standard, and use this to calculate the mass of 3HB in the sample, and then calculate the percentage of 3HB in the sample: 3HB mass/cell dry weight.

Example 1: Using sodium glutamate as a nitrogen source to improve the glucose conversion rate during fermentation production

Halomonas campaniensis LS21 was inserted into 60MM culture media with three different nitrogen sources. The culture medium of control group 1 used 0.05 mol/L ammonium sulfate as the nitrogen source, and the culture medium of control group 2 used 0.025 mol/L. Urea was used as the nitrogen source, and the experimental group used 0.05 mol/L sodium glutamate as the nitrogen source. The quality of nitrogen in the culture media of the control group 1, control group 2, and the experimental group was consistent, and each group contained 60 g/L. of NaCl and 35 g/L glucose. Three parallel samples were set up for each set of experiments, and the results were averaged. The samples were cultured at 37°C, 200 rpm for 48 h, and the glucose conversion rate was calculated. The results are shown in Table 1.

Table 1: Halomonas campaniensis LS21 uses sodium glutamate as nitrogen source to improve the conversion rate of glucose into PHB.

As can be seen from Table 1, compared with using ammonia sulfate or urea as the nitrogen source, using sodium glutamate as the nitrogen source in Halomonas campaniensis LS21 can significantly improve the glucose conversion rate, and the glucose conversion rate reached 0.47±0.01.

The results show that the glucose conversion rate of fermentation using sodium glutamate as a nitrogen source is better than that of ammonia sulfate and urea, and can convert as much glucose as possible into the target product P3HB.

Example 2: Knock out the glnN gene in Halomonas and use sodium glutamate as the nitrogen source to improve the glucose conversion rate during fermentation production.

First, CRISPR/AID technology was used to knock out the glutamine synthetase gene glnN in Halomonas aydingkolgenesis M1. Specifically, a plasmid with pSEVA341 as the backbone was constructed. The plasmid contained the sgRNA sequence: gccagattctgcggcgcattct (SEQ ID NO: 1). Then, the plasmid containing the sgRNA sequence and the CRISPR/AID plasmid were combined and transferred into the Halomonas aydingkolgenesis M1 strain, and a pair of primers F: tgttgaagagaccgggctt (SEQ ID NO: 2) and R: tacagctggctttgagtgg (SEQ ID NO: 3) were designed for colony PCR verification. , the band with the correct size was sent for sequencing, and the 126th codon CAG of the glnN gene of the successfully knocked out strain was mutated into a TAG stop codon. The results are shown in Figure 1. The colonies with successful glnN gene knockout were selected and transferred to 60LB culture medium, placed on a 37°C shaker to drop the plasmid, and after streaking verification, the glnN gene knockout strain was obtained, named M1-Δ glnN.

The above gene knockout strain M1-Δ glnN and the original starting strain Halomonas aydingkolgenesis M1 were inserted into 60MM culture medium, using sodium glutamate as the nitrogen source, and the concentration of sodium glutamate was set to 0.1 mol/L. Each group contained 60g /L NaCl and 35 g/L glucose. Three parallel samples were set up for each set of experiments, and the results were averaged. The samples were cultured at 37°C, 200 rpm for 48 hours, and the glucose conversion rate was calculated. The results are shown in Table 2.

Table 2: Knocking out the glnN gene in Halomonas aydingkolgenesis M1 and using sodium glutamate as a nitrogen source can improve the conversion rate of glucose into P3HB.

As can be seen from the results in Table 2, in the same culture medium using sodium glutamate as the nitrogen source, compared with the wild-type Halomonas aydingkolgenesis M1, the glnN gene-deleted Halomonas aydingkolgenesis M1 strain can significantly increase the glucose conversion rate. The conversion rate reached 0.58±0.02.

This shows that knocking out the glutamine synthetase gene glnN and using sodium glutamate instead of urea as the nitrogen source can significantly improve the glucose conversion rate and convert as much glucose as possible into the target product P3HB, which is very good in industrial microbial production. application prospects.

Example 3: Using wool hydrolyzate (mixed amino acids) as nitrogen source to increase cell dry weight, PHA production and glucose conversion rate during fermentation production

Halomonas bluephagenesis TD01 was used as the starting strain for fermentation culture, and a control group and an experimental group were set up. The medium of the control group used the above-mentioned 60MM medium (0.1 mol/L urea as a nitrogen source); the medium of the experimental group used the medium of the control group. The urea in the 60MM medium was replaced with 0.1 mol/L wool hydrolyzate (mixed amino acids. The main components and proportions of the mixed amino acids are shown in Figure 2 and Table 3. The main component is glutamic acid, and it is also rich in a variety of other Amino acids), both the control group and the experimental group contained 60 g/L NaCl and 35 g/L glucose. Three parallel samples were set up in each group of experiments, and the results were averaged. Fermentation culture was carried out at 37°C, 200 rpm, for 48 h. Afterwards, the bacterial cells were collected and freeze-dried to detect the dry weight of the cells, and gas chromatography was used to detect the PHA content and calculate the glucose conversion rate. The results are shown in Table 4.

Table 3: Amino acid composition and proportions contained in wool hydrolyzate

Table 4: Halomonas bluephagenesis TD01 uses wool hydrolyzate (mixed amino acids) as nitrogen source to produce PHA

As can be seen from the results in Table 4, compared with the control group using urea, the experimental group using wool hydrolyzate (mixed amino acids) as the nitrogen source for PHB production and fermentation in Halomonas bluephagenesis TD01 can significantly increase the cell dry weight and PHA production. , PHA percentage and glucose conversion rate. The dry cell weight reached 15.67 g/L, the weight of PHA reached 12.41 g/L, and the glucose conversion rate reached 0.45.

Example 4 Knocking out the glutamine synthetase gene in Pseudomonas putida and fermenting and producing PHA using sodium glutamate or mixed amino acids as nitrogen source

Using CRISPR/AID technology, the glutamine synthetase gene glnA in the genome of Pseudomonas putida was deleted. A plasmid with pSEVA341 as the backbone was constructed, which contained the sgRNA sequence cagcaccacgtgaccatgc (SEQ ID NO: 4) targeting the glutamine synthetase gene. The plasmid containing the sgRNA sequence and the CRISPR/AID plasmid were transformed into Pseudomonas putida. After sequencing verification, the colonies with successful glnA gene knockout were selected and placed in LB medium for cultivation at 30°C to discard the plasmid. After streaking verification, the glnA gene knockout strain was obtained, named PP-Δ glnA.

The above gene knockout strain PP-Δ glnA was inserted into the MM medium, and three sets of experiments were set up, using 0.1 mol/L urea, sodium glutamate and wool hydrolyzate (mixed amino acids) as nitrogen sources, and the amount of glucose added Both are 35g/L. Three parallel samples were set up for each set of experiments, and the results were averaged. Fermentation culture was carried out at 30°C, 200 rpm, for 48 h. Afterwards, the bacterial cells were collected and freeze-dried to detect the dry weight of the cells, and gas chromatography was used to detect the PHA content and calculate the glucose conversion rate. The experimental results are shown in Table 5.

Table 5: Comparison of fermentation results using urea, sodium glutamate and wool hydrolyzate as nitrogen sources in PP-Δ glnA strain.

As can be seen from Table 5, compared with urea as a nitrogen source, the cell dry weight, PHA percentage and glucose conversion rate of the PP-Δ glnA strain were significantly improved when using sodium glutamate or mixed amino acids as a nitrogen source. .

Example 5 Knocking out the glutamine synthetase gene in recombinant Escherichia coli and using sodium glutamate or mixed amino acids as nitrogen sources can optimize PHA production

The PHB synthesis genes phaA , phaB and phaC genes from Ralstonia eutropha ( Cupriavidus necator ) were integrated into the genome of Escherichia coli to construct a recombinant E. coli capable of producing PHA. Then the CRISPR/AID technology mentioned above was used to knock out the glutamine synthetase gene glnA in its genome. A plasmid with pSEVA341 as the backbone was constructed, which contained the sgRNA sequence caggtgaatgctgaattctt (SEQ ID NO: 5) targeting the glutamine synthetase gene. The plasmid containing the sgRNA sequence and the CRISPR/AID plasmid were transformed into E. coli. After sequencing verification, the colonies with successful glnA gene knockout were selected and placed in LB medium at 37°C to culture the plasmid. After streaking verification, the glnA gene knockout strain was obtained, named EC-Δ glnA.

The above gene knockout strain EC-Δ glnA was inserted into the MM medium, and three sets of experiments were set up, using 0.1 mol/L urea, sodium glutamate and wool hydrolyzate (mixed amino acids) as nitrogen sources, and the amount of glucose added Both are 35g/L. Three parallel samples were set up for each set of experiments, and the results were averaged. Fermentation culture was carried out at 37℃, 200rpm, for 48h. Afterwards, the bacterial cells were collected and freeze-dried to detect the dry weight of the cells, and gas chromatography was used to detect the PHA content and calculate the glucose conversion rate. The experimental results are shown in Table 6.

Table 6: Fermentation results using urea, sodium glutamate and wool hydrolyzate as nitrogen sources respectively in EC-Δ glnA strain

It can be seen from the results in Table 6 that compared with urea as a nitrogen source, the cell dry weight, PHA percentage and glucose conversion rate of the EC-Δ glnA strain when using sodium glutamate or mixed amino acids as a nitrogen source are significantly different. promote.

Example 6 Knocking out the glutamine synthetase gene in Ralstonia eutropha and using sodium glutamate or mixed amino acids as nitrogen sources can optimize PHA production

CRISPR/AID technology was used to knock out the glutamine synthetase gene glnA in the genome of Ralstonia eutropha ( Cupriavidus necator ). A plasmid with pSEVA341 as the backbone was constructed, which contained the sgRNA sequence cgatccaggtggttgacctc (SEQ ID NO: 6) targeting the glutamine synthetase gene. The plasmid containing the sgRNA sequence and the CRISPR/AID plasmid were transformed into E. eutropha roschii. After sequencing verification, the colonies with successful glnA gene knockout were selected and placed in LB medium at 30°C to culture the plasmid. After streaking verification, the glnA gene knockout strain was obtained, named RE-Δ glnA.

The above gene knockout strain RE-Δ glnA was inserted into MM culture medium, and three sets of experiments were set up, using 0.1 mol/L urea, sodium glutamate and wool hydrolyzate (mixed amino acids) as nitrogen sources, and the amount of fructose added Both are 20g/L. Three parallel samples were set up for each set of experiments, and the results were averaged. Fermentation culture was carried out at 30℃, 200rpm, for 48h. Afterwards, the bacterial cells were collected and freeze-dried to detect the dry weight of the cells, and gas chromatography was used to detect the PHA content and calculate the fructose conversion rate. The experimental results are shown in Table 7.

Table 7: Fermentation results using urea, sodium glutamate and wool hydrolyzate as nitrogen sources respectively in RE-Δ glnA strain

It can be seen from the results in Table 7 that compared with urea as a nitrogen source, the cell dry weight, PHA percentage and fructose conversion rate of the RE-Δ glnA strain when using sodium glutamate or mixed amino acids as a nitrogen source are significantly different. promote.

Example 7 Knocking out the glutamine synthetase gene in Aeromonas hydrophila and using sodium glutamate or mixed amino acids as nitrogen sources can optimize PHA production

CRISPR/AID technology was used to knock out the glutamine synthetase gene glnA in the genome of Aeromonas hydrophila . A plasmid with pSEVA341 as the backbone was constructed, which contained the sgRNA sequence ggagcagcacgtctccatcc (SEQ ID NO: 7) targeting the glutamine synthetase gene. The plasmid containing the sgRNA sequence and the CRISPR/AID plasmid were transformed into Aeromonas hydrophila. After sequencing verification, the colonies with successful glnA gene knockout were selected and placed in LB culture medium at 30°C to culture the plasmid. After streaking verification, the glnA gene knockout strain was obtained, named AH-Δ glnA.

The above gene knockout strain AH-Δ glnA was inserted into MM culture medium, and three sets of experiments were set up, using 0.1 mol/L urea, sodium glutamate and mixed amino acids as nitrogen sources respectively, and the addition amount of lauric acid was 10 g/ L. Three parallel samples were set up for each set of experiments, and the results were averaged. Fermentation culture was carried out at 30℃, 200rpm, for 48h. Afterwards, the bacterial cells were collected and freeze-dried to detect the dry weight of the cells, and gas chromatography was used to detect the PHA content and calculate the lauric acid conversion rate. The experimental results are shown in Table 8.

Table 8: Fermentation results using urea, sodium glutamate and wool hydrolyzate as nitrogen sources respectively in AH-Δ glnA strain

As can be seen from Table 8, compared with urea as a nitrogen source, the AH-Δ glnA strain has better cell dry weight, PHA percentage content and lauric acid conversion rate when using sodium glutamate or mixed amino acids as a nitrogen source. Significantly improved.

The above results show that by knocking out the glutamic acid synthesis gene and using amino acids or mixed amino acids as nitrogen sources for fermentation experiments, the cell dry weight, PHA production and carbon source conversion rate are better than using other nitrogen sources alone, which can make the carbon source Convert as much as possible into the target product P3HB while increasing the accumulation of PHA. This fermentation method has great prospects in microbial industrial production.

Comparative Example 1 Compare the effect of using other amino acids on the transformation rate in wild-type Halomonas bluephagenesis TD01 and the recombinant strain Halomonas bluephagenesis TD01-Δ glnN with the glnN gene knocked out.

CRISPR/AID technology was used to knock out the glutamine synthetase gene glnN in Halomonas bluephagenesis TD01. For the specific method of knocking out the glnN gene in H. bluephagenesis , please refer to Example 2. The sgRNA used is cagccagtatggccagctat (SEQ ID NO: 8).

The above gene knockout strain TD-Δ glnN and the original strain Halomonas bluephagenesis TD01 were inserted into 60MM culture medium, and urea, glutamic acid and other amino acids were used as nitrogen sources respectively. The nitrogen source concentrations are shown in Table 9 (concentration design The concentration of N element is 1 mol/L). Three parallel samples were set up for each set of experiments, and the results were averaged. The culture was performed at 37°C, 200rpm for 48 hours, and the glucose conversion rate was calculated. The results are shown in Table 9.

Table 9

As can be seen from the results in Table 9, compared to using urea or other types of amino acids as nitrogen sources, when glutamic acid is used as the nitrogen source, the conversion rate of glucose reaches 0.47, which is significantly higher than the conversion rates of other groups; and, Using glutamate as a nitrogen source to ferment the Halomonas bluephagenesis TD01 strain with the glnN gene knocked out can further improve the transformation rate.

The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention. These simple modifications All belong to the protection scope of the present invention.

In addition, it should be noted that each of the specific technical features described in the above-mentioned specific embodiments can be combined in any suitable manner without conflict. In order to avoid unnecessary repetition, the present invention combines various possible combinations. The combination method will not be further explained.

In addition, any combination of various embodiments of the present invention can also be carried out. As long as they do not violate the idea of the present invention, they should also be regarded as the disclosed content of the present invention.

Claims (17)

1. A recombinant microorganism, characterized in that the glutamic acid synthesis-related protein of the recombinant microorganism is inactivated; The glutamate synthesis-related protein includes one or more of glutamate dehydrogenase GdhA, glutamate synthase GltBD, glutamine synthetase GlnA or glutamine synthetase GlnN. 2. The recombinant microorganism according to claim 1, characterized in that the recombinant microorganism expresses exogenous polyhydroxyalkanoate synthesis-related proteins. 3. The recombinant microorganism according to claim 1, characterized in that the recombinant microorganism is selected from the group consisting of Escherichia , Pseudomonas , Ralstonia , Aeromonas Aeromonas , Corynebacterium or Halomonas . 4. A method for preparing a recombinant microorganism according to any one of claims 1 to 3, characterized in that the preparation method includes inactivating glutamate synthesis-related proteins in the starting strain. 5. The preparation method according to claim 4, characterized in that the preparation method further includes introducing polyhydroxyalkanoate synthesis-related proteins. 6. A culture medium for microorganisms, characterized in that the culture medium includes a nitrogen source, and the nitrogen source includes amino acids or amino acid salts; The method of adding amino acids includes directly adding one or more than two amino acids, or adding substances that produce amino acids after hydrolysis. 7. The culture medium according to claim 6, wherein the substance that produces amino acids after hydrolysis includes one or more of hair hydrolyzate, algae powder hydrolyzate or casein hydrolyzate. 8. The culture medium according to claim 6, wherein the amino acid is selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, methionine, proline, Tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine or groups One or more than two kinds of amino acids, The amino acid salts include one or more of potassium salts, sodium salts, calcium salts, magnesium salts or zinc salts of amino acids. 9. The culture medium according to claim 6, wherein the concentration of amino acids or amino acid salts in the culture medium is 0.01-2 mol/L. 10. The culture medium according to claim 6, wherein the culture medium contains a carbon source, and the concentration of the carbon source in the culture medium is 1-300 g/L. 11. The culture medium according to claim 10, wherein the carbon source is selected from the group consisting of glucose, gluconate, gluconate, sucrose, fructose, lauric acid, acetic acid, propionic acid, butyric acid, and palm oil. Or one or more than two kinds of oleic acid. 12. The culture medium according to claim 6, wherein the culture medium contains inorganic salts, The concentration of inorganic salts in the culture medium is 2.5-100 g/L. 13. The culture medium according to claim 6, wherein the microorganism is selected from the group consisting of Escherichia , Pseudomonas , Ralstonia , Aeromonas Aeromonas , Corynebacterium or Halomonas . 14. The culture medium according to claim 6, wherein the microorganism expresses exogenous polyhydroxyalkanoate synthesis-related proteins. 15. The culture medium according to claim 14, characterized in that the glutamic acid synthesis-related protein of the microorganism is inactivated. 16. A method for cultivating microorganisms, characterized in that the method includes using the culture medium according to any one of claims 6-15. 17. A method for producing polyhydroxyalkanoate, characterized in that the method includes culturing microorganisms using the culture medium of any one of claims 6-15.