CN110857449A

CN110857449A - Improved method for producing polyhydroxyalkanoate

Info

Publication number: CN110857449A
Application number: CN201810971946.0A
Authority: CN
Inventors: 陈国强; 凌晨; 乔冠清; 帅博闻
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2018-08-24
Filing date: 2018-08-24
Publication date: 2020-03-03
Anticipated expiration: 2038-08-24
Also published as: CN110857449B

Abstract

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

Description

Improved method for producing polyhydroxyalkanoate

Technical Field

The invention relates to the field of biotechnology, in particular to an improved method for producing Polyhydroxyalkanoate (PHA). More specifically, the present invention relates to a method for increasing the microbial production of PHA and/or controlling the monomer ratio in PHA copolymer product by adding an amount of acetic acid to the culture medium.

Background

Currently, glucose or sodium gluconate is generally used as a carbon source in industrial mass production, wherein the glucose or sodium gluconate first generates pyruvic acid through glycolysis, then generates acetyl coenzyme a from the pyruvic acid, and then enters a synthesis pathway for synthesizing various polymer monomers from the acetyl coenzyme a. Glycolysis produces a large amount of NADH and ATP, NADH is also produced in the process of producing acetyl coenzyme A from pyruvic acid, NADH accumulation is caused due to the lack of oxygen in high-density cell culture, and product synthesis is further influenced, so that resource waste is caused. Accordingly, there is a need in the art for a method that alleviates or solves this problem.

Polymers can be divided into copolymers (homopolymers) and homopolymers (homopolymers) according to the composition of the monomers, the homopolymers consisting of one monomer and the copolymers consisting of more than two monomers. The material properties of the homopolymers are simple and not adjustable, while the material properties of the copolymers can be adjusted by varying the monomer ratios therein. The copolymer has higher added value due to better material performance, wherein the proportion of the monomer greatly determines the material performance.

In addition, many microorganisms are known to be capable of producing PHA copolymers through metabolic engineering, but the control of the ratio of monomers therein is difficult. The current methods for controlling the monomer ratio are mainly divided into two categories, wherein one category is to improve the ratio of specific monomers by genetically engineering the metabolic pathways of microorganisms, by optimizing the expression of specific genes, or to reduce the ratio of specific monomers by inhibiting the expression of specific genes. The other is to change the ratio of the corresponding monomers in the polymer by directly controlling the addition amount of a specific related precursor, but most of the related precursors of the monomers are expensive, and some of the precursors are toxic even to cells, so that the method is difficult to be applied to industrial production on a large scale. Therefore, it is important to find a method for adjusting the monomer ratio in the copolymer at low cost in industrial production.

Disclosure of Invention

The present invention has been made to solve the foregoing problems of the prior art.

As for the glycolytic pathway for synthesizing PHA polymer by microorganisms, the inventor of the invention has found through repeated experimental research and analysis that the inhibition of the synthesis of polymer product under high cell density fermentation is caused by the inhibition of the pathway of pyruvate to acetyl-CoA due to the accumulation of NADH, and further the inhibition of the synthesis pathway of various PHA polymer monomers starting from acetyl-CoA, and finally the limitation of the synthesis of polymer product. In view of this, finding a cost-effective method to reduce or eliminate the inhibition of pyruvate metabolism is of great importance for the industrial production of polymers.

In order to reduce or eliminate the inhibition on the metabolism of pyruvic acid, the inventor finds that the addition of acetic acid, acetate or other acetic acid derivatives to a culture medium for microbial fermentation can reduce the inhibition of NADH accumulation on the pathway of producing acetyl coenzyme A from pyruvic acid, reduce the accumulation of pyruvic acid and improve the product yield. Furthermore, it has surprisingly been found that the addition of acetic acid, acetate or other acetic acid derivatives even has the effect of adjusting the monomer ratio in the copolymer.

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

(A)

It is an object of the present invention to increase the yield of polymers obtained in microbial synthetic polymers such as Polyhydroxyalkanoate (PHA) processes. The inventor of the invention finds that the aim can be achieved by adding acetic acid, acetate or other acetic acid derivatives into a basic culture medium, and the invention has the advantages of simple production process, low cost and wide application prospect.

Accordingly, in one aspect, the present invention provides an improved method for the production of Polyhydroxyalkanoates (PHAs), comprising the addition of acetic acid, acetate salt, acetate ester or other acetic acid derivative, preferably acetic acid or acetate salt, to a basal medium comprising a carbon source for culturing microorganisms to produce polymers. By the method, the metabolic inhibition of pyruvic acid generated in the process of producing the polymer by the microorganism is relieved, the utilization rate of the carbon source is improved, and the aims of improving the content and the final yield of the polymer in the microbial cells can be fulfilled.

The microorganisms involved in the method of the invention are gram-positive or negative bacteria capable of synthesizing biopolymers. The bacteria include, but are not limited to, bacteria of the genera Escherichia (Escherichia), halophilic (Halophile), Halomonas (Halomonas), Pseudomonas (Pseudomonas), Bacillus (Bacillus), or the like, or combinations thereof. Preferably the microorganism is a bacterium of the genus Halomonas (Halomonas). More specifically, the microorganism may be, for example, Escherichia coli (Escherichia coli), Pseudomonas putida (Pseudomonas putida), Halomonas camphaniensis, or Halomonas bluephaseensis, or the like, or a combination thereof, preferably Halomonas camphaniensis or Halomonas bluephaseensis, or a combination thereof.

The above-mentioned microorganisms involved in the process of the invention may be wild-type (i.e. in their natural form, under suitable culture conditions, capable of synthesizing a polymer, such as PHA, using a suitable substrate), or may be recombinant, including but not limited to bacteria obtained by mutagenesis, genetic engineering, etc. for example, in the case of recombinant, the above-mentioned microorganisms may be recombinant microorganisms engineered in a microorganism which is not itself capable of synthesizing PHA, such as by introduction of genes involved in PHA synthesis, thereby enabling them to synthesize a polymer using suitable substrates under suitable culture conditions, for example, recombinant microorganisms obtained by introduction of the synthetic gene phaC gene of polyhydroxybutyrate in escherichia coli, which may also be obtained by disruption/knock-out of the etf gene on the basis of a wild-type microorganism capable of synthesizing PHA (such as Halomonas genenesogenas 01), for example, by disruption/knock-out of the etf- α or etf-2 gene, more specifically on the basis of the mutagenesis of the Halomonas genes involved in the process of pseudomonas aeruginosa, such as pseudomonas aeruginosa, pseudomonas sp, pseudomonas.

More preferably, the microorganism may 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 microorganism to which the present invention relates may be cultured under appropriate culture conditions (temperature, rotation speed, dissolved oxygen, pH, etc.) as long as the culture enables synthesis of the desired PHA polymer. For example, the temperature and the rotation speed during the culture can be appropriately set by those skilled in the art according to the characteristics of the microorganism or selected by routine optimization experiments.

In the above-mentioned method, a medium for fermentation culture can be obtained by adding a substrate (also referred to as a carbon source) and a regulating substance (such as acetic acid) associated with product synthesis to the basal medium. For example, the final medium may comprise glucose, gluconic acid, gluconate, or a combination thereof as a carbon source for the cultivation of the microorganism. For purposes of differentiation, the carbon sources described herein do not encompass acetic acid. Preferably, the medium comprises glucose as a carbon source. Optionally, the medium may or may not also contain a carbon source other than glucose.

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

In the above process, acetic acid, acetate ester or other acetic acid derivative may be added to the basal medium before the start of the culture of the microorganism. Alternatively, acetic acid, acetate or other acetic acid derivatives may be added to the medium once, in portions or in streams during the cultivation of the microorganism. Alternatively, the acetic acid, acetate ester or other acetic acid derivative may be added to the culture medium at the same time as the microorganism is inoculated into the culture medium. The acetic acid, acetate salt, acetate ester or other acetic acid derivative may be added at a concentration, for example, in the range of about 1-12g/L, about 1-11g/L, about 1-10g/L, about 1-9g/L, or about 1-8 g/L; preferably about 1-8g/L, for example in the range of about 2-8g/L, about 2-7g/L, about 2-6g/L, about 2-5g/L, about 2-4g/L, or 2-3 g/L; more preferably 2-6g/L, for example in the range of about 3-6g/L or about 3-5 g/L. These concentrations may be appropriately adjusted depending on factors such as the composition of the medium and the culture conditions on the basis of the present invention as long as they do not affect the effects of the present invention.

The acetate salt mentioned above may be any one or more acetate salts as long as the addition thereof can be reasonably expected by those skilled in the art not to adversely affect the object of the present invention, for example, the acetate salt may be, but is not limited to: sodium acetate, potassium acetate, calcium acetate, and the like. Likewise, the above-mentioned acetic acid ester may be any one or more acetic acid esters as long as the addition thereof can be reasonably expected by those skilled in the art not to adversely affect the object of the present invention, for example, methyl acetate, ethyl acetate, propyl acetate, etc.

The above-mentioned basal medium means a medium containing nutrients which can be used to support the growth of the microorganism of the present invention. The above-mentioned basic medium may be a medium conventionally used in the art for culturing microorganisms, such as mineral medium, LB medium, MM medium or beef extract peptone, etc., or a medium modified according to the intended purpose on the basis of these media. That is, one skilled in the art can routinely select an appropriate basal medium as long as it is capable of allowing the growth of the microorganism.

In the method of the present invention, the synthesized polymer may be Polyhydroxyalkanoate (PHA), but is not limited thereto. In particular, the PHA can be a copolymer or a homopolymer, or a combination thereof. In the case of a homopolymer, it may include, but is not limited to, polyhydroxypropionate, polyhydroxybutyrate, polyhydroxyvalerate, and the like, for example, poly-3-hydroxybutyrate (P3HB), poly-4-hydroxybutyrate (P4HB), poly-3-hydroxypropionate (P3HP), or poly-3-hydroxyvalerate (P3HV), and the like. In the case of the copolymer, the copolymer may be a dimer, a trimer, but is not limited thereto, and for example, the copolymer may be a copolymer of a hydroxypropionate and a hydroxybutyrate; copolymers of a hydroxy propionate and a hydroxy valerate; a copolymer of hydroxybutyrate and hydroxyvalerate; hydroxy propionate, hydroxy butyrate, hydroxy valerate, and the like. 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), or the like.

According to the technical scheme of the invention, by adding a proper amount of acetic acid into a basal medium containing a carbon source for culturing microorganisms, the metabolic inhibition of pyruvic acid is relieved or eliminated, the capability of the microorganisms for synthesizing polymers is improved, the dry weight of cells is increased, and the yield of the finally obtained polymers is increased.

Based on the foregoing, the present invention also provides a method for increasing the yield of polyhydroxyalkanoate in the microbial synthesis of polyhydroxyalkanoate, comprising adding acetic acid, acetate salt, acetate ester or other acetic acid derivative, preferably acetic acid or acetate salt, to a basal medium comprising a carbon source for culturing a microorganism for the production of a polymer. The definitions for the specific microorganism, carbon source, culture medium, acetic acid (or acetate, acetate or other acetic acid derivative) involved in the process are as described above.

(II)

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

Thus, in a second aspect, the present invention provides an improved method for the production of Polyhydroxyalkanoates (PHAs), the method comprising adding acetic acid, acetate or other acetic acid derivative to a basal medium comprising a source of carbon for the growth and cultivation of the microorganism, the polymer being a copolymeric polyhydroxyalkanoate. By the method, the shunting of the microbial metabolic flow can be changed, the internal environment of the microbe is changed, the metabolic flow of acetyl coenzyme A entering different monomer synthesis paths is influenced, and the proportion of various monomers in the cell accumulated biopolymer is changed. The invention has simple production process, low cost and wide application prospect.

The microorganism to which the method of the invention relates may be of wild type (i.e. in its natural form, under suitable culture conditions, capable of synthesizing a polymer, such as PHA, using a suitable substrate), or of recombinant type, which has been artificially engineered, in the case of recombinant type, to be a recombinant microorganism which is engineered in a microorganism which is not itself capable of synthesizing PHA, such as for example by introducing genes associated with PHA synthesis, and which is in turn capable of synthesizing a polymer using suitable substrates, such as for example a recombinant microorganism obtained by introducing polyhydroxybutyrate synthesis gene phaC gene in escherichia coli, and which may also be obtained by knocking out/knocking out etf genes on the basis of a wild type microorganism capable of synthesizing PHA, such as Halomonas bluegenes TD 5632, for example by knocking out/knocking out etf- α or etf- β genes, more particularly on the basis of Halomonas bluenesensis, Pseudomonas canadensis, Pseudomonas putida (Pseudomonas putida), or the like, such as for example on the basis of Halomonas copy, or the mutagenesis method of the invention, such as Halomonas copy, or the invention, such as the method of knocking out/or the invention, such as Halomonas copy, the invention, the method of mutagenesis of Halomonas copy, such as Halomonas copy, the invention is further described above.

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 microorganism to which the present invention relates may be cultured under appropriate culture conditions (temperature, rotation speed, dissolved oxygen, pH, etc.) as long as the culture enables it to synthesize a desired polymer. For example, the temperature and the rotation speed during the culture can be appropriately set by those skilled in the art according to the characteristics of the microorganism or selected by routine optimization experiments.

In the above-mentioned method, a medium for fermentation culture can be obtained by adding a substrate (also referred to as a carbon source) and a regulating substance (such as acetic acid) associated with product synthesis to the basal medium. For example, the final medium may be glucose, gluconic acid, gluconate, or a combination thereof as a carbon source for the cultivation of the microorganism. Preferably, the medium comprises glucose as a carbon source. For purposes of differentiation, the carbon sources described herein do not encompass acetic acid. Optionally, the medium may or may not also contain a carbon source other than glucose.

In the method of the present invention, the synthesized polymer is a copolymer, which may be a dimer, a trimer, but is not limited thereto, for example, the copolymer may be a copolymer of a hydroxypropionate ester and a hydroxybutyrate ester; copolymers of a hydroxy propionate and a hydroxy valerate; a copolymer of hydroxybutyrate and hydroxyvalerate; hydroxy propionate, hydroxy butyrate, hydroxy valerate, and the like. 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), or the like.

Based on the foregoing, the present invention also provides a method for adjusting the monomer ratio in a polyhydroxyalkanoate copolymer in the microbial synthesis, comprising adding acetic acid, acetate salt, acetate ester or other acetic acid derivative, preferably acetic acid or acetate salt, to a basal medium comprising a carbon source for culturing a microorganism to produce the polymer. The definitions for the specific microorganism, carbon source, culture medium, acetic acid (or acetate, acetate or other acetic acid derivative) involved in the process are as described above.

(III)

In a third aspect, the present invention provides a method of reducing pyruvate accumulated during production of Polyhydroxyalkanoate (PHA) by a microorganism, the method comprising adding acetic acid, acetate salt, acetate ester or other acetic acid derivative at a concentration as described in the first to second aspects above to a basal medium comprising a carbon source for culturing the microorganism. Further, the basal medium, carbon source and the like in the method are also as described in the foregoing first to second aspects.

(IV)

In a fourth aspect, the present invention provides a recombinant bacterium obtained by knocking out/inactivating any or all of the etf- α and etf- β subunits of etf gene on the basis of a bacterium of the genus Halomonas, preferably Halomonas bluephasegenesis, more preferably Halomonas bluephasegenesis TD01 with the accession number of cgmccno. 4353.

The present invention also provides a method for producing poly (3-hydroxybutyrate-co-4-hydroxybutyrate), which comprises using the recombinant bacterium wherein the etf gene is knocked out/inactivated as described above to perform fermentation in a basal medium comprising a carbon source under conditions suitable for culturing the recombinant bacterium to produce poly (3-hydroxybutyrate-co-4-hydroxybutyrate). Preferably, acetic acid, acetate salt, acetate ester or other acetic acid derivative may be added to the basal medium at a concentration as described in the first to second aspects above. Further, the basal medium, carbon source and the like in the method are also as described in the foregoing first to second aspects.

Drawings

FIG. 1 is a diagram showing the agarose gel results of the etf- β subunit knock-outs in example 2.

FIG. 2 is a schematic diagram showing transmission electron micrographs of Halomonas bluephagesis TD01 and TD Delta β accumulating P3HB in example 2, the left graph is a graph of TD01 under the condition of 6g/L acetic acid addition, and the right graph is a graph of TD Delta β under the condition of 3g/L acetic acid addition.

Detailed Description

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

As used herein, PHA is a polyhydroxyalkanoate which can be classified as a homopolymer and a copolymer based on the monomer composition, but is not limited thereto, depending on the number of carbon atoms in the monomer, PHA of the present invention can be a short chain PHA (i.e., a hydroxy fatty acid whose monomer is C3-C5) or a medium chain PHA (i.e., a hydroxy fatty acid whose monomer is C6-C16), but is not limited thereto. in some embodiments of the present invention, PHA can be a homopolymer, including but not limited to a polyhydroxypropionate, a polyhydroxybutyrate, a polyhydroxyvalerate, and the like, e.g., poly-3-hydroxybutyrate (P3HB), poly-4-hydroxybutyrate (P4HB), poly-3-hydroxypropionate (P3HP), or poly-3-hydroxyvalerate (P3 HV). in some embodiments of the present invention, PHA can be a copolymer such as a dimer, a trimer, and the like, but is not limited thereto, e.g., the copolymer can be a copolymer of a hydroxypropionate and a hydroxyvalerate, a copolymer of a hydroxybutyrate with hydroxyvalerate, a hydroxybutyrate, a polyhydroxybutyrate, a copolymer of a polyhydroxybutyrate with a polyhydroxybutyrate, a polyhydroxybutyrate with a polyhydroxybutyrate in some embodiments, a 3-hydroxybutyrate (P3-hydroxybutyrate) or a combination of the present invention, or a combination of a polyhydroxybutyrate with a polyhydroxybutyrate (P3-hydroxybutyrate) (i.e., P3-9, 3-hydroxybutyrate, 3-9, or 3-9).

The content (wt%) of P3HB3HV referred to herein is the mass of P3HB3HV in the ice-dried cells as a percentage of the mass of the ice-dried cells participating in the esterification, wherein the mass of P3HB3HV is the sum of the mass of 3HV obtained after the esterification and the mass of 3HB, and in the examples, "%" means "% by weight" unless otherwise specified.

The P3HB content (wt%) referred to herein is the mass percentage of P3HB to the mass of the ice-dried cells participating in the esterification, where the mass of P3HB is the total mass of 3HB obtained after the esterification. In the examples, "%" means "% by weight" unless otherwise specified.

The 3HV (mol%) referred to herein in example 3 is the percentage of moles of 3HV monomer to the total moles of P3HB3HV monomer, where the total moles of P3HB3HV monomer is the moles of 3HV monomer + moles of 3HB monomer, where 3HV represents 3-hydroxyvalerate and 3HB represents 3-hydroxybutanoic acid (ester). Similarly, the reference to 4HB (mol%) in example 3 herein represents the percentage of moles of 4HB monomer to the total moles of P3HB4HB monomer.

The "basal medium" as referred to herein means a medium suitable for culturing a microorganism and for the microorganism to synthesize polyhydroxyalkanoate using a carbon source added to the medium, such as MM medium, LB medium, mineral medium, and the like, but is not limited thereto. The formulation of these media is routinely known to those skilled in the art, and those skilled in the art can routinely make appropriate adjustments to their components or component concentrations. In this context, unless otherwise specified, the medium or basal medium used for culturing the microorganism to synthesize the desired product is referred to as liquid medium.

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

The general formulation of MM liquid medium is: 0.1-2 ‰ (NH)₄)₂SO₄Or urea, 0.1-1 MgSO₄，5‰-10‰Na₂HPO₄·12H₂O，0.5‰-2‰KH₂PO₄Not more than 0.1% of other trace elements (Fe (III) -NH)₄-Citrate，CaCl₂·2H₂O，ZnSO₄·7H₂O，MnCl₂·4H₂O，H₃BO₃，CoCl₂·6H₂O，CuSO₄·5H₂O，NiCl₂·6H₂O，NaMoO₄·2H₂Trace O) (pH adjusted to about 9.0). Preferably: 0.1% (NH)₄)₂SO₄Or 0.2% urea, 0.02% MgSO₄，1.0％Na₂HPO₄·12H₂O，0.15％KH₂PO₄Not more than 0.1% of other trace elements (Fe (III) -NH)₄-Citrate，CaCl₂·2H₂O，ZnSO₄·7H₂O，MnCl₂·4H₂O，H₃BO₃，CoCl₂·6H₂O，CuSO₄·5H₂O，NiCl₂·6H₂O，NaMoO₄·2H₂O) (pH adjusted to about 9.0).

The "carbon source" is a nutrient substance that provides carbon elements necessary for growth and reproduction of microorganisms. In the present invention, the "carbon source" is the source of substrate for PHA synthesis by the microorganism of the present invention, excluding acetic acid. Thus, "carbon source" may be used interchangeably herein with "substrate". The carbon source may be: glucose, gluconic acid, gluconate ester, starch, sucrose, and the like, or combinations 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 a gene encoding Electron Transfer Flavoprotein (ETF), which is known to exist in, for example, Halomonas and Pseudomonas, and comprises two subunits ETF- α and ETF- β, and NCBI numbers within Halomonas bluephaseensis TD01 are WP _009724031.1 and WP _009724032.1, respectively.

Examples

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Except for special instructions, the enzyme reagents are purchased from Thermo Scientific Fermentas, the kit for extracting plasmids is purchased from Beijing Bomai technical development company, the kit for recovering DNA fragments is purchased from American omega, and the corresponding operation steps are carried out according to product instructions; all culture media are prepared by deionized water if no special instructions exist; the reagents and enzymes involved in PCR were obtained from NEB corporation (USA).

Experimental materials:

the LB medium contained: 5g/L yeast extract (from OXID, U.K., catalog No. LP0021), 10g/L peptone (from OXID, U.K., catalog No. LP0042), 10g/L NaCl, and the balance distilled water; adjusting the pH value to 7.0-7.2; then autoclaved.

The LB60 medium contained: 5g/L yeast extract (from OXID, U.K., catalog No. LP0021), 10g/L peptone (from OXID, U.K., catalog No. LP0042), 60g/L NaCl, and the balance distilled water; adjusting the pH value to 7.0-7.2; then autoclaved.

MM60 medium configuration method: preparing a NaCl solution of yeast extract (available from OXID, UK, catalog No. LP0021) at a yeast extract concentration of 1g/L and a NaCl concentration of 60 g/L; after dissolution, autoclaving; after cooling, 1ml of component I (to 10g (NH) was added per 50ml of solution₄)₂SO₄And 2g MgSO₄Adding distilled water to a volume of 200mL, followed by autoclaving) and 1mL of component II (96.5 g Na to₂HPO₄·12H₂O and 15g KH₂PO₄Adding distilled water to a constant volume of 200ml, and then sterilizing by high-pressure steam); finally, the pH of the system was adjusted to about 9.0 with 5M aqueous NaOH.

(NH) used in the above preparation₄)₂SO₄、NaCl、MgSO₄、Na₂HPO₄·12H₂O、KH₂PO₄From the national pharmaceutical group chemical agents, ltd, catalog numbers 10002992, 10019308, 10034998, 10020392, 1017628, respectively; glucose was purchased from national pharmaceutical group chemical agents limited under catalog number 63005518; sodium gluconatePurchased from SIGMA-ALDRICH, USA under the catalog number S2054-1 KG.

During the culture process, antibiotics with certain concentration, such as 100. mu.g/mL ampicillin, are added to the culture 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:

coli S17-1, described in Simon R, Priefer U, P ü hler A.A broad host licensing organization system for in vivo genetic engineering, transposon mutagenetic engineering [ J ]. Nature biotechnology,1983,1(9):784. presented by the university of Suzus Richardson, Switzerland, Uwe Sauer, publicly available from the university of Qinghua.

Halomonas bluephagene TD 01: the strain is deposited in China general microbiological culture Collection center on 19 th 11 th 2010 with the collection registration number of CGMCCNo.4353 and is classified and named as Halomonas sp.TD01 (also known as Halomonas bluephagenes TD01, see XB Chen et al (2017), Construction of Halomonas bluephagenes capable of high cell density growth for effective PHA production. applied Microbiol. biotechnol. volume 244, Part 1,534 + 541 pages); it is described in patent application publication No. CN 102120973A; the bacteria are available to the public from the university of Qinghua.

Escherichia coli JM109-pBHR68 was prepared by transformation of pBHR68 plasmid into Escherichia coli JM109 (available from Beijing Bomeide Gene technology, Inc.), wherein pBHR68 was a plasmid containing synthetic polyhydroxybutyrate-related genes described in Spiekermann P, Rehm BHA, Kalscheuer R, Baumeister D, Steinb ü gel A (1999) A sensitive, viable-colloidal stabilization synthetic plasmid for direct screening of Bacillus thuringiensis and Escherichia coli, Arch Microbiol 171: 73-80, which plasmid contains polyhydroxybutyrate-related genes derived from Escherichia coli, NADPH-dependent genes, NADPH-linked reductase, NADPH-linked genes, NADPH-linked to Achilles-linked polyhydroxybutyrate-linked to Achilles.

Escherichia coli JM109SG-p68orfZ + pMCSH 5: this strain is described in Li Z J, Shi Z Y, Jian J, et al.production of poly (3-hydroxybutyric-co-4-hydroxybutyric) free transformed carbon sources by Metabolic engineering,2010,12(4): 352-) 359, which is capable of producing P3HB4HB using glucose as a single carbon source, which is stored at the university of Qinghua and which is available to the public from the university of Qinghua.

Halomonas bluephagene TD08 AB: this strain is described in Yin J, Wang H, Fu X Z, et. effects of chromosomal gene copy number and location on polyhydroxyakanoate synthesis by Escherichia coli and Halomonas sp [ J ]. Applied Microbiology and Biotechnology 2015,99(13): 5523. 5534. P3HB3HV can be produced using glucose as a single carbon source, which is stored at the university of Qinghua and is publicly available from the university of Qinghua.

Halomonas bluephagenes TD △ gabD2-D2, which is described in Ye J, Hu D, Che X, et al. Engineering of Halomonas bluephagenes for low cost development of poly (3-hydroxybutyric-co-4-hydroxybutyric) from glucose [ J ]. Metabolic Engineering,2018,47:143-152, is capable of producing P3HB4HB using glucose as a single carbon source, which is stored at Qinghua university and publicly available from Qinghua university.

Halomonas bluephagene TD delta β obtained by knockout of etf- β subunit of Halomonas bluephagene TD01 as a starting strain, which was constructed and deposited at Qinghua university in the experiments of this application, the specific construction process is described in example 2.

Halomonas bluephagene TD delta α obtained by knockout of etf- α subunit of Halomonas bluephagene TD01 as a starting strain, which was constructed and deposited at Qinghua university in the experiments of this application, the specific construction process is described in example 2.

The method for detecting the content of Polyhydroxyalkanoate (PHA) by gas chromatography comprises the following steps:

setting the furnace temperature to 80 ℃, the injector temperature to 200 ℃, the detector temperature to 220 ℃, the column head pressure to 0.25Mpa, and the temperature programming conditions as follows: the temperature was held at 80 ℃ for 1.5 minutes, raised to 140 ℃ at a rate of 30 ℃/min, then raised to 220 ℃ at a rate of 40 ℃/min and held at this temperature for 0.5 minutes. The amount of the sample to be introduced was 1. mu.l, and a microsyringe manufactured by Agilent was used.

Gas phase sample preparation: taking 40-60mg of stem cells of a sample to be detected (bacterial liquid is taken to be centrifuged at 10000rpm at normal temperature for 10 minutes, and after the obtained cell sediment is washed once by water, the stem cells are dried by ice to obtain the stem cells, and the homopolymer is produced in the cells), adding 2ml of chloroform and 2ml of esterification solution (3% (v/v) of concentrated sulfuric acid and 2g/L of benzoic acid in pure methanol are taken as internal standards) into an esterification tube, covering and sealing the esterification tube, and heating the esterification tube at 100 ℃ for 4 hours. After cooling, 1ml of distilled water was added thereto, the mixture was sufficiently shaken and allowed to stand, and after the chloroform phase and the aqueous phase were completely separated, 1. mu.l of the chloroform phase in the lower layer was taken out and injected into a gas chromatograph (Hewlett Packard 6890, HP) to conduct chromatography. The gas chromatograph was operated according to the specifications for the HP Hewlett Packard 6890 gas chromatograph.

Preparation of a standard sample: 10-20mg of standard sample is put into an esterification tube, 2ml of chloroform and 2ml of esterification solution are added, and esterification is carried out at 100 ℃ after capping and sealing.

And (4) analyzing results: taking a standard sample as a reference, if an esterified sample (a sample to be detected) of a cell to be detected has an obvious peak at a standard sample, calculating the mass of each monomer according to the peak area, and then calculating the molar ratio according to the mass fraction of each monomer; the specific gravity (wt%) of the polymer contained in the dry cell weight can be calculated from the amount of the sample added.

Example 1 increasing the yield of P3HB fermentative synthesis by microorganisms by adding acetic acid to the basal Medium

Optimization of culture medium and culture conditions

(1) Optimization of glucose concentration

Experiments were performed with Halomonas bluephagene TD01 as the P3HB producing strain to determine the optimum glucose concentration for P3HB production. Firstly, picking single colony to 20mL LB60 (LB containing 60g/L NaCl) culture medium to culture at 37 ℃ and 200rpm overnight; then, the cells were inoculated into 50mL of MM60 medium (pH8.5-9.0, NaCl concentration 60g/L) containing glucose (see Table 1) of various concentrations at an inoculum size of 5% (v/v), respectively, subjected to shake-flask culture at 37 ℃ and 200rpm for 48 hours to obtain a bacterial solution, and after centrifugation, washing with water and ice-drying, the dry cell weight (CDW; unit g/L) of the sample was measured, and then the percentage (%) of P3HB in the dry cell weight was determined by gas chromatography. Three sets of parallels were set for each condition. The dry cell weight and P3HB content are shown in Table 1.

TABLE 1 Shake flask experimental results of Halomonas TD01 for production of P3HB with different glucose concentrations

As can be seen from the results in Table 1, 30g/L of glucose is the optimal concentration for TD01 to accumulate P3HB, and therefore this concentration was chosen for subsequent experiments.

(2) Optimization of shaking culture rotation speed

The culture rotating speed of the shake flask has direct influence on the oxygen environment for the growth of the microorganism, and the external oxygen environment directly influences the internal environment of the microorganism, so that the optimal environment for the microorganism to accumulate P3HB is found by controlling the rotating speed of the shake flask. Taking Halomonas bluephagene TD01 as a P3HB producing strain, firstly picking out a single strain to be cultured in 20mL LB60 (LB containing 60g/L NaCl) culture medium at 37 ℃ and 200rpm overnight; then inoculating the cells into 50mL of MM60 medium (pH8.5-9.0, NaCl concentration 60g/L) containing 30g/L glucose according to the inoculation amount (v/v) of 5%, setting three rotation speed gradients of 100rpm, 200rpm and 300rpm respectively, performing shake culture at 37 ℃ for 48 hours to obtain a bacterial liquid, performing centrifugation, washing and ice drying to obtain the cell dry weight of the sample, and detecting the content percentage (%) of P3HB in the cell dry weight by gas chromatography. Three sets of parallels were set for each condition. The dry cell weight and P3HB content are shown in Table 2.

TABLE 2 Shake flask experimental results of Halomonas TD01 for P3HB production under different rotation speed conditions

From table 2 we can see that the optimum speed for P3HB production by Halomonas TD01 is 200rpm, so a speed of 200rpm was chosen for subsequent experiments.

Secondly, acetic acid was added to the medium supplemented with carbon source to test its effect on the synthesis of Halomonas bluephasenesis TD01 and P3HB

Acetic acid is known to be a carbon source that is less reducing than glucose, and a number of documents have reported that acetic acid has an inhibitory effect on the growth of microorganisms. In the present application, however, the inventors have found that not only is no inhibitory effect of acetic acid on cell growth and accumulation of P3HB observed by adding acetic acid and controlling the appropriate amount of addition, but also it has been found that it can increase the accumulation amount of P3HB, which is likely to be achieved by regulating the microenvironment inside the microorganism, eliminating metabolic inhibition of pyruvate, and saving carbon sources.

Taking P3HB produced by Halomonas bluephagene TD01 as an example, firstly picking up single colony in 20mL LB60 (LB containing 60g/L NaCl) culture medium, and culturing at 37 ℃ and 200rpm overnight; then, the medium was inoculated at 5% (v/v) into 50mL of MM60 medium (pH8.5-9.0, NaCl concentration 60g/L) to which glucose and acetic acid were added, wherein the glucose concentration was 30g/L and the acetic acid was set between 0 and 12g/L, to conduct a concentration gradient experiment (see Table 3). The culture temperature is set to be 37 ℃, the set rotating speed is set to be 200rpm, the shake flask culture is carried out for 48 hours to obtain bacterial liquid, the cell dry weight of the sample is obtained after centrifugation, water washing and ice drying, and then the content percentage (%) of P3HB in the cell dry weight is detected through gas chromatography. Three sets of parallels were set for each condition. The dry cell weight and P3HB content are shown in Table 3.

TABLE 3 Effect of different concentrations of acetic acid addition on P3HB production by Halomonas TD01

As can be seen from the data in Table 3, supplementation with acetic acid increased the ability of the cells of the microorganism to accumulate P3HB per weight, and within certain limits increased the dry cell weight of the microorganism. Supplementation with 1-8g/L acetic acid, particularly 2-8g/L acetic acid, was calculated to increase the total yield (g/L) of the P3HB product by at least about 12% over the range of 2-8g/L acetic acid addition compared to the control without acetic acid. Wherein, under the condition of acetic acid addition of less than 6g/L, the growth of cells and the accumulation of P3HB are promoted to be larger along with the increase of the acetic acid concentration, but when the acetic acid concentration is increased to be more than 6g/L, the dry weight of the cells and the percentage content of P3HB are not further increased along with the increase of the acetic acid concentration and even begin to decrease.

We also examined the contents of various substances in the fermentation broth after the end of fermentation, including glucose, acetic acid and pyruvic acid (Table 4).

TABLE 4 analysis and detection of substances in fermentation broth after fermentation

According to the results of this example, the addition of acetic acid indeed reduced the inhibition of pyruvate metabolism by NADH in the fermentation system and reduced the pyruvate residue, compared to the case where acetic acid was not added, thereby improving the glucose utilization rate.

By combining the P3HB accumulation and cell dry weight data in Table 3, we can find that the addition of 1-8g/L acetic acid, especially 2-8g/L acetic acid, can significantly improve the cell dry weight and the utilization rate of carbon sources, and effectively improve the synthetic amount of P3 HB; simultaneously reducing glucose and pyruvate residuals, thereby alleviating metabolic inhibition of glucose and pyruvate; and the residual proportion of acetic acid is lower, so that the whole fermentation system is optimized.

Thirdly, adding acetic acid and other carbon sources for comparison

To verify that the addition of acetic acid has a alleviating effect on the inhibition of pyruvate and glucose metabolism rather than the effect of additional carbon source addition, we set up a control experiment with additional pyruvate and glucose addition. In this experiment, the strains, the culture media and the culture conditions used were the same as those in the second part of example 1, except that acetic acid, pyruvic acid or glucose, respectively, was additionally added to the basal medium in amounts shown in Table 5, the amounts added of which would theoretically be converted to the same amount of acetyl-CoA (Table 5).

TABLE 5 Effect of addition of acetic acid, pyruvic acid and glucose on the synthesis of P3HB

From table 5 we can conclude that: the extra addition of carbon sources such as glucose or pyruvate cannot increase the cell dry weight or the P3HB accumulation, and even the addition of pyruvate can reduce the cell dry weight and the P3HB content, which proves that the accumulation of pyruvate can actually inhibit the synthesis of P3 HB; the experiment further proves that the addition of acetic acid reduces the residue of pyruvic acid, and further has the promotion effect on the growth of microbial cells and the synthesis of P3 HB.

Fourth, influence of acetate addition on P3HB Synthesis

Considering that acetic acid exists in the form of ions in the culture medium solution, the inventors expect that the addition of acetate will obtain substantially the same effect as the addition of acetic acid, and the same experiment was performed as in the second part of example 1 except that acetic acid was replaced with acetate (sodium acetate, potassium acetate, calcium acetate, etc.).

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

Example 2 the effect of acetic acid addition to carbon source supplemented media on the synthesis of halomonas phasegenes TD Δ β and P3HB was tested.

Firstly, construction of engineering bacteria TD delta β:

1) construction of the homology arms of the knock-out plasmid pKObeta 23

The genome of Halomonas bluephagene Halomonas TD01 was extracted and PCR-amplified with pfu enzyme (NEB, USA) using it as a template, and left and right homologous arms H1 and H2 of etf-567 (nucleotide sequence shown in SEQ ID NO: 1; amino acid sequence shown in SEQ ID NO:2) and beta-H2-R using beta-H1-F and beta-H1-R, beta-H2-F and beta-H2-R, respectively, and plasmid pSEVA241 as a template (this plasmid was amplified with primers Dh-3641 and dH-19 using plasmid pSEVA241 (this plasmid was recorded in The literature: Silva-Rocha, Rafael, et al. "The Standard European Vector Architecture (SEVA): a genetic plasmid for sR analysis and deploymer of fluorescent proteins), and primer dH-19-12-primer).

etf- β (SEQ ID NO: 1):

ATGCGGCCTGAACAACAGCAAAAACAACAAAAGCCGGGTATCGACGTGGCGGTGCTGGTCTCCATAGGCCGTCACCCCACAACCGGTCGTGCGAGGCGCGCCGAGCAGGATGCACGGGGTCTGGAACTTGCTCTAGCCATGGAAGCAGAACTGCCGGGTAGCCGGATTGACGTACTGCACGCTGGTTCTCAGGATGTCGACAGCGAGGCAGCTCTGCGCAGTTACTTGGGTATGGCCACAGGCGTTGGTATGGGGCTTGAGTCTATGACCTTGCTGGAGCAGCCCGATGGCAGCGATGCCATTCTACCGCTGGTGGAACATTTAGCCGCTACCTCGCCCCAACTAGTGATCACCGGTGCCCGGGCCGAGCGCGGAGAAGGTTCTGGGCTTTTGCCCTATGCGTTGGCTGAGCATCTCGGTTGGCCGTTAGTGAATAGCTTGGCGTCGATAGAGACAGTGGAGAATGGTGTGATAACGCTGCTTCAAGCGTTGCCACGGGGCCAGCGTCGCCGTCTCAAGGTGCGCTTGCCCGCCATCATCAGCGTGGATGAAGCAGCGCCGGCGGCGCGTCAGAGCGCCTTCGGCCCGGCTCGCCGAGCCAGCTTCTCGGTGGCGCCCACTACCTCTGAAGCCGACAGTGAGTTGGCCCAGTGGCACTTGAGCCCCGCACGTAAACGGCCTAAGCGTCTGAAAATCATTAAAGCCGCCTCAGCCAGAGATCGTTTCAAAGCGGCGGCTTCCAAAGCCGAGGGCAAGGGCGGGCAAGTGCTCACCGACGTCACCCCCGAAGAGGGCGCCGAGGCGATCTACAAGCTACTCAAGGAAGAGGACGTGCTGCGCTGA

etf- β (SEQ ID NO: 2):

MRPEQQQKQQKPGIDVAVLVSIGRHPTTGRARRAEQDARGLELALAMEAELPGSRIDVLHAGSQDVDSEAALRSYLGMATGVGMGLESMTLLEQPDGSDAILPLVEHLAATSPQLVITGARAERGEGSGLLPYALAEHLGWPLVNSLASIETVENGVITLLQALPRGQRRRLKVRLPAIISVDEAAPAARQSAFGPARRASFSVAPTTSEADSELAQWHLSPARKRPKRLKIIKAASARDRFKAAASKAEGKGGQVLTDVTPEEGAEAIYKLLKEEDVLR*

primers for H1 were:

beta-H1-F：5’CGAGGCGATCTACAAGCTACT 3’(SEQID NO:3)

beta-H1-R：5’

AGGGTTTTCCCAGTCACGACGCCGCTATCCAGCCATTGAC 3’(SEQ ID NO:4)

primers for H2 were:

beta-H2-F：5’

AGTCGGTGCTTTTTTTGAACCCTGACCTTGGCAGCGTCCATT 3’(SEQ ID NO:5)

beta-H2-R：5’

AGTAGCTTGTAGATCGCCTCGTATGGAGACCAGCACCGCC 3’(SEQ ID NO:6)

the primer of the Donor-1 is as follows:

donor-H-F：5’GTCGTGACTGGGAAAACCCT 3’(SEQ ID NO:7)

donor-H-R：5’GGGTTCAAAAAAAGCACCGACT 3’(SEQ ID NO:8)

and (3) PCR reaction conditions:

pre-denaturation at 95 ℃ for 5 minutes; then denaturation at 95 ℃ for 30 seconds, annealing at 58 ℃ for 30 seconds, extension at 72 ℃ for 30 seconds, and 30 cycles; then, the mixture was stretched at 72 ℃ for 10 minutes.

PCR reaction system (50. mu.L system):

when the PCR amplification system is prepared, DNA polymerase is added finally.

The obtained 504bp PCR product is the homologous arm H1 of the target gene etf- β, the obtained 565bp product is the homologous arm H2 of the target gene etf- β, the obtained 5291bp product is the plasmid skeleton Donor-1 derived from pSEVA241, and then the three amplified products are connected by a Gibson kit (purchased from NEB company in the United states and having the catalog number of E2611L).

Gibson ligation conditions:

the target fragments were mixed, made up to 10. mu.l with water and then treated at 50 ℃ for 1 hour according to the instructions of the Gibson kit. The Gibson ligation system (10. mu.L system) is as follows:

the ligation product was obtained and transformed into E.coli JM109 to obtain transformants, which were sequenced, sorted for correct sequencing and stored and cultured overnight in LB medium and then the plasmid was named pSEVA 241-HR.

2) Knockout of sgRNA insertion into pKObet23 plasmid

The pSEVA241-HR plasmid inserted with homology arms H1 and H2 in the previous step is used as a template, donor-G-F and donor-G-R are used as primers, pfu enzyme is used for PCR amplification to obtain a plasmid skeleton derived from pSEVA241-HR, then the primers sgRNA-F and sgRNA-R are annealed and connected to obtain a DNA double chain with a sticky end, and the plasmid skeleton and the DNA double chain with the sticky end are subjected to enzyme digestion and connection by BsaI to obtain a knockout plasmid pKObet23 for the target gene etf- β.

Amplification was performed 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)

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

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

pre-denaturation at 95 ℃ for 5 minutes; every 3 minutes, 10 ℃ was lowered, and finally 15 ℃ was maintained for 5 minutes.

The primer sequence is as follows:

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

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

annealing system (10 μ L):

sgRNA-F 5μL

sgRNA-R 5μL

the final concentration of the primers was 10 pM.

The obtained plasmid skeleton after enzyme digestion and the annealed DNA double strand are connected by T4DNA ligase, transformed into Escherichia coli JM109, the grown recombinant colony is sequenced, the sequencing is correctly preserved, and a plasmid is extracted, namely a knockout plasmid pKObet23 for knocking out the target gene etf- β.

3) And deletion of etf- β gene in halomonas TD01

Firstly, plasmid pQ08 expressing Cas9 protein is transferred into Halomonas TD01 (the transfer process is shown in Qin, Qin, et al, "CRISPR/Cas9 edition genome of exotrophile Halomonas spp." Metabolic engineering (2018):219-229.), the plasmid contains Cas9 protein from Streptococcus pyogenes, and the strain is stored in university of Qinghua.

The pseudomonas halodurans TD01 containing the Cas9 protein is used as a recipient bacterium, E.coli S17-1 carrying plasmid pKObet23 is used as a donor bacterium to carry out conjugation transformation, and a single clone is screened on a plate containing a spectinomycin resistant (100mg/ml) LB60 solid culture medium (15 g/L of agar powder is additionally added into the formula of an LB60 liquid culture medium, the mixture is autoclaved at 121 ℃ for 20min, and the plate is poured after being cooled). Colony PCR amplification is carried out by using tag enzyme by using a monoclonal genome as a template and using Test-beta-F and Test-beta-R as primers.

The primers are as follows:

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

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

colony PCR reaction conditions:

PCR reaction system (50. mu.L system):

after the PCR amplification system is prepared and subpackaged, a single colony is picked by a toothpick, the colony with the amplified band size of 1338bp is a knockout type, the colony with the amplified band size of 2069bp is a wild type (see figure 1), and finally the etf- β gene knockout type of TD01 is obtained and is named as Halomonas bluetoothgenesis TD delta β.

Secondly, the influence of acetic acid addition on P3HB synthesis by recombinant bacteria TD delta β

To examine the ability of TD Δ β to synthesize P3HB, we performed shake flask experiments using TD01 and TD Δ β, by first picking up single colonies into 20mL of LB60 medium, respectively, culturing overnight at 37 ℃ and 200rpm, and then inoculating 50mL of MM60 medium (pH8.5-9.0, NaCl concentration 60g/L), glucose concentration 30g/L, acetic acid concentration gradient 0g/L, 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8g/L, 9g/L, 12g/L, culture temperature 37 ℃, rotation speed 200rpm, shake flask culture for 48 hours, obtaining bacterial liquid, obtaining cells of the sample after centrifugation, water washing, and ice drying, and then detecting the content of P3 in the cells (% HB) by gas chromatography (dry weight set percentage) and each condition is shown in three sets of parallel HB. see Table 6.

TABLE 6 Effect of acetic acid addition on Halomonas TD01 and TD Δ β

From the data in Table 6 we can conclude that TD Δ β, knocked out etf- β from Halomonas TD01, can synthesize P3HB at a significantly higher content than TD01 under equivalent conditions for Halomonas TD Δ β and that adding acetic acid in a range of concentrations in the medium of Halomonas TD Δ β can also increase the dry cell weight of the microorganism and the content of P3HB accumulated in the cells, similar to the results obtained by adding acetic acid in TD01 culture, wherein the content of P3HB of TD Δ β after adding 3g/L acetic acid is 94% of the dry cell weight, which is the highest content reported in the world so far.

The results of this experiment show that by deleting etf- β, in combination with the addition of acetic acid, the specific gravity of P3HB in the cells can be increased up to 94% and the cell volume also increased (see fig. 2).

Thirdly, constructing engineering bacteria TD delta α and testing the influence of acetic acid on P3HB synthesized by the recombinant bacteria

The engineered bacterium Halomonas bluephagenetics TD Δ α was constructed by knocking out the etf- α subunit of the etf gene in Halomonas bluephagenetics TD01, operating as described in the first part of example 2, and was tested and analyzed as described in the second part of example 2 to determine the effect of acetic acid addition on P3HB synthesis.

etf- α subunit nucleotide sequence (SEQ ID NO: 15):

ATGAGTGAAATTATTCGCCGCGACCCACGTCGTGAGTGGATCGCCCGAAACCGTCTGCACCCCGACAACGCCGCGGTGCTGGCCGCTCTTGGTGTAAACAGGGGCGGCGGAGCGGTCAGCGAATGGGTGGGGCCCAACGGCGTGGTGCGTAAGAATCCTCGCGCTATTGGCTTTATCGGCCCCAATGGCGTTAAACGAATTGATCGTAGCGGCCTCCAGCAAGGAGGGCATTCCAGCGCGGCAACCGCCGTGGCTAGCGATAGCCGCCGTACAGTGACTATTGATCAGCCCGCTTTTCTAGTGGCCGGGGTACCTGACCTGATCGGTGGACGCCTTTCTAGCCATGATAAGGATCTGCTGGGACTAGCCAGACGCGTGGCGGATGCCGACGCCGAACAGCCAGGCGCCGTGGTGGCGATCCTGTTTGGTGAGCATAAAGGCGAGTGGGGCGGCGAGCTTAAGAAGCAAGCGCTGGGCGAAGCTGGGATTGATCGCGTCGTACATCTTGATGATGAGCTTTACGCTGGGTTTGCTCCCGAGGCACGGTTAGCGGTGTTAAGTGCCGTCGAACGGGAGATGACGCCGCGTTTCTGGCTGTTGCCGGATTCACCCCTAGGCGGAGCTGATCTGGGGCGGCGGCTTGCCTTACGCCTGGGTGAACGCGCTGCTACAGGGGTGTGGCAGATTGAAGCTGACGATGAGGCACCTCTGGGCTGGCAGTGCACTGCCCGTGGCGCCGCTGGAAGTTTGGATATCCAGCGCCCTCTGCCACGGGTTGCCCTGGCGTTGGCCGAGTGCGCCGAACCGGTGGACGAGACCCGACATGCGGCCGAACACCTGACCTTGGCAGCGTCCATTCCCACCACTCTATCGCGCATTGAAGATCTGGGTCAGGTGGCGGTGGATCCCGCTGGCGTGGCGTTGGCCGAGGCTGAGTTCATCCTCTCTGGTGGCAATGGTGTTAAACAGTGGGACGCCTTCCATCATGCCGCGAAAGTACTGGGCGCTACCGAAGGGGCCTCGCGTGTTGCGGTAGACGACGGCTTTATGGCCCGCGACCGGCAGGTAGGCGCGACCGGTACCTGGGTAACCGCCCGAGTCTATATGGCGGTGGGGATTTCAGGCGCTATCCAGCACCTGCAGGGCATTCAGCGCTGCGACAAGGTGGTGGCCATCAATCTCGATCCGGGGTGCGACATGATCgAACGCGCAGACCTGGCGGTGATAGGCGACAGCACGCAAATTCTTGCTGCGTTAGTGGCGATGGTGGAACAGCAGCGAGGAGGGCAGCGCGATGCGGCCTGA

etf- α subunit is (SEQ ID NO: 16):

MSEIIRRDPRREWIARNRLHPDNAAVLAALGVNRGGGAVSEWVGPNGVVRKNPRAIGFIGPNGVKRIDRSGLQQGGHSSAATAVASDSRRTVTIDQPAFLVAGVPDLIGGRLSSHDKDLLGLARRVADADAEQPGAVVAILFGEHKGEWGGELKKQALGEAGIDRVVHLDDELYAGFAPEARLAVLSAVEREMTPRFWLLPDSPLGGADLGRRLALRLGERAATGVWQIEADDEAPLGWQCTARGAAGSLDIQRPLPRVALALAECAEPVDETRHAAEHLTLAASIPTTLSRIEDLGQVAVDPAGVALAEAEFILSGGNGVKQWDAFHHAAKVLGATEGASRVAVDDGFMARDRQVGATGTWVTARVYMAVGISGAIQHLQGIQRCDKVVAINLDPGCDMIERADLAVIGDSTQILAALVAMVEQQRGGQRDAA*

as a result, it was found that, compared to the original strain, Halomonas bluephagesis TD01, Halomonas bluephagesis TD Δ α also showed significantly increased P3HB content under the same culture conditions, presumably because either the deletion of etf- α subunit or the deletion of etf- β subunit resulted in the inactivation of etf gene, and thus had substantially the same effect on the P3HB synthesis pathway.

In addition, the addition of acetic acid within a certain concentration range can also improve the cell dry weight and the P3HB content of the knockout strain, and the experimental result and the trend are basically consistent with Halomonas bluephasegenetics TD delta β.

Example 3 control of the proportion of monomers in the copolymer by addition of acetic acid to the basal Medium

The material properties of the copolymers P3HB3HV and P3HB4HB depend to a large extent on the monomer proportions therein, in particular for P3HB3 HV. The biggest problem in producing copolymer by Halomonas bluephagene TD is that the proportion of 3HB monomer is high, and the proportion of 3HV and 4HB is low. Acetic acid is a carbon source that is less reductive than glucose, and it is considered by the inventors that acetic acid and glucose are used together to regulate the internal redox environment of the microorganism, thereby controlling the metabolic pathway of acetyl-coa. Specifically, the copolymers P3HB3HV and P3HB4HB are dimers in which the former is copolymerized from 3-hydroxybutyric acid (3HB) and 3-hydroxyvaleric acid (3HV), and the latter is copolymerized from 3-hydroxybutyric acid (3HB) and 4-hydroxybutyric acid (4 HB). 3HB is synthesized directly from acetyl-CoA by anaerobic fermentation, whereas both 3HV and 4HB are derived from precursors generated from acetyl-CoA into the TCA cycle, and therefore, a key to changing the monomer ratios in P3HB3HV and P3HB4HB is to control the metabolic pathway of acetyl-CoA. This experiment demonstrates that by varying the amount of acetic acid added within the appropriate range, the ratio of monomers in the copolymers P3HB3HV and P3HB4HB can also be adjusted.

Firstly, the content of 3HV in P3HB3HV is adjusted by changing the concentration of added acetic acid

Halomonas bluephagene TD08AB was used as a P3HB3HV synthetic strain, and the strain is described in the following documents: yin J, Wang H, Fu X Z, et al.effects of chromosomal gene copy number and location on polyhydroxyakanoate synthesis by Escherichia coli and Halomonas sp [ J ]. Applied microbiology and biotechnology 2015,99(13): 5523) 5534. in this strain, P3HB3HV can be produced using glucose as the sole carbon source.

Firstly, picking single colony in 20mL LB60 (LB containing 60g/L NaCl) culture medium, culturing at 37 ℃ and 200rpm overnight; then, the medium was inoculated at 5% (v/v) into 50mL of MM60 medium (pH8.5-9.0, NaCl concentration 60g/L), glucose concentration was set at 30g/L, and acetic acid was set at 8 concentration gradients: 0g/L, 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8 g/L. Culturing at 37 deg.C and 200rpm for 48 hr, centrifuging, washing with water, and lyophilizing to obtain cell dry weight, and detecting the polymer content (%) by gas chromatography. Three sets of parallels were set for each condition. The dry cell weight, P3HB3HV content, and 3HV ratio are shown in Table 7.

TABLE 7 Synthesis of P3HB3HV at different acetic acid concentrations by Halomonas bluephagene TD08AB

From the data of table 7 we can conclude that: the monomer ratio of the copolymer P3HB3HV can be changed by adding 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L and 8g/L of acetic acid, particularly after 1g/L and 2g/L of acetic acid are added, the monomer ratio of 3HV is improved by nearly one time compared with the result when no acetic acid is added, and the promotion effect on the improvement of the material performance is very obvious. Furthermore, the results also show that the addition of 1-8g/L acetic acid results in an increase in cell dry weight, resulting in a significant increase in the final P3HB3HV yield as compared to no addition (data not shown).

Secondly, the content of 4HB in P3HB4HB is controlled by changing the concentration of added acetic acid

Halomonas bluephagenesis TD △ gabD2-D2 was used as P3HB4HB synthetic strain described in Ye J, Hu D, Che X, et al.engineering of Halomonas bluepharmacologesis for low cost development of poly (3-hydroxybuytrate-co-4-hydroxybuytrate) from glucose [ J ]. Metabolic engineering,2018,47: 143. 152. the strain was capable of producing P3HB4HB using glucose as a single carbon source, and was maintained at the university of Qing Hua, and publicly available at the university of Qing Hua.

Firstly, picking single colony in 20mL LB60 (LB containing 60g/L NaCl) culture medium, culturing at 37 ℃ and 200rpm overnight; then, the medium was inoculated at 5% (v/v) into 50mL of MM60 medium (pH8.5-9.0, NaCl concentration 60g/L), glucose concentration was fixed at 30g/L, and acetic acid was set at 9 concentration gradients: 0g/L, 1g/L, 2g/L, 3g/L, 4g/L, 5g/L, 6g/L, 7g/L, 8 g/L. Culturing at 37 deg.C and 200rpm for 48 hr, centrifuging, washing with water, and lyophilizing to obtain cell dry weight, and detecting the polymer content (%) by gas chromatography. Three sets of parallels were set for each condition. The dry cell weight, P3HB4HB content, and 4HB ratio are shown in Table 8.

TABLE 8 Synthesis of P3HB4HB at different acetic acid concentrations by Halomonas bluephagene TD △ gabD2-D2

The experimental results show that the cell content and the yield of the copolymer P3HB4HB can be improved by adding acetic acid, compared with a control group without adding acetic acid (the cell dry weight: 8.79 +/-0.07 g/L; the content of P3HB4 HB: 66.89 +/-0.37%), the cell dry weight and the content of P3HB4HB are improved within the range of adding 1g/L-8g/L of acetic acid, wherein the yield improvement effect under the condition of adding 6g/L of acetic acid is most remarkable, the cell dry weight is measured to be 9.11 +/-0.28 g/L, and the content of P3HB4HB is measured to be 69.91 +/-1.93%. Furthermore, from the data of table 8 we can also conclude that: the monomer proportion of the copolymer P3HB4HB can be changed by adding acetic acid, and particularly after 4g/L of acetic acid is added, the 3HV monomer proportion is increased by more than 50%, so that the improvement of the material performance is remarkably promoted.

The two groups of experiments prove that the invention can really adjust the internal environment of microbial cells and control the metabolic flow of acetyl coenzyme A entering 3HV and 4HB by adding a proper amount of acetic acid in a basic culture medium, thereby changing the proportion of each monomer in the polymer synthesized by the microbes. In a specific embodiment, the proportion of 3HV monomer in P3HB3HV polymer can be significantly increased (from 4% to 8%) by adjusting the concentration of acetic acid added; the proportion of 4HB monomer in the P3HB4HB polymer can also be increased significantly (from 7.8% to 12%).

Sequence listing

<110> Qinghua university

<120> an improved method for producing polyhydroxyalkanoate

<130>CGCNL181110W

<160>16

<170>SIPOSequenceListing 1.0

<210>1

<211>843

<212>DNA

<213>Halomonas bluephagenesis TD01

<400>1

atgcggcctg aacaacagca aaaacaacaa aagccgggta tcgacgtggc ggtgctggtc 60

tccataggcc gtcaccccac aaccggtcgt gcgaggcgcg ccgagcagga tgcacggggt 120

ctggaacttg ctctagccat ggaagcagaa ctgccgggta gccggattga cgtactgcac 180

gctggttctc aggatgtcga cagcgaggca gctctgcgca gttacttggg tatggccaca 240

ggcgttggta tggggcttga gtctatgacc ttgctggagc agcccgatgg cagcgatgcc 300

attctaccgc tggtggaaca tttagccgct acctcgcccc aactagtgat caccggtgcc 360

cgggccgagc gcggagaagg ttctgggctt ttgccctatg cgttggctga gcatctcggt 420

tggccgttag tgaatagctt ggcgtcgata gagacagtgg agaatggtgt gataacgctg 480

cttcaagcgt tgccacgggg ccagcgtcgc cgtctcaagg tgcgcttgcc cgccatcatc 540

agcgtggatg aagcagcgcc ggcggcgcgt cagagcgcct tcggcccggc tcgccgagcc 600

agcttctcgg tggcgcccac tacctctgaa gccgacagtg agttggccca gtggcacttg 660

agccccgcac gtaaacggcc taagcgtctg aaaatcatta aagccgcctc agccagagat 720

cgtttcaaag cggcggcttc caaagccgag ggcaagggcg ggcaagtgct caccgacgtc 780

acccccgaag agggcgccga ggcgatctac aagctactca aggaagagga cgtgctgcgc 840

tga 871

<210>2

<211>280

<212>PRT

<213>Halomonas bluephagenesis TD01

<400>2

Met Arg Pro Glu Gln Gln Gln Lys Gln Gln Lys Pro Gly Ile Asp Val

1 5 10 15

Ala Val Leu Val Ser Ile Gly Arg His Pro Thr Thr Gly Arg Ala Arg

20 25 30

Arg Ala Glu Gln Asp Ala Arg Gly Leu Glu Leu Ala Leu Ala Met Glu

35 40 45

Ala Glu Leu Pro Gly Ser Arg Ile Asp Val Leu His Ala Gly Ser Gln

50 55 60

Asp Val Asp Ser Glu Ala Ala Leu Arg Ser Tyr Leu Gly Met Ala Thr

65 70 75 80

Gly Val Gly Met Gly Leu Glu Ser Met Thr Leu Leu Glu Gln Pro Asp

85 90 95

Gly Ser Asp Ala Ile Leu Pro Leu Val Glu His Leu Ala Ala Thr Ser

100 105 110

Pro Gln Leu Val Ile Thr Gly Ala Arg Ala Glu Arg Gly Glu Gly Ser

115 120 125

Gly Leu Leu Pro Tyr Ala Leu Ala Glu His Leu Gly Trp Pro Leu Val

130 135 140

Asn Ser Leu Ala Ser Ile Glu Thr Val Glu Asn Gly Val Ile Thr Leu

145 150 155 160

Leu Gln Ala Leu Pro Arg Gly Gln Arg Arg Arg Leu Lys Val Arg Leu

165 170 175

Pro Ala Ile Ile Ser Val Asp Glu Ala Ala Pro Ala Ala Arg Gln Ser

180 185 190

Ala Phe Gly Pro Ala Arg Arg Ala Ser Phe Ser Val Ala Pro Thr Thr

195 200 205

Ser Glu Ala Asp Ser Glu Leu Ala Gln Trp His Leu Ser Pro Ala Arg

210 215 220

Lys Arg Pro Lys Arg Leu Lys Ile Ile Lys Ala Ala Ser Ala Arg Asp

225 230 235 240

Arg Phe Lys Ala Ala Ala Ser Lys Ala Glu Gly Lys Gly Gly Gln Val

245 250 255

Leu Thr Asp Val Thr Pro Glu Glu Gly Ala Glu Ala Ile Tyr Lys Leu

260 265 270

Leu Lys Glu Glu Asp Val Leu Arg

275 280

<210>3

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

cgaggcgatc tacaagctac t 21

<210>4

<211>40

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

agggttttcc cagtcacgac gccgctatcc agccattgac 40

<210>5

<211>42

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

agtcggtgct ttttttgaac cctgaccttg gcagcgtcca tt 42

<210>6

<211>40

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

agtagcttgt agatcgcctc gtatggagac cagcaccgcc 40

<210>7

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

gtcgtgactg ggaaaaccct 20

<210>8

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

gggttcaaaa aaagcaccga ct 22

<210>9

<211>50

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

ctagggtctc agttttagag ctagaaatag caagttaaaa taaggctagt 50

<210>10

<211>43

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

ctagggtctc aactagtatt atacctagga ctgagctagc tgt 43

<210>11

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

tagttgggct tttgccctat gcgt 24

<210>12

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>12

aaacacgcat agggcaaaag ccca 24

<210>13

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>13

gggtgtggca gattgaagct 20

<210>14

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>14

gtgccgctaa gaacgttgtt 20

<210>15

<211>1305

<212>DNA

<213>Halomonas bluephagenesis TD01

<400>15

atgagtgaaa ttattcgccg cgacccacgt cgtgagtgga tcgcccgaaa ccgtctgcac 60

cccgacaacg ccgcggtgct ggccgctctt ggtgtaaaca ggggcggcgg agcggtcagc 120

gaatgggtgg ggcccaacgg cgtggtgcgt aagaatcctc gcgctattgg ctttatcggc 180

cccaatggcg ttaaacgaat tgatcgtagc ggcctccagc aaggagggca ttccagcgcg 240

gcaaccgccg tggctagcga tagccgccgt acagtgacta ttgatcagcc cgcttttcta 300

gtggccgggg tacctgacct gatcggtgga cgcctttcta gccatgataa ggatctgctg 360

ggactagcca gacgcgtggc ggatgccgac gccgaacagc caggcgccgt ggtggcgatc 420

ctgtttggtg agcataaagg cgagtggggc ggcgagctta agaagcaagc gctgggcgaa 480

gctgggattg atcgcgtcgt acatcttgat gatgagcttt acgctgggtt tgctcccgag 540

gcacggttag cggtgttaag tgccgtcgaa cgggagatga cgccgcgttt ctggctgttg 600

ccggattcac ccctaggcgg agctgatctg gggcggcggc ttgccttacg cctgggtgaa 660

cgcgctgcta caggggtgtg gcagattgaa gctgacgatg aggcacctct gggctggcag 720

tgcactgccc gtggcgccgc tggaagtttg gatatccagc gccctctgcc acgggttgcc 780

ctggcgttgg ccgagtgcgc cgaaccggtg gacgagaccc gacatgcggc cgaacacctg 840

accttggcag cgtccattcc caccactcta tcgcgcattg aagatctggg tcaggtggcg 900

gtggatcccg ctggcgtggc gttggccgag gctgagttca tcctctctgg tggcaatggt 960

gttaaacagt gggacgcctt ccatcatgcc gcgaaagtac tgggcgctac cgaaggggcc 1020

tcgcgtgttg cggtagacga cggctttatg gcccgcgacc ggcaggtagg cgcgaccggt 1080

acctgggtaa ccgcccgagt ctatatggcg gtggggattt caggcgctat ccagcacctg 1140

cagggcattc agcgctgcga caaggtggtg gccatcaatc tcgatccggg gtgcgacatg 1200

atcgaacgcg cagacctggc ggtgataggc gacagcacgc aaattcttgc tgcgttagtg 1260

gcgatggtgg aacagcagcg aggagggcag cgcgatgcgg cctga 1305

<210>16

<211>434

<212>PRT

<213>Halomonas bluephagenesis TD01

<400>16

Met Ser Glu Ile Ile Arg Arg Asp Pro Arg Arg Glu Trp Ile Ala Arg

1 5 10 15

Asn Arg Leu His Pro Asp Asn Ala Ala Val Leu Ala Ala Leu Gly Val

20 25 30

Asn Arg Gly Gly Gly Ala Val Ser Glu Trp Val Gly Pro Asn Gly Val

35 40 45

Val Arg Lys Asn Pro Arg Ala Ile Gly Phe Ile Gly Pro Asn Gly Val

50 55 60

Lys Arg Ile Asp Arg Ser Gly Leu Gln Gln Gly Gly His Ser Ser Ala

65 70 75 80

Ala Thr Ala Val Ala Ser Asp Ser Arg Arg Thr Val Thr Ile Asp Gln

85 90 95

Pro Ala Phe Leu Val Ala Gly Val Pro Asp Leu Ile Gly Gly Arg Leu

100 105 110

Ser Ser His Asp Lys Asp Leu Leu Gly Leu Ala Arg Arg Val Ala Asp

115120 125

Ala Asp Ala Glu Gln Pro Gly Ala Val Val Ala Ile Leu Phe Gly Glu

130 135 140

His Lys Gly Glu Trp Gly Gly Glu Leu Lys Lys Gln Ala Leu Gly Glu

145 150 155 160

Ala Gly Ile Asp Arg Val Val His Leu Asp Asp Glu Leu Tyr Ala Gly

165 170 175

Phe Ala Pro Glu Ala Arg Leu Ala Val Leu Ser Ala Val Glu Arg Glu

180 185 190

Met Thr Pro Arg Phe Trp Leu Leu Pro Asp Ser Pro Leu Gly Gly Ala

195 200 205

Asp Leu Gly Arg Arg Leu Ala Leu Arg Leu Gly Glu Arg Ala Ala Thr

210 215 220

Gly Val Trp Gln Ile Glu Ala Asp Asp Glu Ala Pro Leu Gly Trp Gln

225 230 235 240

Cys Thr Ala Arg Gly Ala Ala Gly Ser Leu Asp Ile Gln Arg Pro Leu

245 250 255

Pro Arg Val Ala Leu Ala Leu Ala Glu Cys Ala Glu Pro Val Asp Glu

260 265 270

Thr Arg His Ala Ala Glu His Leu Thr Leu Ala Ala Ser Ile Pro Thr

275280 285

Thr Leu Ser Arg Ile Glu Asp Leu Gly Gln Val Ala Val Asp Pro Ala

290 295 300

Gly Val Ala Leu Ala Glu Ala Glu Phe Ile Leu Ser Gly Gly Asn Gly

305 310 315 320

Val Lys Gln Trp Asp Ala Phe His His Ala Ala Lys Val Leu Gly Ala

325 330 335

Thr Glu Gly Ala Ser Arg Val Ala Val Asp Asp Gly Phe Met Ala Arg

340 345 350

Asp Arg Gln Val Gly Ala Thr Gly Thr Trp Val Thr Ala Arg Val Tyr

355 360 365

Met Ala Val Gly Ile Ser Gly Ala Ile Gln His Leu Gln Gly Ile Gln

370 375 380

Arg Cys Asp Lys Val Val Ala Ile Asn Leu Asp Pro Gly Cys Asp Met

385 390 395 400

Ile Glu Arg Ala Asp Leu Ala Val Ile Gly Asp Ser Thr Gln Ile Leu

405 410 415

Ala Ala Leu Val Ala Met Val Glu Gln Gln Arg Gly Gly Gln Arg Asp

420 425 430

Ala Ala

Claims

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.