阅读说明：本技术 一种改进的生产聚羟基脂肪酸酯的方法 (Improved method for producing polyhydroxyalkanoate ) 是由 陈国强 凌晨 乔冠清 帅博闻 于 2018-08-24 设计创作，主要内容包括：本发明提供一种改进的微生物生产聚羟基脂肪酸酯(PHA)的方法,所述方法包括向包含碳源以供微生物合成PHA的基础培养基中添加乙酸、乙酸盐、乙酸酯或其他的乙酸衍生物；优选添加乙酸或乙酸盐。该方法可以用于提高微生物合成PHA聚合物的产量和/或控制PHA共聚物产物中的单体比例。(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.)

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.

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 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 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.

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 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.

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 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.