CN117701486B - A recombinant bacterium for producing PHA and its construction method and application - Google Patents

Abstract

The invention relates to the technical field of microorganisms, in particular to recombinant bacteria for producing PHA, and a construction method and application thereof. The invention discovers that the reduced expression quantity and/or activity of the Pel polysaccharide synthesis gene or the encoded protein thereof can obviously improve the PHA production performance of PHA producing bacteria, the growth rate and the biomass of recombinant bacteria constructed by reducing or losing the expression quantity and/or activity of the Pel polysaccharide synthesis gene or the encoded protein thereof are obviously improved, and the yield and the production strength of PHA are also obviously improved. The discovery of the new function of the Pel polysaccharide synthesis gene provides a new transformation target point and strategy for constructing PHA producing bacteria, is beneficial to reducing the cost of PHA industrial production, and further improves the competitiveness of PHA in the market of traditional plastics and bio-based degradable plastics and the commercial application value thereof.

Description

A recombinant bacterium for producing PHA and its construction method and application

Technical Field

The present invention relates to the field of microbial technology, and in particular to a recombinant bacterium for producing PHA and a construction method and application thereof.

Background technique

Polyhydroxyalkanoates (PHA) are a class of high-molecular polyesters synthesized by microorganisms. They have good biodegradability and can replace traditional plastics in a variety of application scenarios. However, the current production cost of PHA is still relatively high compared to traditional plastics and other bio-based degradable plastics, which to a certain extent limits its commercial application. The PHA production performance of PHA-producing bacteria is a key factor affecting the PHA production cost. Therefore, in order to reduce the production cost of PHA, it is necessary to improve the production performance of PHA-producing bacteria, such as PHA yield and production intensity.

Summary of the invention

The present invention provides a recombinant bacterium for producing PHA and a construction method and application thereof

The prior art genetic modification and optimization of PHA production bacteria are mostly focused on the PHA synthesis pathway, and rarely involve the modification of the background genome of the chassis bacteria. During the research and development process, the present invention found that the expression and/or activity of the bacterial Pel polysaccharide synthesis gene and its encoded protein have a significant effect on the bacterial PHA production performance. Reducing the expression and/or activity of the Pel polysaccharide synthesis gene and its encoded protein can not only significantly promote bacterial growth and increase its biomass, but also significantly increase the bacterial PHA yield and production intensity.

Pel polysaccharide is a bacterial polysaccharide rich in N-acetylgalactosamine (GalNAc). Studies have shown that it is related to the structure and function of Pseudomonas aeruginosa biofilm. Pel polysaccharide synthesis genes include pelA , pelB , pelC , pelD , pelE , pelF , and pelG , which exist in the bacterial genome in the form of pelABCDEFG operon. This operon participates in the synthesis of extracellular polysaccharides related to biofilm development in the early and late stages of biofilm development, producing a biofilm matrix rich in glucose. The pelABCDEFG operon has been reported in bacteria such as Cupriavidus and Pseudomonas , and the PelABCDEFG operon is highly conserved in different bacterial species. Therefore, Pel polysaccharide may be a biofilm determinant that is widely present in bacteria. Patent application CN108060111A discloses that the expression change of pelABCDEFG is related to the rhamnolipid production in Pseudomonas, as follows: The patent application discloses a Pseudomonas aeruginosa strain for improving rhamnolipid production and a construction method thereof, wherein the gene expression in group A of the strain is reduced or lost while the gene expression in group B is increased; the genes in group A are selected from one or a combination of any several genes in the pslABCDEFGHIJKLMNO extracellular polysaccharide synthesis gene cluster, the pelABCDEFG polysaccharide synthesis gene cluster, the alginate synthesis gene cluster algD-alg8-alg44 - algKEGXLIJF and the polyhydroxyalkanoic acid PHA synthesis key gene cluster phaC1 - D - C2 ; the genes in group B are selected from one or a combination of any several genes in rmlACBD , rmlBDAC , rhlYZ , algC , fadD4 , lipC and estA . The strain has the advantages of significantly improving the production of rhamnolipid, stable inheritance and being conducive to the separation and purification of downstream products. However, there are no reports on the correlation between Pel polysaccharide biosynthesis genes and bacterial growth rate and PHA production.

Specifically, the present invention provides the following technical solutions:

In a first aspect, the present invention provides a recombinant bacterium for producing PHA, wherein the expression level and/or activity of the Pel polysaccharide synthesis gene or the protein encoded by the recombinant bacterium is reduced or lost, thereby improving the PHA production performance.

The above-mentioned recombinant bacteria producing PHA refer to engineered bacteria that are capable of synthesizing and accumulating PHA.

In some embodiments of the present invention, bacteria capable of synthesizing and accumulating PHA are used as starting bacteria, and the starting bacteria are modified so that the expression level and/or activity of the Pel polysaccharide synthesis gene or its encoded protein is reduced or lost.

There is no particular limitation on the starting bacteria in the present invention, as long as it can synthesize and accumulate PHA and contains the Pel polysaccharide synthesis gene, reducing or losing the expression level and/or activity of the Pel polysaccharide synthesis gene or its encoded protein can in principle improve its PHA production performance.

The Pel polysaccharide synthesis gene mentioned above is one or more selected from pelA , pelB , pelC , pelD , pelE , pelF , and pelG .

Specifically, the Pel polysaccharide synthesis gene is any one of pelA , pelB , pelC , pelD , pelE , pelF , pelG, or a combination of any 2 to 7 of them.

In some embodiments of the present invention, the Pel polysaccharide synthesis genes are pelA , pelB , pelC , pelD , pelE , pelF and pelG .

Preferably, the Pel polysaccharide synthesis gene is part of or all of the genes in the pelABCDEFG polysaccharide synthesis gene cluster, that is, one or more essential genes on the Pel polysaccharide synthesis pathway are intervened in expression, thereby regulating the Pel polysaccharide synthesis pathway.

As a scheme with the best effect, in some embodiments of the present invention, the Pel polysaccharide synthesis gene is a pelABCDEFG polysaccharide synthesis gene cluster, that is, the expression of 7 essential genes on the Pel polysaccharide synthesis pathway is intervened simultaneously.

The aforementioned reduction or loss of expression level and/or activity includes weakening the expression level and/or activity of the gene or protein, or making the gene or protein non-expressed or inactivated.

The present invention has no particular limitations on the methods and technical means for achieving reduction or loss of expression level and/or activity. For example, commonly used genetic engineering means and gene editing methods can be used to modify the protein, its encoding gene, its regulatory element and/or its regulatory gene or protein so that the expression level and/or activity of the gene or protein is reduced or lost.

In some embodiments of the present invention, the reduction or loss of the expression level and/or activity of the gene or protein is achieved by a combination of any one or more of the following (1) to (3):

(1) Mutating the amino acid sequence of a protein to reduce or eliminate the expression and/or activity of a gene or protein;

(2) Mutating the nucleotide sequence of a gene so that the expression level and/or activity of the gene or protein is reduced or lost;

(3) Replacing the transcriptional and/or translational regulatory elements of a gene with elements with weaker activity to reduce the expression of the gene or protein.

The mutation of the amino acid sequence described above includes deletion, insertion or substitution of one or more amino acids.

The mutation of the nucleotide sequence described above includes deletion, insertion or substitution of one or more nucleotides.

The transcription and translation regulatory elements mentioned above include promoters, ribosome binding sites, etc.

In some embodiments of the present invention, the reduction or loss of the expression level and/or activity of the gene or protein is achieved by inactivating the gene or protein. The inactivation can be achieved by knocking out the entire gene or a portion of its sequence.

In some embodiments of the present invention, the pelA , pelB , pelC , pelD , pelE , pelF and pelG genes or their encoded proteins of the recombinant bacteria are inactivated.

In some embodiments of the present invention, the pelABCDEFG polysaccharide synthesis gene cluster of the recombinant bacteria is knocked out.

It should be understood that in the specific embodiments of the present invention, inactivating pelA , pelB , pelC , pelD , pelE , pelF and pelG genes to construct PHA-producing recombinant bacteria is only for exemplary purposes. On the basis of the purpose of reducing the expression level and/or activity of the Pel polysaccharide synthesis gene or its encoded protein known to those skilled in the art, other technical means that can also achieve the above-mentioned purpose are equivalent variations of the technical means of the present invention and are therefore within the scope of protection of the present invention.

In principle, there is no special restriction on the bacterial species to which the recombinant bacteria belongs, as long as it can synthesize and accumulate PHA and contains the Pel polysaccharide synthesis gene, including but not limited to Ralstonia bacteria (such as Ralstonia eutropha), Pseudomonas bacteria (such as Pseudomonas putida, Pseudomonas aeruginosa), Alcaligenes bacteria (such as Alcaligenes eutrophus), Aeromonas bacteria (such as Aeromonas hydrophila), Escherichia bacteria (such as Escherichia coli), Bacillus bacteria (such as Bacillus subtilis), Corynebacterium bacteria (such as Corynebacterium glutamicum), halophilic bacteria and yeast, etc.

In some embodiments of the present invention, the recombinant bacteria are Ralstonia bacteria or Pseudomonas bacteria. Preferably, the recombinant bacteria are Eutropha rhodochii, Pseudomonas putida or Pseudomonas aeruginosa.

The present invention verifies through experiments that the production performance of PHA-producing recombinant bacteria obtained by modifying, reducing or losing the expression amount and/or activity of the Pel polysaccharide synthesis gene or its encoded protein of the PHA-producing bacteria is significantly improved, which is specifically manifested in the increase in the growth rate of the strain, the increase in biomass, and the increase in PHA yield and production intensity.

In a second aspect, the present invention provides a method for preparing the above-mentioned recombinant bacteria, the method comprising: modifying the PHA-producing bacteria so that the expression level and/or activity of the Pel polysaccharide synthesis gene or the protein encoded by it is reduced or lost.

Preferably, the reduction or loss of the expression level and/or activity of the Pel polysaccharide synthesis gene or its encoded protein is achieved by knocking out the pelABCDEFG polysaccharide synthesis gene cluster.

In a third aspect, the present invention provides the use of the above-mentioned recombinant bacteria in PHA fermentation production.

Preferably, the use comprises the steps of culturing the recombinant bacteria and collecting the culture containing PHA.

The above-mentioned culture can be carried out using common substrates for PHA production (including carbon sources, nitrogen sources, etc.), wherein the carbon source includes but is not limited to biomass such as vegetable oil, waste cooking oil, and sugar substances, and the nitrogen source includes but is not limited to inorganic nitrogen sources such as ammonium salts or organic nitrogen sources such as yeast powder and peptone. Inorganic salts (including but not limited to disodium hydrogen phosphate, potassium dihydrogen phosphate, etc.) and trace elements (including but not limited to magnesium, calcium, zinc, manganese, cobalt, boron, copper, nickel, molybdenum, etc.) can also be added to the culture medium.

In some embodiments of the present invention, the culture is carried out using vegetable oil (including but not limited to a mixture of one or more of palm oil, palm kernel oil, peanut oil, soybean oil, linseed oil, rapeseed oil, cottonseed oil, castor oil, and corn oil) as a carbon source.

In a fourth aspect, the present invention provides a method for improving the PHA production performance of a PHA producing bacterium, the method comprising: modifying the PHA producing bacterium so that the expression level and/or activity of the Pel polysaccharide synthesis gene or its encoded protein is reduced or lost.

Preferably, the PHA production performance is selected from one or more of strain growth rate, biomass, PHA yield, and PHA production intensity.

In a fifth aspect, the present invention provides an application of reducing or losing the expression level and/or activity of a Pel polysaccharide synthesis gene or a protein encoded therein to improve the PHA production performance of a PHA producing bacterium.

In the present invention, the Pel polysaccharide synthesis gene is one or more selected from pelA , pelB , pelC , pelD , pelE , pelF , and pelG .

Reducing the expression level and/or activity of one or more of the Pel polysaccharide synthesis genes pelA , pelB , pelC , pelD , pelE , pelF , pelG or their encoded proteins will affect the synthesis of Pel polysaccharide, thereby affecting the production performance of PHA producing bacteria. Based on this, the Pel polysaccharide synthesis gene of the present invention can be selected from one or more of pelA , pelB , pelC , pelD , pelE , pelF , pelG .

Specifically, the Pel polysaccharide synthesis gene is any one of pelA , pelB , pelC , pelD , pelE , pelF , pelG, or a combination of any 2 to 7 of them.

The pelA , pelB , pelC , pelD , pelE , pelF , and pelG genes in bacteria are mostly present in the form of gene clusters. Therefore, the Pel polysaccharide synthesis gene is a partial gene or all genes in the pelABCDEFG polysaccharide synthesis gene cluster.

In some embodiments of the present invention, the Pel polysaccharide synthesis genes are pelA , pelB , pelC , pelD , pelE , pelF and pelG .

In some embodiments of the present invention, the Pel polysaccharide synthesis gene is a pelABCDEFG polysaccharide synthesis gene cluster.

For the sequences of Pel polysaccharide synthesis genes pelA , pelB , pelC , pelD , pelE , pelF , pelG and their encoded proteins in bacteria, those skilled in the art can obtain them through databases such as Genbank.

In the present invention, the PHA-producing bacteria refers to bacteria that can synthesize and accumulate PHA. PHA-producing bacteria include, but are not limited to, bacteria of the genus Ralstonia (e.g., Eutropha rothrips), bacteria of the genus Pseudomonas (e.g., Pseudomonas putida, Pseudomonas aeruginosa), bacteria of the genus Alcaligenes (e.g., Alcaligenes eutrophus), bacteria of the genus Aeromonas (e.g., Aeromonas hydrophila), bacteria of the genus Escherichia (e.g., Escherichia coli), bacteria of the genus Bacillus (e.g., Bacillus subtilis), bacteria of the genus Corynebacterium (e.g., Corynebacterium glutamicum), halophiles, and yeasts.

In some embodiments of the present invention, the PHA-producing bacteria are Ralstonia bacteria or Pseudomonas bacteria. Preferably, the PHA-producing bacteria are Eutropha rhodochii, Pseudomonas putida or Pseudomonas aeruginosa.

Ralstonia eutropha (also known as Cupriavidus necator ) is an important model bacterium for studying PHA synthesis, and is also a strain that is currently being studied more for PHA industrial production. Ralstonia eutropha H16 and its derivative strains can be used as a platform chassis to achieve high cell density batch fed fermentation under industrial conditions, and produce molecular products including different types of PHA using biomass raw materials such as sugars and oils as substrates. In the specific embodiments of the present invention, Ralstonia eutropha is used as an example to verify the effect of the Pel polysaccharide synthesis gene on the growth rate, biomass, PHA yield and production intensity of the strain. Based on the representative effect of the model bacteria on bacteria of the same genus and PHA-producing bacteria of other genera, those skilled in the art can infer that the above-mentioned effect is also applicable to other bacteria of the genus Ralstonia that contain the Pel polysaccharide synthesis gene and can synthesize and accumulate PHA, and PHA-producing bacteria of other genera.

In some embodiments of the present invention, the PHA-producing bacteria is Eutropha rosea. For Eutropha rosenbergii, the GenBanklocus_tags of the Eutropha rosenbergii genes corresponding to the Pel polysaccharide synthesis genes pelA , pelB , pelC , pelD , pelE , pelF , and pelG are H16_B2198~B2204, respectively. Specifically, H16_B2198 is pelG (the amino acid sequence of the protein is shown in SEQID NO.1, and the gene sequence is shown in SEQ ID NO.2), H16_B2199 is pelF (the amino acid sequence of the protein is shown in SEQID NO.3, and the gene sequence is shown in SEQ ID NO.4), H16_B2200 is pelE (the amino acid sequence of the protein is shown in SEQID NO.5, and the gene sequence is shown in SEQ ID NO.6), H16_B2201 is pelD (the amino acid sequence of the protein is shown in SEQID NO.7, and the gene sequence is shown in SEQ ID NO.8), and H16_B2202 is pelC (the amino acid sequence of the protein is shown in SEQID NO.9, and the gene sequence is shown in SEQ ID NO.10). NO.9, and the gene sequence is shown in SEQ ID NO.10), H16_B2203 is pelB (the amino acid sequence of the protein is shown in SEQ ID NO.11, and the gene sequence is shown in SEQ ID NO.12), and H16_B2204 is pelA (the amino acid sequence of the protein is shown in SEQ ID NO.13, and the gene sequence is shown in SEQ ID NO.14).

In the above applications, the reduction or loss of expression level and/or activity includes weakening the expression level and/or activity of the gene or protein, or making the gene or protein non-expressed or inactivated.

The present invention has no particular limitations on the methods and technical means for achieving reduced expression levels and/or activity. For example, commonly used genetic engineering means and gene editing methods can be used to modify the protein, its encoding gene, its regulatory elements and/or its regulatory genes or proteins so that the expression level and/or activity of the gene or protein is reduced or lost.

In some embodiments of the present invention, the reduction or loss of the expression level and/or activity of the gene or protein is achieved by a combination of any one or more of the following (1) to (3):

(1) Mutating the amino acid sequence of a protein to reduce or eliminate the expression and/or activity of a gene or protein;

(2) Mutating the nucleotide sequence of a gene so that the expression level and/or activity of the gene or protein is reduced or lost;

(3) Replacing the transcriptional and/or translational regulatory elements of a gene with elements with weaker activity to reduce the expression of the gene or protein.

The mutation of the amino acid sequence described above includes deletion, insertion or substitution of one or more amino acids.

The mutation of the nucleotide sequence described above includes deletion, insertion or substitution of one or more nucleotides.

The transcription and translation regulatory elements mentioned above include promoters, ribosome binding sites, etc.

In some embodiments of the present invention, the reduction or loss of the expression level and/or activity of the gene or protein is achieved by inactivating the gene or protein. The inactivation can be achieved by knocking out the entire gene or a portion of its sequence.

In a sixth aspect, the present invention provides a method for producing PHA, comprising: culturing the recombinant bacteria described above, and collecting the culture containing PHA.

The beneficial effects of the present invention include at least: the present invention found that although the Pel polysaccharide synthesis pathway and the PHA synthesis pathway do not have the same substrates or intermediate metabolites, the reduction in the expression and/or activity of the Pel polysaccharide synthesis gene or its encoding protein can significantly improve the PHA production performance of the PHA-producing bacteria, and the growth rate and biomass of the recombinant bacteria constructed by reducing or losing the expression and/or activity of the Pel polysaccharide synthesis gene or its encoding protein are significantly improved, and the yield and production intensity of PHA are also significantly improved. The discovery of the new function of the above-mentioned Pel polysaccharide synthesis gene provides a new transformation target and strategy for the construction of PHA-producing bacteria, which is conducive to reducing the cost of PHA industrial production, thereby enhancing the competitiveness of PHA in the traditional plastic and bio-based degradable plastic markets and its commercial application value.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

FIG1 is a comparison of the growth curves of the recombinant strain W01 and the control strain H16 in Example 1 of the present invention; wherein the circular data point curve represents the control strain H16, and the square data point curve represents the recombinant strain W01; t-test statistical test: p<0.05, with Indicates; p<0.01, with/> express.

Detailed ways

In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

The materials and reagents used in the following examples, unless otherwise specified, can be obtained from commercial sources. The kit used to extract the plasmid was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. The corresponding operation steps were strictly carried out in accordance with the product instructions, and all culture media were prepared with deionized water unless otherwise specified.

The culture medium formula used in the following examples is as follows:

Seed culture medium I: 10 g/L peptone, 5 g/L yeast extract, 3 g/L fructose.

Seed culture medium II: 0.15% palm oil, 10 g/L peptone, 5 g/L yeast extract.

Production medium: 1.0% palm oil, 9.85g/L Na ₂ HPO ₄ ·12H ₂ O, 1.5g/L KH ₂ PO ₄ , 3.0g/L NH ₄ Cl, 10mL/L trace element solution I and 1mL/L trace element solution II. The composition of trace element solution I is: 20g/L MgSO ₄ , 2g/L CaCl ₂ . The composition of trace element solution II is: 100mg/L ZnSO ₄ ·7H ₂ O, 30mg/L MnCl ₂ ·4H ₂ O, 300mg/L H ₃ BO ₃ , 200mg/L CoCl ₂ ·6H ₂ O, 10mg/L CuSO ₄ ·5H ₂ O, 20mg/L NiCl ₂ ·6H ₂ O, 30mg/L NaMoO ₄ ·2H ₂ O. The above reagents were purchased from Sinopharm Chemical Reagent Company.

The experimental data in the following examples were obtained from 3 or more parallel group experiments.

The calculation formula of the average growth rate described in the following examples is as follows:

Average growth rate = final OD/growth time; the final OD is the OD value of the strain detected at 600nm absorbance at the end of strain culture.

The PHA content (PHA%) described in the following examples refers to the mass percentage of PHA in the dry weight of cells.

The calculation formula for the PHA yield described in the following examples is as follows:

PHA yield = CDW × PHA%; where CDW is cell dry weight and PHA% is the percentage of PHA in cell dry weight.

The calculation formula of the production intensity described in the following examples is as follows:

Production intensity = PHA output/time; PHA output is calculated as described above, and the time is the whole fermentation process time of 54h.

Example 1: Construction and growth test of Eutropha rosea ReΔB2198~B2204

In this example, the H16 strain of Eutropha roeblingii (H16 for short) was used as the starting strain, and a common gene editing method in the field was used to knock out a total of 7 genes on the genome, including H16_B2198, H16_B2199, H16_B2200, H16_B2201, H16_B2202, H16_B2203, and H16_B2204. The resulting multi-gene knockout recombinant strain was Eutropha roeblingii W01, and the recombinant strain was tested for strain growth curves. The specific methods and results are as follows:

Step 1: Construction of H16_B2198~B2204 multi-gene knockout recombinant strain W01

1.1 The genome of Eutropha rosenbergii H16 was used as a template for PCR amplification to obtain the upstream homology arm of B2198~B2204 B2198~B2204-H1 and the downstream homology arm of B2198~B2204 B2198~B2204-H2; the modified plasmid pK18mob (Orita I, Iwazawa R, Nakamura S, et al. Identification of mutation pointsin Cupriavidus necator NCIMB 11599 and genetic reconstitution of glucose-utilization ability in wild strain H16 for polyhydroxyalkanoate production[J]. Journal of Bioscience&Bioengineering, 2012, 113(1):63-69) was used as a template to obtain the vector fragment by PCR amplification. B2198~B2204-H1 and B2198~B2204-H2 were connected to the vector fragment by the Gibson Assembly method to obtain the editing plasmid pKO-ΔB2198~B2204, and the subcloning construction of the editing plasmid was completed by WuXi Biologics Co., Ltd. The sequences of the homology arms B2198~B2204-H1 and B2198~B2204-H2 are shown in SEQ ID NO.15 and SEQ ID NO.16, respectively.

1.2 The recombinant plasmid pKO-ΔB2198~B2204 was transformed into Escherichia coli S17-1, and then into E. rothrips H16 by conjugation transformation. Taking advantage of the fact that the suicide plasmid cannot replicate in the host bacteria, the positive clones were screened on LB plates containing 250μg/mL kanamycin and 100μg/mL apramycin. The recombinant plasmid carrying the homologous fragment in the positive clone was integrated into the specific position of the genome, thus obtaining the first homologous recombinant strain. The first homologous recombinant strain was streaked out as a single clone on an LB plate containing 100 mg/mL sucrose, and clones without kanamycin resistance were screened out from these single clones, and PCR identification was performed. Sequencing confirmed that the recombinant strain was correctly edited. The resulting recombinant strain was Eutropha rhodii ReΔB2198~B2204 , named recombinant strain W01, in which a total of 7 genes, including H16_B2198, H16_B2199, H16_B2200, H16_B2201, H16_B2202, H16_B2203, and H16_B2204, were knocked out.

Step 2: Growth test of strain W01 using palm oil as the sole carbon source

The recombinant strains H16 and W01 preserved in the glycerol tube were streaked on LB plates. After obtaining a single clone, a 24-deep-well plate was used for subsequent seed culture and fermentation culture. The single clone was inoculated into seed culture medium I (2 mL) for 15 hours of primary seed culture to obtain seed culture solution I; then the seed culture solution I was inoculated into seed culture medium II (2 mL) at an inoculation volume of 10% (v/v) for secondary seed culture, and cultured for 5 hours to obtain seed culture solution II; then the seed culture solution II was inoculated into a 24-deep-well plate containing 3 mL of production culture medium at an inoculation volume of 15% (v/v) for micro-fermentation. The fermentation incubator temperature was 30°C and the speed was 450rpm for continuous culture for 24 hours. During this period, the absorbance value at 600nm was sampled and tested every 2 hours, and the growth OD of the strain at that time was calculated. A total of 10 sampling time points were taken for the growth test, and 10 strain growth ODs were obtained. The growth curve was prepared, and the results are shown in Figure 1. The results showed that compared with the control strain H16, the recombinant strain W01 could achieve a higher OD turbidity in a 24-hour growth period, and the average growth rate was also higher than that of the control strain H16.

Example 2: Fermentation performance test of Eutropha rosea ReΔB2198~B2204

In this example, Eutropha rosea H16 (H16 for short) was used as a control strain, and palm oil was used as the sole carbon source to test the production performance of Eutropha rosea ReΔB2198~B2204 (W01 strain) in shake flask fermentation and fermenter fermentation.

Step 1: Performance test of recombinant strain W01 in producing PHA by shake flask fermentation

1.1 The control strain H16 stored in the glycerol tube and the recombinant strain W01 obtained in step 1 of Example 1 were streaked on LB plates to obtain a single clone. The single clone was inoculated into seed culture medium I (3 mL) and seed activation was performed for 15 hours to obtain an activated seed solution; then the activated seed solution was inoculated into seed culture medium I (10 mL) at an inoculation amount of 10% (v/v) for primary seed culture, cultured for 8 hours, and seed culture solution I was obtained; then the seed culture solution I was inoculated into seed culture medium II (15 mL) at an inoculation amount of 10% (v/v) for secondary seed culture, cultured for 15 hours, and seed culture solution II was obtained; then the seed culture solution II was inoculated into a 250 mL conical flask containing 30 mL of production culture medium at an inoculation amount of 15% (v/v), and the fermentation incubator temperature was 30°C and the rotation speed was 220 rpm for continuous culture for 24 hours.

1.2 Take the above fermentation liquid and centrifuge it to obtain bacterial cells. Dry the bacterial cells to constant weight. Measure the weight of the dried bacterial cells and record it as dry weight. Add 3.3 mL of chloroform to the obtained dry bacterial cells, stir at room temperature for a day and night, and extract the polyester in the bacterial cells. After filtering out the bacterial residue, use an evaporator to concentrate to a total volume of about 1 mL, then slowly add about 3 mL of hexane and let it stand for 1 hour under slow stirring. After filtering out the precipitated polyester, vacuum dry it for 3 hours. Measure the mass of the dry polyester and calculate the polyester content in the bacterial cells.

The results are shown in Table 1. Compared with the control strain H16, the dry weight of the W01 strain was significantly increased by 8.3%, the PHA% was significantly increased by 4.8%, and the PHA yield was significantly increased by 13.6%.

Table 1

Step 2: Performance test of PHA production by recombinant strain W01 in reactor

2.1 The recombinant strain W01 and the control strain H16 (1000 μL) constructed in Example 1 preserved in the glycerol tube were inoculated into the seed culture medium I (20 mL) respectively, and the seed culture was carried out for 12 hours to obtain the seed culture solution I; then, the seed culture solution I was inoculated into the seed culture medium II (100 mL) at an inoculation amount of 1% (v/v), and the secondary seed culture was carried out for 13 hours to obtain the seed culture solution II; then, the seed culture solution II was inoculated into a 500 mL small fermentation tank containing 250 mL of production culture medium at an inoculation amount of 10% (v/v). The operating conditions were a culture temperature of 30°C, a stirring speed of 800 rpm, a ventilation volume of 1 L/min, and the pH was controlled between 6.7 and 6.8. A 28% ammonia solution was used for pH control. During the culture process, palm oil was continuously used as a carbon source, and the culture time was 54 hours.

2.2 Take the above fermentation liquid and centrifuge it to obtain bacterial cells. Dry the bacterial cells to constant weight. Measure the weight of the dried bacterial cells and record it as dry weight. Add 25mL of chloroform to the obtained dry bacterial cells, stir at room temperature for a day and night, and extract the polyester in the bacterial cells. After filtering out the bacterial residue, use an evaporator to concentrate to a total volume of about 7.5mL, then slowly add about 22.5mL of hexane and leave it for 1 hour under slow stirring. After filtering out the precipitated polyester, vacuum dry it for 3 hours. Measure the mass of the dry polyester and calculate the polyester content in the bacterial cells.

The results are shown in Table 2. The following three PHA production performance indicators of the recombinant strain W01 are significantly better than those of the control strain H16. Compared with the control strain H16, the dry weight of the recombinant strain W01 increased significantly by 10.02%, the PHA yield increased significantly by 11.77%, and the production intensity increased significantly by 11.74%.

Table 2

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (7)

1. A recombinant bacterium for producing PHA, characterized in that the H16_B2198, H16_B2199, H16_B2200, H16_B2201, H16_B2202, H16_B2203 and H16_B2204 genes of the recombinant bacterium are knocked out, thereby improving the PHA production performance; The starting strain of the recombinant bacteria is Eutropha rosea H16. 2. The method for preparing the recombinant bacteria according to claim 1, characterized in that the method comprises: modifying the Eutropha rhodii H16 to knock out the H16_B2198, H16_B2199, H16_B2200, H16_B2201, H16_B2202, H16_B2203 and H16_B2204 genes. 3. Use of the recombinant bacteria according to claim 1 in PHA fermentation production. 4. A method for improving the PHA production performance of a PHA producing bacterium, characterized in that the method comprises: modifying the PHA producing bacterium to knock out the H16_B2198, H16_B2199, H16_B2200, H16_B2201, H16_B2202, H16_B2203 and H16_B2204 genes; The PHA producing bacteria is Eutropha rosea H16. 5. The method according to claim 4, characterized in that the PHA production performance is PHA yield and/or PHA production intensity. 6. Application of knockout of H16_B2198, H16_B2199, H16_B2200, H16_B2201, H16_B2202, H16_B2203 and H16_B2204 genes in improving the PHA production performance of PHA producing bacteria; The PHA producing bacteria is Eutropha rosea H16. 7. The use according to claim 6, characterized in that the PHA production performance is PHA yield and/or PHA production intensity.