Recombinant engineered bacteria which improve polyhydroxyalkanoate yield and application of recombinant engineered bacteria

Abstract

The present invention relates to the technical field of microorganisms, and relates in particular to recombinant engineered bacteria which improve polyhydroxyalkanoate yield and an application of the recombinant engineered bacteria. The present invention has found that a reduction in H16_A3043 protein and / or H16_A3044 protein expression and / or activity can significantly improve the PHA production performance of PHA-producing bacteria; the recombinant engineered bacteria constructed thereby has significant improvement in aspects such as PHA yield, substrate conversion rate, and growth rate, effectively reducing the costs of industrial PHA production, and improving the competitiveness and commercial application value of PHA in the traditional plastics and biobased degradable plastics markets.

Description

Recombinant engineering bacteria for increasing polyhydroxyalkanoate production and its application

[0001] Cross-references

[0002] This application claims priority to Chinese Patent Application No. 202410019579X, filed on January 5, 2024, entitled “Recombinant engineered bacteria for improving the production of polyhydroxyalkanoates and their applications,” the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to the field of microbial technology, in particular to recombinant engineering bacteria for increasing the yield of polyhydroxyalkanoate and applications thereof. Background Art

[0004] Polyhydroxyalkanoates (PHAs) are a class of high-molecular-weight polyesters synthesized by microorganisms. They have good biodegradability and can replace traditional plastics in a variety of scenarios. Ralstonia eutropha (also known as Cupriavidus necator) is an important model bacterium for studying PHA synthesis and is currently the most studied bacterial species for industrial PHA production. Commonly used Ralstonia eutropha H16 and its derivative strains can be used as a platform chassis to achieve high cell density batch fermentation under industrial conditions, using biomass raw materials such as sugars and oils as substrates to efficiently produce products including different types of PHA.

[0005] The current production cost of PHA remains relatively high compared to other traditional plastics and biodegradable plastics, limiting its commercial application to a certain extent. Improving the productivity of PHA-producing strains is key to reducing PHA production costs. However, current efforts to optimize PHA synthesis have primarily focused on screening and modifying PHA synthesis pathways, with few reports on genome modification of the basement bacterial background. Summary of the Invention

[0006] The present invention provides recombinant engineering bacteria for increasing the yield of polyhydroxyalkanoate and application thereof.

[0007] During the research and development process of improving PHA production performance, the present invention discovered two new targets related to PHA synthesis - H16_A3043 and H16_A3044. Experimental verification shows that weakening or inactivating H16_A3043 and H16_A3044 can significantly increase PHA yield and substrate conversion rate, thereby improving the PHA production performance of the strain.

[0008] Specifically, the present invention provides the following technical solutions:

[0009] In a first aspect, the present invention provides the use of reducing the expression and / or activity of H16_A3043 protein and / or H16_A3044 protein in improving the PHA production performance of PHA-producing bacteria.

[0010] In a second aspect, the present invention provides the use of reducing the expression and / or activity of a homologous protein of the H16_A3043 protein and / or a homologous protein of the H16_A3044 protein in improving the PHA production performance of a PHA-producing bacterium.

[0011] In the present invention, the PHA-producing bacteria refer to microorganisms that can synthesize and accumulate PHA, including but not limited to Ralstonia bacteria (e.g., Ralstonia eutropha), Alcaligenes bacteria (e.g., Alcaligenes eutrophus), Escherichia bacteria (e.g., Escherichia coli), Bacillus bacteria (e.g., Bacillus subtilis), Corynebacterium bacteria (e.g., Corynebacterium glutamicum), halophiles and yeasts.

[0012] Preferably, the PHA-producing bacteria are Ralstonia bacteria.

[0013] Further preferably, the PHA-producing bacteria is Eutropha rosea.

[0014] In the present invention, H16_A3043 and H16_A3044 are locus tags of protein encoding genes in GenBank. Those skilled in the art can obtain the sequences of H16_A3043 and H16_A3044 proteins and their encoding genes from GenBank.

[0015] Specifically, the amino acid sequence of the H16_A3043 protein is shown in SEQ ID NO.1, the nucleotide sequence of the H16_A3043 gene is shown in SEQ ID NO.2, the amino acid sequence of the H16_A3044 protein is shown in SEQ ID NO.3, and the nucleotide sequence of the H16_A3044 gene is shown in SEQ ID NO.4.

[0016] In the present invention, the homologous protein of the H16_A3043 protein is a protein derived from Ralstonia bacteria and having at least 70% homology (preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%) with the amino acid sequence of the H16_A3043 protein. The homologous protein of the H16_A3044 protein is a protein derived from Ralstonia bacteria and has at least 70% (preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.5%) homology with the amino acid sequence of the H16_A3044 protein.

[0017] In some embodiments of the present invention, there is provided the use of reducing the expression and / or activity of the H16_A3043 protein in improving the PHA production performance of Eutropha rosea.

[0018] In some embodiments of the present invention, there is provided the use of reducing the expression and / or activity of the H16_A3044 protein in improving the PHA production performance of Eutropha rosea.

[0019] In some embodiments of the present invention, there is provided the use of reducing the expression and / or activity of H16_A3043 and H16_A3044 proteins in improving the PHA production performance of Eutropha rosea.

[0020] In the present invention, the PHA production performance includes PHA yield and / or substrate conversion rate.

[0021] The substrate conversion rate refers to the conversion rate of the substrate used in the fermentation production of PHA into PHA. Preferably, the substrate conversion rate is the biomass substrate conversion rate.

[0022] In the above applications, reducing expression and / or activity includes weakening the expression and / or activity of the protein, or causing the protein to not be expressed or to be inactivated.

[0023] The present invention has no particular limitations on the methods and technical means for achieving reduced expression 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 to reduce the expression and / or activity of the protein.

[0024] In some embodiments of the present invention, the reduction in expression and / or activity of the protein is achieved by a combination of any one or more of the following methods (1) to (3):

[0025] (1) Mutating the amino acid sequence of a protein to reduce the expression and / or activity of the protein;

[0026] (2) mutating the nucleotide sequence of the protein-encoding gene to reduce the expression and / or activity of the protein;

[0027] (3) Replacing the transcriptional and / or translational regulatory elements of the protein-coding gene with elements with weaker activity to reduce protein expression.

[0028] The mutation of the amino acid sequence mentioned above includes deletion, insertion or substitution of one or more amino acids.

[0029] The mutation of the nucleotide sequence mentioned above includes deletion, insertion or substitution of one or more nucleotides.

[0030] The transcription and translation regulatory elements mentioned above include promoters, ribosome binding sites, etc.

[0031] In some embodiments of the present invention, the reduction of expression and / or activity of the protein is achieved by inactivating the protein.

[0032] In some embodiments of the present invention, the expression and / or activity of the H16_A3043 protein is reduced by mutating the amino acid at position 57 to proline and / or mutating the amino acid at position 173 to proline.

[0033] As an example, the present invention has experimentally verified that H16_A3043 inactivation, H16_A3044 inactivation, and H16_A3043 and H16_A3044 inactivation can significantly improve the PHA production performance of the strain. Moreover, compared with H16_A3044 inactivation, H16_A3043 and H16_A3044 inactivation, the H16_A3043 inactivated strain has better PHA production performance, specifically higher PHA yield and higher substrate conversion rate.

[0034] In a third aspect, the present invention provides a mutant of the H16_A3043 protein, wherein, compared with the H16_A3043 protein, the mutant comprises a mutation in which the amino acid at position 57 is mutated to proline and / or the amino acid at position 173 is mutated to proline.

[0035] The present invention found that mutating the amino acid at position 57 of the H16_A3043 protein to proline and mutating the amino acid at position 173 to proline can significantly increase the PHA yield and substrate conversion rate of the strain, effectively promoting the synthesis of PHA. Moreover, the mutation of the amino acid at position 57 to proline has a significantly better effect on improving the PHA yield and substrate conversion rate.

[0036] In some embodiments of the present invention, the mutation of the mutant compared to the H16_A3043 protein is that the amino acid at position 57 is mutated to proline.

[0037] In some embodiments of the present invention, the mutation of the mutant compared to the H16_A3043 protein is that the amino acid at position 173 is mutated to proline.

[0038] In a fourth aspect, the present invention provides a nucleic acid molecule encoding the mutant.

[0039] Based on the amino acid sequence and codon rules of the above mutant, those skilled in the art can obtain the nucleotide sequence of the nucleic acid molecule encoding the mutant. Due to the degeneracy of codons, the nucleotide sequence of the nucleic acid molecule encoding the mutant is not unique, and all nucleic acid molecules that can encode the mutant are within the scope of protection of the present invention.

[0040] In a fifth aspect, the present invention provides biological materials comprising the nucleic acid molecule or expressing the mutant.

[0041] The biological materials mentioned above include expression cassettes, vectors or host cells.

[0042] The expression cassette is a recombinant nucleic acid molecule obtained by operably linking the nucleic acid molecule with a transcription and / or translation regulatory element.

[0043] The vector includes but is not limited to a plasmid vector, a viral vector, and a transposon.

[0044] The host cell includes a microbial cell, preferably Escherichia coli or Eutropha rosea.

[0045] In a sixth aspect, the present invention provides use of the mutant, the nucleic acid molecule, or the biological material described above in constructing a recombinant engineered bacterium that produces PHA.

[0046] In a seventh aspect, the present invention provides a recombinant engineered bacterium, wherein the recombinant engineered bacterium is modified so that the expression and / or activity of the H16_A3043 protein and / or the H16_A3044 protein is reduced.

[0047] In some embodiments of the present invention, the recombinant engineered bacteria are modified so that the expression and / or activity of the H16_A3043 protein is reduced.

[0048] In some embodiments of the present invention, the recombinant engineered bacteria is modified so that the expression and / or activity of the H16_A3044 protein is reduced.

[0049] In some embodiments of the present invention, the recombinant engineered bacteria are modified so that the expression and / or activity of the H16_A3043 and H16_A3044 proteins are reduced.

[0050] The aforementioned reduction of expression and / or activity includes weakening the expression and / or activity of the protein, or causing the protein to not be expressed or to be inactivated.

[0051] In some embodiments of the present invention, the reduction in expression and / or activity of the protein is achieved by a combination of any one or more of the following methods (1) to (3):

[0052] (1) Mutating the amino acid sequence of a protein to reduce the expression and / or activity of the protein;

[0053] (2) mutating the nucleotide sequence of the protein-encoding gene to reduce the expression and / or activity of the protein;

[0054] (3) Replacing the transcriptional and / or translational regulatory elements of the protein-coding gene with elements with weaker activity to reduce protein expression.

[0055] The mutation of the amino acid sequence mentioned above includes deletion, insertion or substitution of one or more amino acids.

[0056] The mutation of the nucleotide sequence mentioned above includes deletion, insertion or substitution of one or more nucleotides.

[0057] The transcription and translation regulatory elements mentioned above include promoters, ribosome binding sites, etc.

[0058] Preferably, the recombinant engineered bacteria contains any of the following modifications:

[0059] (1) Inactivation of H16_A3043 protein and / or H16_A3044 protein;

[0060] (2) no expression of H16_A3043 protein and / or H16_A3044 protein;

[0061] (3) A mutant that does not express the H16_A3043 protein and expresses the H16_A3043 protein.

[0062] It should be understood that the present invention uses the above means for exemplary purposes in the specific embodiments. Based on the purpose of reducing the expression and / or activity of the H16_A3043 protein and / or the H16_A3044 protein of the present invention 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.

[0063] The PHA production performance of the recombinant engineered bacteria was significantly improved, specifically manifested in a significant increase in PHA yield and substrate conversion rate.

[0064] Preferably, the recombinant engineered bacteria are PHA-producing recombinant engineered bacteria, and the PHA production and / or substrate conversion rate of the recombinant engineered bacteria are increased compared to the starting strain.

[0065] Furthermore, the present invention found that, on the basis of reducing the expression and / or activity of H16_A3043 protein and / or H16_A3044 protein, enhancing the expression and / or activity of the proteins encoded by the Calvin cycle-related genes cbbL and cbbS can further improve the PHA yield and substrate conversion rate. The above-mentioned combined modification has a significantly better effect on improving PHA yield and substrate conversion rate than the modification of each target alone; at the same time, the recombinant engineered bacteria can use different carbon sources (for example: vegetable oil, waste cooking oil, etc.) as substrates to efficiently synthesize PHA.

[0066] Specifically, the recombinant engineered bacteria described above may also contain the following modifications: enhancing the expression and / or activity of proteins encoded by genes related to the Calvin cycle;

[0067] The Calvin cycle-related genes include cbbL, and preferably the expression and / or activity of proteins encoded by cbbL and cbbS genes are enhanced.

[0068] In the present invention, enhancing the expression and / or activity of proteins encoded by the cbbL and cbbS genes can be achieved by any one or more of the following methods (1) to (4):

[0069] (1) modifying the regulatory protein of the gene;

[0070] (2) modifying the transcriptional regulatory elements and / or translational regulatory elements of the gene;

[0071] (3) modifying the sequence of the gene;

[0072] (4) Increasing the copy number of the gene.

[0073] In the above (1), the regulatory protein includes the cbbR encoding protein. Modifying the regulatory protein of the gene is to mutate cbbR so that the regulation mode of the cbbR encoding protein on the gene is changed, thereby increasing the expression of the gene.

[0074] In some embodiments of the invention, expression of the gene is increased by inactivating cbbR.

[0075] In the above (2), the transcriptional regulatory elements include promoters, terminators, enhancers, etc. The translational regulatory elements include ribosome binding sites, etc. The modified transcriptional regulatory elements and / or translational regulatory elements are to change the sequence of the regulatory elements responsible for the transcription and translation of the gene, for example: inserting other transcriptional regulatory elements and translational regulatory elements upstream of the cbbL gene coding region, or mutating the original transcriptional regulatory elements and translational regulatory elements (such as mutating the binding region sequence of cbbR in the promoter so that the promoter is no longer regulated by cbbR), or replacing the original transcriptional regulatory elements and translational regulatory elements with other transcriptional regulatory elements and translational regulatory elements, etc.

[0076] In some embodiments of the present invention, the wild-type promoter preceding cbbL is replaced with a constitutive promoter to increase the expression levels of the cbbL and cbbS genes.

[0077] In some embodiments of the present invention, the wild-type promoter preceding cbbL is replaced with a constitutive promoter, and the original chromosomal cbbR gene is inactivated.

[0078] The constitutive promoter is preferably a p52 promoter (SEQ ID NO. 5), a p53 promoter (p53 promoter is SEQ ID NO: 53 of patent CN108977890B) or a p68 promoter (p68 promoter is SEQ ID NO: 68 of patent CN108977890B).

[0079] In some embodiments of the present invention, the promoter is p52 promoter (SEQ ID NO. 5).

[0080] In the above (3), the expression of the gene can be increased by changing the sequence of the gene, or the activity of the protein encoded by the gene can be increased by changing the sequence of the gene.

[0081] In the above (4), increasing the copy number of the gene can be achieved by increasing the copy number of the gene on the chromosome and / or endogenous plasmid, or by introducing an exogenous plasmid containing the gene.

[0082] It should be understood that the present invention adopts the above means for exemplary illustration in specific embodiments. On the basis of the purpose of improving the expression and / or activity of the cbbL gene known to those skilled in the art, the use of other technical means that can also achieve the above purpose are equivalent variations of the technical means of the present invention and are therefore within the scope of protection of the present invention.

[0083] In an eighth aspect, the present invention provides the use of the above-mentioned recombinant engineered bacteria in the fermentation production of PHA.

[0084] Preferably, the application comprises the steps of culturing the recombinant engineered bacteria and collecting the culture containing PHA.

[0085] In some embodiments of the present invention, the culture is carried out using plant 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.

[0086] In other embodiments of the present invention, the culture is carried out using waste cooking oil as a carbon source.

[0087] The culture medium used in the above-mentioned culture may also contain nitrogen sources (including but not limited to ammonium salts, etc.), 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.).

[0088] In a ninth aspect, the present invention provides a method for producing PHA, comprising: culturing the recombinant engineered bacteria described above, and collecting the culture containing PHA.

[0089] In a tenth aspect, the present invention provides a method for increasing PHA production and / or substrate conversion rate of Eutropha rosea, the method comprising: modifying Eutropha rosea to reduce the expression and / or activity of H16_A3043 protein and / or H16_A3044 protein.

[0090] Preferably, the method further comprises: modifying the Eutropha roseae to enhance the expression and / or activity of the protein encoded by the cbbL gene.

[0091] The beneficial effects of the present invention include at least: the present invention discovered that reducing the expression and / or activity of the H16_A3043 protein and / or the H16_A3044 protein can significantly improve the PHA production performance of the strain, and the recombinant engineered bacteria constructed thereby have significantly improved PHA yield, substrate conversion rate, growth rate, etc., effectively reducing the cost of PHA industrial production and enhancing the competitiveness of PHA in the traditional plastics and bio-based degradable plastics markets and its commercial application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0092] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction is given below to 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 any creative work.

[0093] Figure 1 is a comparison of the mass conversion rates of PHA fermented by the gene-knockout recombinant engineered bacteria using palm oil as a carbon source in Example 1, wherein the abscissa represents the control strain H16 of Eutropha rosea and the gene-knockout modified test bacteria XX01, XX02, and XX03, and the ordinate represents the mass conversion rate. t-test statistical test: p<0.05, indicated by *; p<0.01, indicated by **; p<0.001, indicated by ***.

[0094] Figure 2 shows a comparison of the improvement in PHA production by the gene-knockout recombinant engineered bacteria compared to the control strain H16 in Example 1, wherein the horizontal axis represents the gene-knockout modified eutropha strains XX01, XX03, and XX02, and the vertical axis represents the relative proportion of the increase in PHA production; t-test statistical test: p<0.05, indicated by *; p<0.01, indicated by **; p<0.001, indicated by ***.

[0095] Figure 3 shows a comparison of the improvement in PHA production by the recombinant engineered bacteria with single-base mutations compared to the control strain H16 in Example 2, wherein the horizontal axis represents the test bacteria XX04 and XX05 of Roebuck's eutrophic bacteria modified with single-base mutations, and the vertical axis represents the relative proportion of the increase in PHA production; t-test statistical test: p < 0.05, indicated by *; p < 0.01, indicated by **; p < 0.001, indicated by ***.

[0096] Figure 4 shows a comparison of the growth curves of the recombinant engineered bacteria XX06 with H16_A3043 gene knockout and cbbL modification in Example 4 and the control strain H16. The growth curves in Figure 4 were obtained by using waste cooking oil as a carbon source for 24 hours of well plate growth, and measuring the OD value of the bacterial solution at an absorbance of 600 nm every 2 hours. The circular data point curve represents the control strain H16, and the square data point curve represents the recombinant strain XX06. t-test statistical test: p < 0.05, indicated by *; p < 0.01, indicated by **; p < 0.001, indicated by ***, and p < 0.0001, indicated by ****. DETAILED DESCRIPTION

[0097] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0098] Unless otherwise specified, the materials and reagents used in the following examples were commercially available. Enzyme reagents were purchased from New England Biolabs (NEB), plasmid extraction kits were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., and DNA fragment recovery kits were purchased from Omega Corporation (USA). All procedures were performed strictly according to the product specifications. All culture media were prepared with deionized water unless otherwise specified.

[0099] The culture medium formula used in the following examples is as follows:

[0100] Seed medium I: 10 g / L peptone, 5 g / L Yeast Extract, 3 g / L Fructose.

[0101] Seed medium II: 0.15% palm oil, 10 g / L peptone, 5 g / L Yeast Extract.

[0102] Production medium: 1.0% palm oil, 9.85 g / L Na₂HPO₄·12H₂O, 1.5 g / L KH₂PO₄, 3.0 g / L NH₄Cl, 10 mL / L trace element solution I, and 1 mL / L trace element solution II. Trace element solution I consists of 20 g / L MgSO₄ and 2 g / L CaCl₂. Trace element solution II consists of 100 mg / L ZnSO₄·7H₂O, 30 mg / L MnCl₂·4H₂O, 300 mg / L H₃BO₃, 200 mg / L CoCl₂·6H₂O, 10 mg / L CuSO₄·5H₂O, 20 mg / L NiCl₂·6H₂O, and 30 mg / L NaMoO₄·2H₂O. All reagents were purchased from Sinopharm Chemical Reagent Company.

[0103] The calculation formula for PHA yield described in the following examples is as follows:

[0104] PHA yield = CDW × PHA%; where CDW is the cell dry weight and PHA% is the percentage of PHA in the cell dry weight.

[0105] The calculation formula for the PHA yield improvement percentage described in the following examples is as follows: PHA yield improvement percentage = (PHA yield of the test strain - PHA yield of the control strain) / PHA yield of the control strain × 100%.

[0106] The calculation formula for the mass conversion rate of substrate to PHA described in the following examples is as follows:

[0107] Mass conversion rate = total amount of PHA produced by fermentation (g) / palm oil consumed by fermentation (g) × 100%.

[0108] Example 1: Construction and performance testing of recombinant engineering bacteria with H16_A3043 and H16_A3044 gene knockout

[0109] In this example, the H16 strain of Eutropha rothenbergii (abbreviated as H16) was used as the starting strain. The H16_A3043 and H16_A3044 genes on the genome were knocked out using gene editing methods commonly used in the field. The resulting double-gene knockout recombinant engineered bacteria was named Eutropha rothenbergii XX01; the H16-A3043 gene on the genome was knocked out, and the resulting single-gene knockout recombinant engineered bacteria was named Eutropha rothenbergii XX02; the H16-A3044 gene on the genome was knocked out, and the resulting single-gene knockout recombinant engineered bacteria was named Eutropha rothenbergii XX03. The strain performance of each recombinant engineered bacteria was further tested. The specific methods and results are as follows:

[0110] Step 1: Construction of H16_A3043 and H16_A3044 double gene knockout recombinant engineering bacteria XX01

[0111] 1.1 PCR amplification was performed using the genome of Eutropha rosenbergii H16 as a template to obtain the upstream homology arm A3043+A3044 A3043+A3044-H1 and the downstream homology arm A3043+A3044 A3043+A3044-H2 of A3043+A3044; the vector fragment was obtained by PCR amplification using the modified plasmid pK18mob (Orita I, Iwazawa R, Nakamura S, et al. Identification of mutation points in 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) as a template. A3043+A3044-H1 and A3043+A3044-H2 were ligated with the vector fragment using the Gibson Assembly method to generate the editing plasmid pKO-ΔA3043+A3044. Subcloning of the editing plasmid was completed by WuXi Biologics Co., Ltd. The sequences of the homology arms A3043+A3044-H1 and A3043+A3044-H2 are shown in SEQ ID NOs. 6 and 7, respectively.

[0112] 1.2 The recombinant plasmid pKO-ΔA3043+A3044 was transformed into Escherichia coli S17-1 and then into E. coli H16 by conjugation. Utilizing the property that the suicide plasmid cannot replicate in the host bacteria, positive clones were screened using LB plates containing 250 μg / mL kanamycin and 100 μg / mL apramycin. The recombinant plasmid carrying the homologous fragments in the positive clones was integrated into the specific locations of A3043-H1 and A3043-H2 in the genome, thereby obtaining the first homologous recombinant strain. The first homologous recombinant strain was streaked onto LB plates containing 100 mg / mL sucrose to culture single clones. From these single clones, clones without kanamycin resistance were screened, and PCR identification was performed. Sequencing confirmed that the recombinant strain was correctly edited, resulting in E. coli ReΔA3043+A3044 with double gene knockout of H16_A3043 and H16_A3044, designated XX01 in the present invention.

[0113] Step 2: Construction of H16_A3043 gene knockout recombinant engineering bacteria XX02

[0114] According to the homologous recombination method of step 1 in step 1.1 above, the homology arms A3043-H1 and A3043-H2 were designed, and the modified plasmid pK18mob was used as a template to obtain the vector fragment by PCR amplification. The homology arms were then connected to the vector fragment using the Gibson Assembly method to obtain the editing plasmid pKO-ΔA3043. The subcloning construction of the editing plasmid was completed by WuXi Biologics Co., Ltd. The sequences of the homology arms A3043-H1 and A3043-H2 are shown in SEQ ID NO.8 and SEQ ID NO.9. The correctly edited recombinant strain was obtained according to the conjugation transformation method of step 1 in step 1.2 above, and the H16_A3043 gene-knockout E. rothenbergii ReΔA3043 was obtained, which is named XX02 in the present invention.

[0115] Step 3: Construction of H16_A3044 gene knockout recombinant engineering bacteria XX03

[0116] According to the homologous recombination method of step 1 in step 1.1 above, the homology arms A3044-H1 and A3044-H2 were designed, and the modified plasmid pK18mob was used as a template to obtain the vector fragment by PCR amplification. The homology arms were then connected to the vector fragment using the Gibson Assembly method to obtain the editing plasmid pKO-ΔA3044. The subcloning construction of the editing plasmid was completed by WuXi Biologics Co., Ltd. The sequences of the homology arms A3044-H1 and A3044-H2 are shown in SEQ ID NO.10 and SEQ ID NO.11. The correctly edited recombinant strain was obtained by the conjugation transfer method of step 1 in step 1.2 above, and the H16_A3044 gene-knockout E. rothenbergii ReΔA3044 was obtained, which is named XX03 in the present invention.

[0117] Step 4: Performance test of PHA production by recombinant engineered bacteria

[0118] In this example, Eutropha rosea H16 was used as a control strain to test the fermentation performance of the recombinant engineered bacteria XX01, XX02, and XX03.

[0119] 4.1 Each strain constructed in Example 1, stored in a glycerol tube (1000 μL), was inoculated into seed culture medium I (20 mL) for 12 hours of primary seed culture. Then, 1 v / v% of seed culture solution I was inoculated into seed culture medium II (100 mL) for secondary seed culture, which was cultured for 13 hours. Then, 10 v / v% of seed culture solution II was inoculated into a 500 mL small fermenter (Dibil Company) containing 250 mL of production culture medium. The operating conditions were a culture temperature of 30°C, a stirring speed of 800 rpm, an aeration 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.

[0120] 4.2 Take the fermentation broth and centrifuge 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 25 mL of chloroform to the obtained dry bacterial cells and stir at room temperature for one day and one night to extract the polyester in the bacterial cells. After filtering out the bacterial residue, use an evaporator to concentrate to a total volume of approximately 7.5 mL. Then, slowly add approximately 22.5 mL of hexane and let it stand with slow stirring for 1 hour. After filtering out the precipitated polyester, vacuum dry it at 50°C for 3 hours. Measure the mass of the dried polyester and calculate the polyester content in the bacterial cells.

[0121] 4.3 Results As shown in Figures 1 and 2, the mass conversion efficiency of the starting strain H16 was 82.69%; XX01 was 89.66%; XX02 was 92.66%; and XX03 was 90.84%. Compared to the starting strain H16, PHA yields of XX01 increased by 19.53%, XX02 by 28%, and XX03 by 21.67%. This indicates that the increase in PHA yield in XX02 was significantly greater than that in XX03 and XX01.

[0122] Example 2: Construction and performance testing of H16_A3043 single base mutation recombinant engineering bacteria

[0123] In this example, the H16 (H16 for short) strain was used as the starting strain. The 170th base of the H16_A3043 gene coding region on the genome was mutated from T to C using common gene editing methods in the field, so that the leucine encoded at this site was mutated to proline (i.e., the 57th leucine was mutated to proline). The resulting single-base mutation recombinant engineered bacterium was H16_A3043 XX04; the 518th base of the H16-A3043 gene coding region on the genome was mutated from T to C, so that the leucine encoded at this site was changed to proline (i.e., the 173rd leucine was mutated to proline). The resulting single-base mutation recombinant engineered bacterium was H16_A3043 XX05; and the recombinant engineered bacteria were subjected to strain performance testing.

[0124] Step 1: Construction of recombinant engineered bacteria XX04 with H16_A3043-L57P single base mutation

[0125] The A3043-L57P homology arm was designed according to the homologous recombination method of step 1 in Example 1; the vector fragment was obtained by PCR amplification using the modified plasmid pK18mob as a template. The homology arm was then connected to the vector fragment using the Gibson Assembly method to obtain the editing plasmid pKO-ΔA3043-L57P. The subcloning construction of the editing plasmid was completed by WuXi Biologics Co., Ltd. The sequence of the homology arm A3043-L57P is shown in SEQ ID NO.12. The correctly edited recombinant strain was obtained according to the conjugation transfer method of step 1 in Example 1. The resulting recombinant engineered bacterium is Eutropha rothiae Re_A3043-L57P, which is named XX04 in the present invention.

[0126] Step 2: Construction of recombinant engineered bacteria XX05 with H16_A3043-L173P single base mutation

[0127] The A3043-L173P homology arm was designed according to the homologous recombination method of step 1 in Example 1; the vector fragment was obtained by PCR amplification using the modified plasmid pK18mob as a template. The homology arm was then connected to the vector fragment using the Gibson Assembly method to obtain the edited plasmid pKO-ΔA3043-L173P. The subcloning construction of the point mutation plasmid was completed by WuXi Biologics Co., Ltd. The sequence of the homology arm A3043-L173P is shown in SEQ ID NO.13. The correctly edited recombinant strain was obtained according to the conjugation transformation method of step 1 in Example 1. The resulting recombinant engineered bacterium is Eutropha roqueforti Re_A3043-L173P, which is named XX05 in the present invention.

[0128] Step 3: Performance test of PHA production by recombinant engineered bacteria

[0129] In this example, the fermentation performance of recombinant engineered strains XX04 and XX05 was tested, using E. rothrips H16 as a control strain. The specific steps were the same as steps 4.1 and 4.2 in Example 1. The results, shown in Figure 3, show a 28% increase in PHA yield for XX04 and a 17% increase for XX05 compared to the starting strain H16. This indicates that strain XX04 significantly outperformed strain XX05 in terms of PHA yield.

[0130] Example 3: Construction and performance testing of recombinant engineered bacteria with H16_A3043 gene knockout and cbbL and cbbS modifications

[0131] In this example, using E. rothenbergii XX02 as the starting strain, we silenced the cbbR gene using gene editing methods commonly used in the art. The wild-type promoter preceding cbbL was replaced with a medium-strength constitutive promoter (SEQ ID NO: 52 from patent CN108977890B, hereinafter referred to as p52); the sequence of this constitutive promoter p52 is shown in SEQ ID NO. 5. The resulting recombinant strain, H16_A3043 knockout and cbbL and cbbS modifications, was designated E. rothenbergii XX06, and strain performance testing was performed on the recombinant strain.

[0132] Step 1: Construction of H16_A3043 gene knockout superimposed cbbL and cbbS modified recombinant engineering bacteria XX06

[0133] The p52-cbbL-H1 and p52-cbbL-H2 homology arms were designed using the homologous recombination method described in step 1 of Example 1. The vector fragment was amplified by PCR using the modified plasmid pK18mob as a template. The homology arms were then ligated to the vector fragment using the Gibson Assembly method to generate the edited plasmid pKO-p52-cbbL. Subcloning of the edited plasmid was performed by WuXi Biologics Co., Ltd. The sequences of the homology arms p52-cbbL-H1 and p52-cbbL-H2 are shown in SEQ ID NOs. 14 and 15, respectively. A correctly edited recombinant strain was obtained using the conjugative transfer method described in step 1 of Example 1. The resulting recombinant strain is E. rhodesi ReΔA3043_ΔcbbR-p52-cbbL, designated XX06 herein. This recombinant strain silences the genes H16_A3043 and cbbR, and replaces the cbbL promoter with the moderate constitutive promoter p52.

[0134] Step 2: Performance test of PHA production by recombinant engineered bacteria with H16_A3043 gene knockout and cbbL and cbbS modification

[0135] In this example, the fermentation performance of recombinant strains XX01 and XX06 was tested using the Re 03 strain constructed in patent CN202310277052.2 (using H16 as the starting strain, silencing the cbbR gene, and replacing the wild-type promoter before cbbL with the medium-strength constitutive promoter p52) as a control.

[0136] 2.1 Each strain constructed in Example 1, stored in a glycerol tube (1000 μL), was inoculated into seed culture medium I (100 mL) for 12 hours of primary seed culture. Then, 10 v / v% of seed culture medium I was inoculated into seed culture medium II (50 L) for secondary seed culture, which was cultured for 13 hours. Then, 15 v / v% of seed culture medium II was inoculated into a 75 L medium-sized fermenter containing 50 L of production medium. The operating conditions were a culture temperature of 30°C, a stirring speed of 500 rpm, an aeration volume of 50 L / min, and a pH 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 48 hours.

[0137] 2.2 The PHA polyester content in the bacteria was detected according to the method in step 4.2 of Example 1, and the mass conversion rate of substrate to PHA was calculated. The results are shown in Table 1. The mass conversion rate of the test strain XX06 was 98.0%, which was much higher than the mass conversion rate of the control strain Re 03 (86.7%) and slightly higher than the mass conversion rate of the control strain XX01 (95.9%).

[0138] Table 1

[0139] Example 4: Performance testing of recombinant engineered bacteria using different carbon sources as substrates

[0140] In this example, E. rothripsii H16 was used as a control strain, and the recombinant strain XX06 constructed in Example 3 was used as an experimental strain to test the growth curves and fermentation performance of the recombinant engineered bacteria of the present invention under different biomass substrate conditions (waste cooking oil). The waste cooking oil was provided by Shangao Environmental Energy Group Co., Ltd. and was obtained from urban kitchen wastewater through three-phase separation. The quality control test data of the waste cooking oil were: moisture: 0.9% (test standard: GB 5009.236-2016), impurities: 0.1% (test standard: GB 5009.236-2016), acid value: 10.7 (mgKOH / g, test standard: ISO 660:2009), total sulfur: 56 mg / kg (test standard: ASTM D 5453-19a), total chlorine: 29 mg / kg (test standard: UOP 779-2008), iodine value: 101glz / 100g (test standard: GB / T5532), saponification value: 195mgKOH / g (test standard: GB / T5534), unsaponifiable matter: 0.81% (test standard: GB / T5535.1).

[0141] Step 1: Growth curve test of strain XX06 using waste cooking oil as the sole carbon source

[0142] The H16 strain preserved in the glycerol tube and the recombinant engineered bacteria XX06 of Example 3 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; then 10 v / v% of seed culture solution I was inoculated into seed culture medium II (2 mL) for secondary seed culture and cultured for 5 hours; then 15 v / v% of seed culture solution II was inoculated into a 24-deep-well plate containing 3 mL of production culture medium for micro-fermentation. The fermentation incubator temperature was 30°C and the speed was 450 rpm for continuous culture for 24 hours. During this period, samples were taken every 2 hours to test the absorbance value at 600 nm, and the growth OD of the strain at that time was calculated. The growth curve test in the present invention included 12 sampling time points, and 12 strain growth OD values ​​were obtained. The results are shown in FIG4 . Using waste cooking oil as a carbon source for micro-fermentation, the growth rate of the XX06 strain was significantly higher than that of the H16 strain. This indicates that XX06 has a significant growth advantage over H16.

[0143] Step 2: Performance test of PHA production by strain XX06 using waste cooking oil as the sole carbon source

[0144] In this example, the fermentation performance of the recombinant engineered strain XX06 was tested using E. rothrips H16 as a control strain and waste cooking oil as the sole carbon source. The specific steps were the same as steps 4.1 and 4.2 of Example 1 above, except that the 1.0% palm oil in the production medium was replaced with 1.0% waste cooking oil. The results, shown in Table 2, show that after full fermentation, the control strain H16 had a PHA percentage of 49.86% and a PHA yield of 36.04 g / L; the recombinant strain XX06 had a PHA percentage of 71.60% and a PHA yield of 101.76 g / L. Compared to the starting strain H16, the PHA percentage of XX06 increased by 43.6% and the PHA yield by 182.4%.

[0145] Table 2

[0146] These results demonstrate that the recombinant engineered strain XX06 constructed in the present invention exhibits faster growth, higher PHA production, and higher biomass substrate conversion efficiency compared to the original strain H16. This demonstrates that the inventive concept of the present invention is not limited by the specific choice of carbon source and can achieve higher PHA production and substrate conversion efficiency using common biomass carbon sources.

[0147] 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 various embodiments of the present invention. Industrial Applicability

[0148] The present invention provides a recombinant engineered bacterium for increasing polyhydroxyalkanoate (POHA) production and its application. The present invention discovered that reducing the expression and / or activity of the H16_A3043 protein and / or the H16_A3044 protein significantly improves the PHA production performance of PHA-producing bacteria. The recombinant engineered bacteria constructed in this manner significantly improve PHA yield, substrate conversion rate, and growth rate, effectively reducing the cost of PHA industrial production and enhancing PHA's competitiveness and commercial application value in the traditional plastics and biodegradable plastics markets. The bacteria possess excellent economic value and application prospects.

Claims

Application of reduced expression and / or activity of H16_A3043 protein or its homologous protein and / or H16_A3044 protein or its homologous protein in improving PHA production performance of PHA-producing bacteria.

2. The application according to claim 1, characterized in that, The PHA-producing bacteria are bacteria of the genus Ralstonia; Preferably, the PHA-producing bacteria are Ralstonia eutropha.

3. The application according to claim 1 or 2, characterized in that, The PHA production performance includes PHA yield and / or substrate conversion rate.

4. Mutants of the H16_A3043 protein, characterized in that, Compared with the H16_A3043 protein, the mutant contains a mutation in which the 57th amino acid is mutated to proline and / or the 173rd amino acid is mutated to proline.

5. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the mutant according to claim 4.

6. A biological material, characterized in that, The biological material contains the nucleic acid molecule according to claim 5 or expresses the mutant according to claim 4.

7. Recombinant engineering bacteria, characterized in that, The recombinant engineered bacteria are modified to reduce the expression and / or activity of H16_A3043 protein and / or H16_A3044 protein therein.

8. The recombinant engineered bacterium according to claim 7, characterized in that, The recombinant engineered bacteria contain any one of the following modifications: (1) Inactivation of H16_A3043 protein and / or H16_A3044 protein; (2) Non-expression of H16_A3043 protein and / or H16_A3044 protein; (3) Non-expression of H16_A3043 protein and expression of the mutant according to claim 4; Preferably, the recombinant engineered bacteria further contain the following modification: enhancing the expression and / or activity of the protein encoded by the Calvin cycle-related gene; The Calvin cycle-related gene includes cbbL.

9. Application of the recombinant engineered bacteria according to claim 7 or 8 in PHA fermentation production.

10. A method for increasing the PHA yield and / or conversion rate of PHA-producing bacteria, characterized in that, Reduce the expression and / or activity of the endogenous H16_A3043 protein and its homologous protein and / or H16_A3044 protein and its homologous protein of the PHA-producing bacteria.

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