CN117701488A - Recombinant bacterium for producing PHA and method for improving PHA yield - Google Patents

Recombinant bacterium for producing PHA and method for improving PHA yield Download PDF

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CN117701488A
CN117701488A CN202410161633.4A CN202410161633A CN117701488A CN 117701488 A CN117701488 A CN 117701488A CN 202410161633 A CN202410161633 A CN 202410161633A CN 117701488 A CN117701488 A CN 117701488A
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CN117701488B (en
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饶驰通
谭婷婷
蒋天宇
周小雪
范丁丁
李腾
张浩千
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Shanghai Blue Crystal Microbial Technology Co ltd
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Abstract

The invention relates to the technical field of microorganisms, in particular to a recombinant bacterium for producing PHA and a method for improving PHA yield. The present invention has found that the decrease or loss of the expression level and/or activity of the h16_b1231 gene or its encoded protein or the homologous gene of the h16_b1231 gene or its encoded protein can significantly improve the PHA productivity of PHA-producing bacteria. The discovery of the new function of the H16_B1231 gene provides a new transformation target point and strategy for constructing PHA producing bacteria, is beneficial to reducing the industrial production cost of PHA and further improves the commercial application value of PHA.

Description

Recombinant bacterium for producing PHA and method for improving PHA yield
Technical Field
The invention relates to the technical field of microorganisms, in particular to a recombinant bacterium for producing PHA and a method for improving PHA yield.
Background
Polyhydroxyalkanoates (PHAs) are intracellular carbon sources or energy storage substances accumulated by prokaryotic microorganisms, and belong to high molecular polyesters. The PHA can be completely degraded into carbon dioxide and water in natural environment, has thermoplasticity and biocompatibility, is more sustainable than the traditional plastic, and is an ideal traditional plastic substitute. However, the production costs of PHAs are still high compared to traditional plastics and other bio-based degradable plastics, limiting to some extent their commercial application. In order to reduce the production cost of PHA, it is necessary to further improve the productivity of PHA-producing bacteria.
Disclosure of Invention
The invention provides a recombinant bacterium for producing PHA and a method for improving PHA yield.
The current genetic engineering and optimization of PHA-producing bacteria has focused on PHA synthesis pathways, with less involvement in engineering the background genome of Chaetoceros. In the research and development process, the invention discovers that the expression quantity and/or activity of the H16_B1231 gene or the encoded protein thereof has obvious influence on the PHA production performance of bacteria, and the reduction of the expression quantity and/or activity of the H16_B1231 gene or the encoded protein thereof can not only obviously promote the growth of bacteria and improve the biomass thereof, but also obviously improve the PHA yield and the production strength of the bacteria. H16_b1231 is a putative protein, and no study about its function is reported.
Specifically, the invention provides the following technical scheme:
in a first aspect, the present invention provides a recombinant bacterium for producing PHA, which has reduced or lost expression level and/or activity of the H16_B1231 gene or its encoded protein or the homologous gene of the H16_B1231 gene or its encoded protein, and thus has improved PHA production performance.
The recombinant PHA-producing bacteria mentioned above are engineered bacteria capable of synthesizing and accumulating PHA.
In some embodiments of the present invention, a bacterium capable of synthesizing and accumulating PHA is used as a starting bacterium, which is modified such that the expression level and/or activity of the h16_b1231 gene or a protein encoded thereby or a homologous gene of the h16_b1231 gene or a protein encoded thereby is reduced or lost.
As for the starting strain, the present invention is not particularly limited as long as it is capable of synthesizing and accumulating PHA, and the decrease or loss of the expression level and/or activity of the H16_B1231 gene or its encoding protein or the homologous gene of the H16_B1231 gene or its encoding protein can in principle enhance its PHA productivity.
The bacterial species to which the recombinant bacteria belong is not particularly limited in principle as long as it is capable of synthesizing and accumulating PHA and contains the H16_B1231 gene or a homologous gene thereof, and includes, but is not limited to, bacteria of the genus Ralstonia (e.g., ralstonia rogowski), 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, yeast, and the like.
In some embodiments of the invention, the recombinant bacterium is a ralstonia bacterium. Preferably, the recombinant bacterium is a eutrophic bacterium of rochanterium.
The homologous gene described above is preferably a gene derived from a bacterium of the genus Ralstonia and having at least 70% homology with the amino acid sequence of the protein encoded by the H16_B1231 gene and the same function.
The above homology is 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%.
The reduction or loss of the expression amount and/or activity described above includes weakening the expression amount and/or activity of the gene or protein or causing the gene or protein not to be expressed or inactivated.
The means and technical means for achieving the reduction of the expression level and/or activity are not particularly limited, and for example, the protein, its encoding gene, its regulatory element and/or its regulatory gene or protein may be modified by a usual genetic engineering means and genetic editing method so that the expression level and/or activity of the gene or protein is reduced.
In some embodiments of the present invention, the reduction or loss of expression level and/or activity of the gene or protein is achieved by a combination of any one or more of the following (1) - (3):
(1) Mutating the amino acid sequence of the protein such that the expression level and/or activity of the gene or protein is reduced or lost;
(2) Mutating the nucleotide sequence of the gene so that the expression amount and/or activity of the gene or protein is reduced or lost;
(3) The transcriptional and/or translational regulatory elements of the gene are replaced with less active elements such that the expression level of the gene or protein is reduced.
Mutations in the amino acid sequences described above include deletions, insertions or substitutions of one or more amino acids.
Mutations in the nucleotide sequences described above include deletions, insertions or substitutions of one or more nucleotides.
The transcriptional and translational regulatory elements described above include promoters, ribosome binding sites, and the like.
In some embodiments of the invention, the reduced or lost expression of the gene or protein is achieved by inactivating the gene or protein. The inactivation may be achieved by knocking out the gene entirely or by knocking out a portion of its sequence.
In some embodiments of the invention, the h16_b1231 gene of the recombinant bacterium or a homologous gene thereof is knocked out.
It should be understood that, in the present invention, constructing PHA-producing recombinant bacteria by inactivating the h16_b1231 gene is merely illustrative, and other technical means that can achieve the above-mentioned purpose are equally modified in the technical means of the present invention on the basis that the person skilled in the art knows the purpose of reducing or losing the expression level and/or activity of the h16_b1231 gene or its encoding protein or the homologous gene of the h16_b1231 gene or its encoding protein, and therefore, all the technical means are within the scope of the present invention.
The invention realizes verification that the expression quantity and/or activity of the H16_B1231 gene or the coded protein thereof or the homologous gene of the H16_B1231 gene or the coded protein thereof of the PHA-producing strain is reduced or lost through modification, and the production performance of the obtained recombinant strain for producing PHA is obviously improved, and the invention is particularly characterized in that the growth rate of the strain is improved, the biomass is improved and the PHA yield and the production intensity are improved.
In a second aspect, the present invention provides a method for preparing the recombinant bacterium described above, the method comprising: the PHA-producing bacteria are modified such that the H16_B1231 gene or its encoded protein, or the homologous gene of the H16_B1231 gene or its encoded protein, is reduced or lost in expression and/or activity.
Preferably, the reduction or loss of the expression level and/or activity of the h16_b1231 gene or a protein encoded thereby, or the gene homologous to the h16_b1231 gene or a protein encoded thereby, is achieved by knocking out the h16_b1231 gene or a gene homologous thereto.
In a third aspect, the invention provides the use of the recombinant bacterium described above in PHA fermentation production.
Preferably, the application comprises: culturing the recombinant engineering bacteria, and collecting a culture containing PHA.
The culture can be carried out by using common substrates (including carbon sources, nitrogen sources and the like) for PHA production, wherein the carbon sources comprise biomass such as vegetable oil, kitchen waste oil, saccharides and the like, and the nitrogen sources comprise inorganic nitrogen sources such as ammonium salt and the like or organic nitrogen sources such as yeast powder, peptone and the like. 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 into the culture medium.
In some embodiments of the invention, the culturing is performed with a vegetable oil (including, but not limited to, palm oil, palm kernel oil, peanut oil, soybean oil, linseed oil, rapeseed oil, cottonseed oil, castor oil, a mixture of one or more of corn oils) as a carbon source.
In a fourth aspect, the present invention provides a method of improving PHA-producing performance of a PHA-producing bacterium, the method comprising: the PHA-producing bacterium is modified so that the expression level and/or activity of the H16_B1231 gene or its encoded protein, or the homologous gene of the H16_B1231 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, PHA production intensity.
In a fifth aspect, the present invention provides the use of the h16_b1231 gene or a protein encoded thereby, or a gene homologous to the h16_b1231 gene or a protein encoded thereby, for reducing or losing the expression level and/or activity of the gene or the protein encoded thereby, in improving the PHA-producing performance of a PHA-producing bacterium.
In the present invention, the PHA-producing bacteria are bacteria capable of synthesizing and accumulating PHA. PHA-producing bacteria include, but are not limited to, bacteria of the genus Rockwell (e.g., eutrophic bacteria), 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, yeast, and the like.
In some embodiments of the invention, the PHA-producing bacterium is a ralstonia bacterium. Preferably, the PHA-producing bacteria are Eutrophic bacteria.
The fungus eutrophic (Ralstonia eutropha, also called Cupriavidus necator) is an important model of bacterial research on PHA synthesis and is also a strain currently being studied for PHA industrial production. The eutrophic bacteria H16 and the derivative strains thereof can be used as a platform chassis to realize fed-batch fermentation with high cell density under industrial conditions, and biomass raw materials such as saccharides, grease and the like are used as substrates to produce molecular products including different types of PHAs. In the invention, the effect of the H16_B1231 protein and the encoding gene thereof on the growth rate, biomass, PHA yield and production intensity of the strain is verified by taking the eutrophic bacteria of Roche as an example in the specific embodiment. Based on the typical action of model bacteria on bacteria of the same genus and PHA-producing bacteria of other genus, those skilled in the art can infer that the above action is equally applicable to PHA-producing bacteria of other genus of the genus Ralstonia and PHA-producing bacteria of other genus which contain the homologous gene of the H16_B1231 gene and are capable of synthesizing and accumulating PHA.
In the invention, the H16_B1231 is derived from the eutrophic bacteria of Roche and is the locus_tag of the encoding gene of the protein in GenBank, and the sequences of the H16_B1231 protein and the encoding gene thereof can be obtained from the GenBank by a person skilled in the art.
Specifically, the amino acid sequence of the H16_B1231 is shown as SEQ ID NO.1, and the nucleotide sequence of the H16_B1231 gene is shown as SEQ ID NO. 2.
In the present invention, the gene homologous to the H16_B1231 gene is derived from bacteria of the genus Ralstonia, and the encoded protein thereof has at least 70% homology with the amino acid sequence of the encoded protein of the H16_B1231 gene and has the same function.
The above homology is 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%.
The PHA production performance is one or more selected from the group consisting of strain growth rate, biomass, PHA yield and PHA production intensity.
In such applications, reducing or losing the amount of expression and/or activity includes reducing the amount of expression and/or activity of the gene or protein, or alternatively, causing the gene or protein not to be expressed or inactivated.
The means and technical means for achieving the reduction of the expression level and/or activity are not particularly limited, and for example, the protein, its encoding gene, its regulatory element and/or its regulatory gene or protein may be modified by a usual genetic engineering means and genetic editing method so that the expression level and/or activity of the gene or protein is reduced.
In some embodiments of the present invention, the reduction in 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) - (3):
(1) Mutating the amino acid sequence of the protein such that the expression level and/or activity of the gene or protein is reduced or lost;
(2) Mutating the nucleotide sequence of the gene so that the expression amount and/or activity of the gene or protein is reduced or lost;
(3) The transcriptional and/or translational regulatory elements of the gene are replaced with less active elements such that the expression level of the gene or protein is reduced.
Mutations in the amino acid sequences described above include deletions, insertions or substitutions of one or more amino acids.
Mutations in the nucleotide sequences described above include deletions, insertions or substitutions of one or more nucleotides.
The transcriptional and translational regulatory elements described above include promoters, ribosome binding sites, and the like.
In some embodiments of the invention, the reduced or lost expression of the gene or protein is achieved by inactivating the gene or protein. The inactivation may be achieved by knocking out the gene entirely or by knocking out a portion of its sequence.
In some embodiments of the invention, the reduced expression level and/or activity or loss of the gene or protein is achieved by knocking out the h16_b1231 gene or a homologous gene thereof.
In a sixth aspect, the present invention provides a method for producing PHA, said method comprising: culturing the recombinant engineering bacteria, and collecting a culture containing PHA.
The beneficial effects of the invention at least comprise: the invention discovers that the reduction of the expression quantity and/or activity of the H16_B1231 gene or the encoded protein thereof can obviously improve the PHA production performance of PHA producing bacteria, and the growth rate and the biomass of recombinant engineering bacteria constructed by reducing the expression quantity and/or activity of the H16_B1231 gene or the encoded protein thereof can also obviously improve the yield and the production intensity of PHA. The discovery of the new function of the H16_B1231 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.
Drawings
In order to more clearly illustrate the invention or the technical solutions of the prior art, the following description will briefly explain the drawings used in the embodiments or the description of the prior art, and it is obvious that the drawings in the following description are some embodiments of the invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a comparison of growth curves of recombinant strain W02 and 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 W02; t-test statistical test: p is p<0.05 toA representation; p is p<0.01, in->A representation; p is p<0.001, in->Representing p<0.0001, in->And (3) representing.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The kit for extracting the plasmids is purchased from Tiangen Biochemical technology (Beijing) limited company, the corresponding operation steps are strictly carried out according to the specifications of the products, and all culture media are prepared by deionized water if no special description exists.
The medium formulation used in the following examples was as follows:
seed culture medium i: 10g/L Peptone (Peptone), 5g/L Yeast Extract (Yeast Extract), 3g/L Fructose (Fructose).
Seed medium II: 0.15% palm oil, 10g/L Peptone (Peptone), 5g/L Yeast Extract (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. Wherein the trace element solution I comprises the following components: 20g/L MgSO 4 ，2g/L CaCl 2 . The trace element solution II comprises the following components: 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 national drug group chemical reagent company.
The experimental data in the following examples were obtained from 3 or more parallel sets of experiments.
The calculation formula for the average growth rate described in the following examples is as follows:
average growth rate = final OD/growth time; wherein the final OD is the OD of the strain detected at 600nm absorbance at the end of the strain culture.
The PHA content (PHA%) described in the examples below is the mass percent of PHA based on the dry weight of the cells.
The calculation formula for PHA yields described in the examples below is as follows:
PHA yield = cdw×pha; wherein CDW is the dry weight of the cells, and PHA% is the percentage of PHA in the dry weight of the cells.
The calculation formula of the production intensity described in the following examples is as follows:
production intensity = PHA yield/time; wherein PHA production was calculated as described above, for a total fermentation run time of 54h.
Example 1: construction and growth test of eutrophic bacteria Re delta B1231 of Roche
In this embodiment, the H16 strain of eutrophic rochanterium H16 (abbreviated as H16) is used as an initial strain, the h16_b1231 gene on the genome is knocked out by using a gene editing method commonly used in the art, the obtained gene knocked-out recombinant strain is eutrophic rochanterium Re Δb1231, and is named as recombinant strain W02, and the recombinant strain is subjected to growth test.
Step 1: construction of H16B 1231 Gene knockout recombinant Strain W02
1.1 PCR amplification is carried out by taking a genome of the eutrophic bacteria H16 of Roche as a template to obtain an H16-B1231 upstream homologous arm B1231-H1 and an H1231 downstream homologous arm B1231-H2; the vector fragment was amplified by PCR 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. The B1231-H1 and B1231-H2 were ligated to the vector fragment by Gibson Assembly method to obtain the edited plasmid pKO-. DELTA.B1231, and subcloning of the edited plasmid was completed by Ministry of Biotechnology. Wherein the sequences of homologous arms B1231-H1 and B1231-H2 are shown as SEQ ID NO.3 and SEQ ID NO. 4.
1.2 The recombinant plasmid pKO-delta B1231 is transformed into escherichia coli S17-1, and then transferred into the eutrophic rochanterium H16 by a conjugation transfer method, and positive clones are screened by using an LB plate simultaneously containing 250 mug/mL kanamycin and 100 mug/mL apramycin by utilizing the characteristic that the suicide plasmid cannot replicate in host bacteria. The recombinant plasmid carrying the homologous fragment in the positive clone is integrated into the specific position of the genome, thereby obtaining the first homologous recombinant strain. The first homologous recombinant strain is subjected to monoclonal culture on an LB plate containing 100mg/mL sucrose, clones which do not have kanamycin resistance are selected from the monoclonal strains, PCR identification is carried out, sequencing is carried out to confirm that the recombinant strain is correctly edited, and the obtained recombinant strain is the eutrophic rocarpium Re delta B1231，Designated as W02.
Step 2: w02 strain growth test with palm oil as sole carbon source
And (3) carrying out LB plate streaking on the control strain H16 and the recombinant strain W02 stored in the glycerol pipe, obtaining a monoclonal, and carrying out subsequent seed culture and fermentation culture by using a 24 deep hole plate. Inoculating the monoclonal into a seed culture medium I (2 mL), and performing seed primary culture for 15 hours to obtain a seed culture solution I; then inoculating the seed culture solution I into a seed culture medium II (2 mL) according to the inoculum size of 10% (v/v), and carrying out secondary seed culture for 5h to obtain a seed culture solution II; then inoculating the seed culture solution II into a 24-deep hole plate filled with 3mL of production culture medium according to the inoculum size of 15% (v/v), carrying out micro-fermentation, culturing for 24 hours at the temperature of a culture box of 30 ℃ at the rotating speed of 450rpm, sampling and testing absorbance value of 600nm every 2 hours during the culture, and calculating to obtain the growth OD of the strain at the time. For a total of 10 sampling time points, 10 strain growth OD were obtained, and a growth curve was prepared, and the results are shown in fig. 1. The results show that the recombinant strain W02 can reach a higher OD turbidity at 24 hours growth cycle than the control strain H16, and the average growth rate is also higher than that of the control strain H16.
Example 2: fermentation performance test of eutrophic bacteria Re delta B1231
In the embodiment, the performance test of shake flask fermentation and fermenter fermentation of the eutrophic rogowski bacterium Re delta B1231 (recombinant strain W02) is performed by taking the eutrophic rogowski bacterium H16 (H16 for short) as a control strain and palm oil as a unique carbon source.
Step 1: performance test of PHA production by shake flask fermentation of recombinant strain W02
1.1 The control strain H16 stored in glycerol and the recombinant strain W02 obtained in step 1 of example 1 were streaked on LB plates to obtain monoclonal antibodies. Inoculating the monoclonal into a seed culture medium I (3 mL), and performing seed activation for 15 hours to obtain a seed activation solution; then inoculating the seed activating solution into a seed culture medium I (10 mL) according to the inoculum size of 10% (v/v), and performing primary seed culture for 8h to obtain a seed culture solution I; then inoculating the seed culture solution I into a seed culture medium II (15 mL) according to the inoculum size of 10% (v/v) to perform secondary seed culture for 15h to obtain a seed culture solution II; then, the seed culture solution II was inoculated in an inoculum size of 15% (v/v) into a 250mL Erlenmeyer flask containing 30mL of the production medium, and the fermentation incubator was continuously cultured at 30℃and 220rpm for 24 hours.
1.2 And centrifuging the fermentation liquor to obtain thalli. And drying the thalli to constant weight. The weight of the dried cells was measured and recorded as dry weight. To the dried cells obtained, 3.3mL of chloroform was added, and the mixture was stirred at room temperature for one day and night to extract the polyester in the cells. After the cell residue was filtered off, the mixture was concentrated to a total volume of about 1mL by an evaporator, and then about 3mL of hexane was slowly added thereto, followed by standing under slow stirring for 1 hour. The precipitated polyester was filtered off and dried in vacuo for 3 hours. The mass of the dried polyester was measured, and the polyester content in the cells was calculated.
The results are shown in table 1, with a significant increase in dry weight of 6.02%, 4.21% in PHA% and 10.53% in PHA yield compared to control strain H16.
TABLE 1
Step 2: performance test of PHA production by fermentation of recombinant strain W02 fermentation tank
2.1 Inoculating the recombinant strain W02 constructed in the embodiment 1 and the control strain H16 (1000 mu L) stored in an glycerol pipe into a seed culture medium I (20 mL) respectively, and carrying out seed primary culture for 12 hours to obtain a seed culture solution I; then, inoculating the seed culture solution I into a seed culture medium II (100 mL) according to the inoculum size of 1% (v/v), and culturing the seeds for 13h to obtain a seed culture solution II; then inoculating the seed culture solution II into a 500mL small-sized fermentation tank filled with 250mL of production culture medium according to the inoculation amount of 10% (v/v); the operation conditions are that the culture temperature is 30 ℃, the stirring speed is 800rpm, the aeration rate is 1L/min, and the pH is controlled to be 6.7-6.8. 28% aqueous ammonia was used for pH control. During the cultivation, palm oil was continuously used as a carbon source for 54 hours.
2.2 And centrifuging the fermentation liquor to obtain thalli. And drying the thalli to constant weight. The weight of the dried cells was measured and recorded as dry weight. To the dried cells thus obtained, 25mL of chloroform was added, and the mixture was stirred at room temperature for one day and night to extract the polyester in the cells. After the cell residue was filtered off, the mixture was concentrated to a total volume of about 7.5mL by an evaporator, and then about 22.5mL of hexane was slowly added thereto, followed by standing under slow stirring for 1 hour. The precipitated polyester was filtered off and dried in vacuo for 3 hours. The mass of the dried polyester was measured, and the polyester content in the cells was calculated.
As a result, the following 3 strains of recombinant strain W02 all had better performance indicators than the control strain H16, as shown in Table 2. Compared with the control strain H16, the dry weight of the W02 strain is obviously improved by 8.59%, the PHA yield is obviously improved by 9.72%, and the production strength is obviously improved by 9.85%.
TABLE 2
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (10)

1. A recombinant bacterium for producing PHA, characterized in that the expression level of the h16_b1231 gene or its homologous gene of the recombinant bacterium is reduced or lost, and the PHA productivity is improved;
the recombinant bacteria are bacteria of the genus Ralstonia.
2. The recombinant bacterium according to claim 1, wherein the homologous gene is a gene derived from a bacterium of the genus ralstonia and having at least 70% homology with the amino acid sequence of the protein encoded by the h16_b1231 gene and the same function.
3. The recombinant bacterium according to claim 1 or 2, wherein the h16_b1231 gene or a homologous gene thereof of the recombinant bacterium is knocked out.
4. A method for producing a recombinant bacterium according to any one of claims 1 to 3, comprising: PHA-producing bacteria are modified so that the amount of H16_B1231 gene or its homologous gene expressed is reduced or lost.
5. The method according to claim 4, wherein the decrease or loss of the expression level of the H16_B1231 gene or its homologous gene is achieved by knocking out the H16_B1231 gene or its homologous gene.
6. Use of the recombinant bacterium of any one of claims 1-3 in PHA fermentation production.
7. A method of improving PHA-producing performance of a PHA-producing bacterium, the method comprising: the PHA-producing bacterium is modified so that the expression level of its H16_B1231 gene or its homologous gene is reduced or lost.
8. The method of claim 7, wherein the PHA production performance is selected from one or more of strain growth rate, biomass, PHA yield, PHA production intensity.
Use of the h16_b1231 gene or its homologous gene to reduce or eliminate its expression level for improving PHA productivity of a PHA-producing bacterium.
10. The use according to claim 9, characterized in that said PHA-producing bacteria are bacteria of the genus ralstonia.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200087687A1 (en) * 2017-05-25 2020-03-19 Kaneka Corporation Pha-producing microorganism in which glycerol kinase activity is enhanced, and pha production method using same
CN114381415A (en) * 2022-03-22 2022-04-22 深圳蓝晶生物科技有限公司 Gene recombination strain for high-yield PHA and construction method thereof
CN115976088A (en) * 2022-07-21 2023-04-18 深圳蓝晶生物科技有限公司 Low endotoxin-containing fungus Roche and application thereof
WO2023193353A1 (en) * 2022-04-06 2023-10-12 深圳蓝晶生物科技有限公司 Engineered microorganism expressing acetoacetyl coenzyme a reductase variant and method for increasing pha yield

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200087687A1 (en) * 2017-05-25 2020-03-19 Kaneka Corporation Pha-producing microorganism in which glycerol kinase activity is enhanced, and pha production method using same
CN114381415A (en) * 2022-03-22 2022-04-22 深圳蓝晶生物科技有限公司 Gene recombination strain for high-yield PHA and construction method thereof
WO2023193353A1 (en) * 2022-04-06 2023-10-12 深圳蓝晶生物科技有限公司 Engineered microorganism expressing acetoacetyl coenzyme a reductase variant and method for increasing pha yield
CN115976088A (en) * 2022-07-21 2023-04-18 深圳蓝晶生物科技有限公司 Low endotoxin-containing fungus Roche and application thereof
WO2024017318A1 (en) * 2022-07-21 2024-01-25 深圳蓝晶生物科技有限公司 Ralstonia eutropha having low endotoxin content, and use thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
MENGYI LI等: "Engineering Pseudomonas entomophila for synthesis of copolymers with defined fractions of 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoates", METAB ENG, vol. 52, 21 December 2018 (2018-12-21), pages 253 - 262 *
SHAO-PING OUYANG: "Macromol Biosci", CONSTRUCTION OF PHA-OPERON-DEFINED KNOCKOUT MUTANTS OF PSEUDOMONAS PUTIDA KT2442 AND THEIR APPLICATIONS IN POLY(HYDROXYALKANOATE) PRODUCTION, vol. 07, no. 02, 12 February 2007 (2007-02-12), pages 227 - 233 *
李正军等: "生产聚羟基脂肪酸酯的微生物细胞工厂", 生物工程学报, no. 10, 25 October 2010 (2010-10-25), pages 109 - 118 *
王露等: "重组菌Ralstonia eutropha W50-EAB D-木糖代谢相关的限速靶点", 微生物学报, no. 02, 4 February 2015 (2015-02-04), pages 49 - 60 *

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