CN117701486B - Recombinant bacterium for producing PHA and construction method and application thereof - Google Patents
Recombinant bacterium for producing PHA and construction method and application thereof Download PDFInfo
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
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
Technical Field
The invention relates to the technical field of microorganisms, in particular to recombinant bacteria for producing PHA, and a construction method and application thereof.
Background
Polyhydroxyalkanoate (PHA) is a high molecular polyester synthesized by microorganisms, has good biodegradability, and can replace traditional plastics in various application scenes. 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. Since the PHA productivity of PHA-producing bacteria is a key factor affecting the PHA production cost, it is necessary to improve the productivity such as the PHA yield and the production strength of PHA-producing bacteria in order to reduce the PHA production cost.
Disclosure of Invention
The invention provides a recombinant bacterium for producing PHA, a construction method and application thereof
The genetic modification and optimization of PHA-producing bacteria in the prior art have focused on PHA synthesis pathways, and are less involved in the modification of the background genome of Chaetomium. In the research and development process, the invention discovers that the expression quantity and/or activity of the bacterial Pel polysaccharide synthesis gene and the encoded protein thereof have obvious influence on the PHA production performance of bacteria, and the reduction of the expression quantity and/or activity of the Pel polysaccharide synthesis gene and the encoded protein thereof not only can obviously promote the growth of bacteria and improve the biomass thereof, but also can obviously improve the PHA yield and the production strength of the bacteria.
The Pel polysaccharide is a bacterial polysaccharide rich in N-acetylgalactosamine (GalNAc), and researches show that the Pel polysaccharide is related to the structure and the function of a pseudomonas aeruginosa biological film. The Pel polysaccharide synthesis genes include pelA, pelB, pelC, pelD, pelE, pelF, pelG, which exist in the bacterial genome in the form of the pelABCDEFG operon, which is involved in the synthesis of extracellular polysaccharides associated with biofilm development both early and late in biofilm development, resulting in a glucose-rich biofilm matrix. pelABCDEFG the operon is reported in bacteria of the genus copper (Cupriavidus), pseudomonas (Pseudomonas), etc., and PelABCDEFG the operon is highly conserved among different bacterial species, so Pel polysaccharide may be a biofilm determinant widely present in bacteria. Patent application CN108060111a discloses that the variation of the expression level of pelABCDEFG is related to the yield of rhamnolipid in pseudomonas, specifically as follows: the patent application discloses a pseudomonas aeruginosa for improving the yield of rhamnolipid and a construction method thereof, wherein the gene expression quantity in group A of the strain is reduced or lost and the gene expression quantity in group B is increased; the genes in the A group are selected from one or a combination of a plurality of genes in pslABCDEFGHIJKLMNO extracellular polysaccharide synthesis gene clusters, pelABCDEFG polysaccharide synthesis gene clusters, alginate synthesis gene clusters algD-alg8-alg44-algKEGXLIJF and polyhydroxyalkanoate PHA synthesis key gene clusters phaC 1-D-C2; the genes in the B group are selected from one gene or a combination of any several genes in rmlACBD, rmlBDAC, rhlYZ, algC, fadD, lipC and estA. The strain has the advantages of remarkably improving the yield of rhamnolipid, stabilizing inheritance, being beneficial to the separation and purification of downstream products and the like. However, there are no reports of the association of Pel polysaccharide synthesis genes with bacterial growth rate and PHA production.
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 Pel polysaccharide synthesis gene or the encoded protein thereof, 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 invention, the bacterium capable of synthesizing and accumulating PHA is used as a starting bacterium, and the starting bacterium is modified so that the expression level and/or activity of the Pel polysaccharide synthesis gene or the 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 contains therein the Pel polysaccharide synthesis gene, and reducing or losing the expression level and/or activity of the Pel polysaccharide synthesis gene or the encoded protein thereof can in principle enhance the PHA production performance thereof.
The Pel polysaccharide synthesis genes are one or more selected from pelA, pelB, pelC, pelD, pelE, pelF, pelG.
Specifically, the Pel polysaccharide synthesis genes are any one or a combination of any 2-7 of pelA, pelB, pelC, pelD, pelE, pelF, pelG.
In some embodiments of the 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 genes in pelABCDEFG polysaccharide synthesis gene clusters, i.e. one or more essential genes on the Pel polysaccharide synthesis pathway are subjected to expression intervention, so that the Pel polysaccharide synthesis pathway is regulated.
As an optimal solution, in some embodiments of the present invention, the Pel polysaccharide synthesis genes are pelABCDEFG polysaccharide synthesis gene clusters, i.e. the expression intervention is performed on 7 essential genes on the Pel polysaccharide synthesis pathway at the same time.
Such a reduction or loss of expression level and/or activity includes weakening the expression level 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 or loss 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 or lost.
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 pelA, pelB, pelC, pelD, pelE, pelF and pelG genes of the recombinant bacterium or their encoded proteins are inactivated.
In some embodiments of the invention, the pelABCDEFG polysaccharide synthesis gene cluster of the recombinant bacterium is knocked out.
It should be understood that, in the specific embodiment, the construction of the PHA-producing recombinant bacteria by inactivating pelA, pelB, pelC, pelD, pelE, pelF and pelG genes is only exemplified, and on the basis that the purpose of reducing the expression level and/or activity of the Pel polysaccharide synthesis gene or the encoded protein thereof is known to those skilled in the art, other technical means capable of achieving the above purpose are adopted, and all the technical means belong to equivalent variants of the technical means of the present invention, so all the technical means are included in the protection scope of the present invention.
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 therein Pel polysaccharide synthesis genes, including but not limited to, bacteria of the genus Ralstonia (e.g., pseudomonas stutzeri), 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), halophilum, yeast, and the like.
In some embodiments of the invention, the recombinant bacterium is a bacteria of the genus ralstonia or pseudomonas. Preferably, the recombinant bacterium is eutrophic bacterium, pseudomonas putida or pseudomonas aeruginosa.
Experiments prove that the PHA-producing recombinant bacterium obtained by modifying and reducing or losing the expression quantity and/or activity of the Pel polysaccharide synthetic gene or the encoding protein of the PHA-producing bacterium has obviously improved production performance, and is particularly characterized by improved growth rate, biomass and PHA yield and production intensity of the strain.
In a second aspect, the present invention provides a method for preparing the recombinant bacterium described above, the method comprising: PHA-producing bacteria are modified such that their Pel polysaccharide synthesis genes or their encoded proteins are reduced or lost in expression and/or activity.
Preferably, the reduction or loss of expression level and/or activity of the Pel polysaccharide synthesis gene or the encoded protein thereof is achieved by knocking out pelABCDEFG polysaccharide synthesis gene cluster.
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 strain, 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: PHA-producing bacteria are modified so that the expression level and/or activity of the Pel polysaccharide synthesis gene or the protein encoded thereby 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 a Pel polysaccharide synthesis gene or a protein encoded thereby, the expression level and/or activity of which is reduced or lost, for improving the PHA production performance of PHA-producing bacteria.
In the invention, the Pel polysaccharide synthesis gene is one or more selected from pelA, pelB, pelC, pelD, pelE, pelF, pelG.
The reduction of the expression level and/or activity of one or more of the Pel polysaccharide synthesis genes pelA, pelB, pelC, pelD, pelE, pelF, pelG or the encoded proteins thereof affects the synthesis of Pel polysaccharide and thus the productivity of PHA-producing bacteria. Based on this, the Pel polysaccharide synthesis genes described in the present invention may be selected from one or more of pelA, pelB, pelC, pelD, pelE, pelF, pelG.
Specifically, the Pel polysaccharide synthesis genes are any one or a combination of any 2-7 of pelA, pelB, pelC, pelD, pelE, pelF, pelG.
The pelA, pelB, pelC, pelD, pelE, pelF, pelG genes in bacteria are often present in gene clusters. Therefore, the Pel polysaccharide synthesis gene is part of or all of the genes in pelABCDEFG polysaccharide synthesis gene clusters.
In some embodiments of the invention, the Pel polysaccharide synthesis genes are pelA, pelB, pelC, pelD, pelE, pelF and pelG.
In some embodiments of the invention, the Pel polysaccharide synthesis gene is pelABCDEFG polysaccharide synthesis gene cluster.
For the Pel polysaccharide synthesis gene pelA, pelB, pelC, pelD, pelE, pelF, pelG and its coding protein sequences in bacteria, those skilled in the art can obtain it from Genbank et al databases.
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 or a pseudomonas bacterium. Preferably, the PHA-producing bacteria are Eutrophic bacteria, pseudomonas putida or Pseudomonas aeruginosa.
The fungus Eutrophic Roche (Ralstonia eutropha, also known as Cupriavidus necator) is an important model of bacterial research on PHA synthesis, and is also a strain currently being studied more 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 real bacteria of Roche are taken as an example in the specific embodiment, and the effect of the Pel polysaccharide synthesis genes on the growth rate, biomass, PHA yield and production intensity of the strain is verified. 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 Ralstonia and PHA-producing bacteria of other genus which contain the Pel polysaccharide synthesis gene and are capable of synthesizing and accumulating PHA.
In some embodiments of the invention, the PHA-producing bacteria is eutrophic rochanterium. For the eutrophic bacteria of Roche, genBank locus_tag of the eutrophic bacteria gene corresponding to Pel polysaccharide synthesis gene pelA, pelB, pelC, pelD, pelE, pelF, pelG is H16_B2198-B2204, specifically, H16_B2198 is pelG (the amino acid sequence of the protein is shown as SEQ ID NO.1, the gene sequence is shown as SEQ ID NO. 2), H16_B2199 is pelF (the amino acid sequence of the protein is shown as SEQ ID NO.3, the gene sequence is shown as SEQ ID NO. 4), H16_B2200 is pelE (the amino acid sequence of the protein is shown as SEQ ID NO.5, the gene sequence is shown as SEQ ID NO. 6), H16_B2201 is pelD (the amino acid sequence of the protein is shown as SEQ ID NO.7, the gene sequence is shown as SEQ ID NO. 8), H16_B2202 is pelC (the amino acid sequence of the protein is shown as SEQ ID NO.9, the gene sequence is shown as SEQ ID NO. 10), H16_B2203 is pelB (the amino acid sequence of the protein is shown as SEQ ID NO.11, the gene sequence is shown as SEQ ID NO. 12), and H16_B2204 is pelA (the amino acid sequence of the protein is shown as SEQ ID NO.13, and the gene sequence is shown as SEQ ID NO. 14).
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 or lost.
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 a sixth aspect, the present invention provides a method for producing PHA, said method comprising: culturing the recombinant strain described above, and collecting the culture containing PHA.
The beneficial effects of the invention at least comprise: the invention discovers that although the Pel polysaccharide synthesis pathway and the PHA synthesis pathway do not have the same substrate or intermediate metabolite, the expression quantity and/or activity of the Pel polysaccharide synthesis gene or the coded protein thereof are reduced, but the PHA production performance of PHA producing bacteria can be obviously improved, the growth rate and biomass of recombinant bacteria constructed by reducing or losing the expression quantity and/or activity of the Pel polysaccharide synthesis gene or the coded protein thereof are obviously improved, and the yield and the production intensity 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.
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 W01 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 W01; t-test statistical test: p <0.05 toA representation; p <0.01, to/>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 Na2HPO4·12H2O,1.5g/L KH2PO4,3.0g/L NH4Cl,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 CaCl2. The composition of the trace element solution II is ：100mg/L ZnSO4·7H2O,30mg/L MnCl2·4H2O,300mg/L H3BO3,200mg/L CoCl2·6H2O,10mg/L CuSO4·5H2O,20mg/L NiCl2·6H2O,30mg/L NaMoO4·2H2O. the above reagents are all purchased from national pharmaceutical 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 B2198-B2204 of Ralstonia
In this embodiment, H16-eutrophic strain (abbreviated as H16) is used as an initial strain, and 7 genes of h16_b2198, h16_b2199, h16_b2200, h16_b2201, h16_b2202, h16_b2203 and h16_b2204 on the genome are knocked out by using a common gene editing method in the field, and the obtained multi-gene knocked-out recombinant strain is eutrophic strain W01, and strain growth curve test is performed on the recombinant strain. The specific method and the results are as follows:
step 1: construction of H16-B2198-B2204 multiple-gene knockout recombinant strain W01
1.1 Performing PCR amplification by taking genome of the eutrophic Rogowski bacterium H16 as a template to obtain upstream homology arms B2198-B2204-H1 of B2198-B2204 and downstream homology arms B2198-B2204-H2 of B2198-B2204; 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) is used as a template, and a carrier fragment is obtained through PCR amplification. B2198-B2204-H1 and B2198-B2204-H2 are connected with the vector segment by a Gibson Assembly method to obtain editing plasmids pKO-delta B2198-B2204, and subcloning construction of the editing plasmids is completed by the pharmaceutical and biological company. Wherein the sequences of the homology arms B2198-B2204-H1 and B2198-B2204-H2 are respectively shown as SEQ ID NO.15 and SEQ ID NO. 16.
1.2 The recombinant plasmid pKO-delta B2198-B2204 is transformed into escherichia coli S17-1, and then transferred into the eutrophic rochanterium H16 by a joint transformation method, and positive clones are screened out by using an LB plate simultaneously containing 250 mug/mL kanamycin and 100 mug/mL apramycin by utilizing the characteristic that suicide plasmids cannot replicate in host bacteria. The recombinant plasmid carrying the homologous fragment in the positive clone is integrated into the genome at a specific position, thereby obtaining a 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, correct editing of the recombinant strain is confirmed, the obtained recombinant strain is a recombinant strain W01, wherein 7 genes of H16B 2198, H16B 2199, H16B 2200, H16B 2201, H16B 2202, H16B 2203 and H16B 2204 are knocked out.
Step 2: w01 strain growth test by using palm oil as sole carbon source
And (3) carrying out LB plate streaking on the recombinant strains H16 and W01 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, continuously culturing for 24 hours at the temperature of a fermentation incubator of 30 ℃ and the rotating speed of 450rpm, sampling and testing the absorbance value of 600nm every 2 hours during the continuous culture, and calculating to obtain the growth OD of the strain at the time. The total of 10 sampling time points for the growth test, 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 W01 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 B2198-B2204
In the embodiment, the production performance test of shake flask fermentation and fermenter fermentation of the eutrophic rogowski bacteria Re delta B2198-B2204 (W01 strain) is carried out by taking the eutrophic rogowski bacteria H16 (H16 for short) as a control strain and palm oil as a unique carbon source.
Step 1: performance test of recombinant strain W01 in shake flask fermentation production of PHA
1.1 The control strain H16 stored in glycerol and the recombinant strain W01 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 an activated seed liquid; then inoculating the activated seed 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. 3.3 mL chloroform was added to the dried cells obtained, 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, it was concentrated to a total volume of about 1 mL by an evaporator, and hexane of about 3: 3 mL was slowly added thereto, and left for 1 hour with slow stirring. 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 8.3% increase in dry weight of the W01 strain, a significant 4.8% increase in PHA% and a significant 13.6% increase in PHA production compared to the control strain H16.
TABLE 1
Step 2: performance test of recombinant strain W01 reactor for fermentation production of PHA
2.1 Inoculating the recombinant strain W01 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; seed culture II was then inoculated at an inoculum size of 10% (v/v) into a 500mL mini-fermentor containing 250mL of production medium. The operating conditions were a culture temperature of 30℃and a stirring speed of 800rpm, an aeration rate of 1L/min, and the pH was controlled to be between 6.7 and 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.
The results are shown in Table 2, the following 3 PHA production performance indicators of recombinant strain W01 are all significantly better than control strain H16. Compared with the control strain H16, the dry weight of the recombinant strain W01 is obviously improved by 10.02 percent, the PHA yield is obviously improved by 11.77 percent, and the production intensity is obviously improved by 11.74 percent.
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 (7)
1. Recombinant strain 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 strain are knocked out, whereby the PHA production performance is improved;
The original strain of the recombinant bacterium is eutrophic bacterium H16.
2. The method for producing a recombinant bacterium according to claim 1, wherein the method comprises: the eutrophic roller H16 was modified 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 bacterium of claim 1 in PHA fermentation production.
4. A method of improving PHA-producing performance of a PHA-producing bacterium, the method comprising: modification of PHA-producing bacteria to knock out the H16_B2198, H16_B2199, H16_B2200, H16_B2201, H16_B2202, H16_B2203 and H16_B2204 genes;
the PHA producing strain is Eutrophic bacteria H16.
5. The method of claim 4, wherein the PHA production performance is PHA yield and/or PHA production intensity.
Use of knockout of h16_b2198, h16_b2199, h16_b2200, h16_b2201, h16_b2202, h16_b2203, and h16_b2204 genes to improve PHA-producing performance of PHA-producing bacteria;
the PHA producing strain is Eutrophic bacteria H16.
7. The use according to claim 6, characterized in that the PHA-producing performance is PHA yield and/or PHA-producing intensity.
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