CN117701486A - 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 PDF

Info

Publication number
CN117701486A
CN117701486A CN202410157848.9A CN202410157848A CN117701486A CN 117701486 A CN117701486 A CN 117701486A CN 202410157848 A CN202410157848 A CN 202410157848A CN 117701486 A CN117701486 A CN 117701486A
Authority
CN
China
Prior art keywords
pha
bacteria
gene
producing
polysaccharide synthesis
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202410157848.9A
Other languages
Chinese (zh)
Inventor
饶驰通
谭婷婷
蒋天宇
周小雪
范丁丁
李腾
张浩千
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bluepha Co ltd
Original Assignee
Bluepha Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bluepha Co ltd filed Critical Bluepha Co ltd
Priority to CN202410157848.9A priority Critical patent/CN117701486A/en
Publication of CN117701486A publication Critical patent/CN117701486A/en
Pending legal-status Critical Current

Links

Landscapes

  • 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

Recombinant bacterium for producing PHA and construction method and application thereof
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 binds with a pseudomonas aeruginosa biological filmStructure and function are related. The Pel polysaccharide synthesis gene comprisespelA、pelB、pelC、pelD、pelE、pelF、pelGIn the bacterial genomepelABCDEFGThe operon exists in the form of an operon that is involved in the synthesis of extracellular polysaccharides associated with biofilm development at early and late stages of biofilm development, resulting in a glucose-rich biofilm matrix.pelABCDEFGOperon in genus Pacific copperCupriavidus) Genus PseudomonasPseudomonas) All of these bacteria are reported, andPelABCDEFGoperons are highly conserved among different bacterial species, and therefore Pel polysaccharide may be a biofilm determinant that is widely present in bacteria. Patent application CN108060111A disclosespelABCDEFGThe expression level of (2) is related to the yield of rhamnolipid in Pseudomonas, and is 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 group A are selected frompslABCDEFGHIJKLMNOAn extracellular polysaccharide synthesis gene cluster,pelABCDEFGPolysaccharide synthetic gene cluster and alginate synthetic gene clusteralgD-alg8-alg44-algKEGXLIJFPolyhydroxyalkanoate PHA synthesis key gene clusterphaC1-D-C2A combination of one or any of several genes; the genes in group B are selected fromrmlACBD、rmlBDAC、rhlYZ、algC、fadD4、lipCAndestAOr a combination of any of several genes. 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 above-mentioned Pel polysaccharide synthetic gene is selected frompelA、pelB、pelC、pelD、pelE、pelF、pelGOne or more of the following.
Specifically, the Pel polysaccharide synthesis gene ispelA、pelB、pelC、pelD、pelE、pelF、pelGAny one or a combination of any 2 to 7.
In some embodiments of the invention, the Pel polysaccharide synthesizing gene ispelA、pelB、pelC、pelD、pelE、pelFAndpelG。
preferably, the Pel polysaccharide synthesis gene ispelABCDEFGAnd (3) performing expression intervention on part or all of genes in the polysaccharide synthesis gene cluster, namely one or more essential genes on the Pel polysaccharide synthesis pathway, so as to regulate the Pel polysaccharide synthesis pathway.
As an optimal solution, in some embodiments of the present invention, the Pel polysaccharide synthesis gene ispelABCDEFGThe polysaccharide synthesis gene cluster is used for simultaneously carrying out expression intervention on 7 essential genes on the Pel polysaccharide synthesis pathway.
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 recombinant bacteriumpelA、pelB、pelC、pelD、pelE、pelFAndpelGthe gene or its encoded protein is inactivated.
In some embodiments of the invention, the recombinant bacteriumpelABCDEFGThe polysaccharide synthesis gene cluster is knocked out.
It should be understood that the present invention is in the specific embodiments described by deactivationpelA、pelB、pelC、pelD、pelE、pelFAndpelGthe PHA-producing recombinant bacterium is only exemplified, and other technical means which can achieve the aim are adopted on the basis of the aim of reducing the expression quantity and/or activity of the Pel polysaccharide synthetic gene or the encoded protein thereof known by the person skilled in the art, and the PHA-producing recombinant bacterium belongs to the equivalent deformation of the technical means of the invention, so the PHA-producing recombinant bacterium is within the protection scope of the 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 expression level and/or activity of the Pel polysaccharide synthesis gene or the encoded protein thereof is reduced or lost by knockoutpelABCDEFGPolysaccharide 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 present invention, the Pel polysaccharide synthesis gene is selected from the group consisting ofpelA、pelB、pelC、pelD、pelE、pelF、pelGOne or more of the following.
Synthesis of Pel polysaccharide into GenepelA、pelB、pelC、pelD、pelE、pelF、pelGReduced expression level and/or activity of one or more of the proteins encoded therebyCan influence the synthesis of the Pel polysaccharide, thereby influencing the productivity of PHA producing bacteria. Based on this, the Pel polysaccharide synthesis gene of the present invention may be selected frompelA、pelB、pelC、pelD、pelE、pelF、pelGOne or more of the following.
Specifically, the Pel polysaccharide synthesis gene ispelA、pelB、pelC、pelD、pelE、pelF、pelGAny one or a combination of any 2 to 7.
In bacteriapelA、pelB、pelC、pelD、pelE、pelF、pelGGenes are often present in the form of gene clusters. Therefore, the Pel polysaccharide synthesis gene ispelABCDEFGPart or all of the genes in the polysaccharide synthesis gene cluster.
In some embodiments of the invention, the Pel polysaccharide synthesizing gene ispelA、pelB、pelC、pelD、pelE、pelFAndpelG。
in some embodiments of the invention, the Pel polysaccharide synthesizing gene ispelABCDEFGPolysaccharide synthesis gene clusters.
Pel polysaccharide synthesis genes in bacteriapelA、pelB、pelC、pelD、pelE、pelF、pelGAnd the sequences encoding the proteins thereof, are available to those skilled in the art 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 Ruo's eutrophic bacteriaRalstonia eutrophaAlso calledCupriavidus necator) Is an important mode of bacteria for researching PHA synthesis, and is also a strain for researching more PHA industrial production at present. 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. Pel polysaccharide synthesis gene for eutrophic bacteria of RochepelA、pelB、pelC、pelD、pelE、pelF、pelGThe corresponding GenBank locus_tag of the eutrophic rogowski gene is H16_B2198-B2204 respectively, specifically, H16_B2198 ispelG(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 ispelF(the amino acid sequence of the protein is shown as SEQ ID NO.3, the gene sequence is shown as SEQ ID NO. 4), and H16_B2200 ispelE(the amino acid sequence of the protein is shown as SEQ ID NO.5, the gene sequence is shown as SEQ ID NO. 6), and H16_B2201 ispelD(the amino acid sequence of the protein is shown as SEQ ID NO.7, the gene sequence is shown as SEQ ID NO. 8), and H16_B2202 ispelC(the amino acid sequence of the protein is shown as SEQ ID NO.9, the gene sequence is shown as SEQ ID NO. 10), and H16_B2203 ispelB(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 ispelA(protein)The amino acid sequence of (a) 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 is p<0.05 toA representation; p is p<0.01, 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 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 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. 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 to confirm that the recombinant strain is correctly edited, and the obtained recombinant strain is the eutrophic rocarpium Re delta B2198-B2204，Designated as recombinant strain W01, in which a total of 7 genes, h16_b2198, h16_b2199, h16_b2200, h16_b2201, h16_b2202, h16_b2203, h16_b2204, were 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, 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 1mL by an evaporator, and then hexane of about 3mL 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 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 (10)

1. A recombinant bacterium for producing PHA, characterized in that the expression level of the Pel polysaccharide synthesis gene of the recombinant bacterium is reduced or lost, and the PHA productivity is improved;
the recombinant bacteria are bacteria of the genus Ralstonia or Pseudomonas.
2. The recombinant bacterium according to claim 1, wherein the Pel polysaccharide synthesizing gene ispelABCDEFGPart or all of the genes in the polysaccharide synthesis gene cluster.
3. The recombinant bacterium according to claim 1 or 2, wherein the recombinant bacteriumpelABCDEFGThe polysaccharide synthesis gene cluster 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 their expression level of the Pel polysaccharide synthesis gene is reduced or lost.
5. The method according to claim 4, wherein the reduction or loss of expression of the Pel polysaccharide synthesis gene is by knockoutpelABCDEFGPolysaccharide synthesis gene cluster.
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: PHA-producing bacteria are modified so that the expression level of the Pel polysaccharide synthesis 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.
The use of reduction or loss of expression of the pel polysaccharide synthesis gene in enhancing PHA productivity of PHA-producing bacteria.
10. The use according to claim 9, characterized in that said PHA-producing bacteria are bacteria of the genus ralstonia or pseudomonas.
CN202410157848.9A 2024-02-04 2024-02-04 Recombinant bacterium for producing PHA and construction method and application thereof Pending CN117701486A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202410157848.9A CN117701486A (en) 2024-02-04 2024-02-04 Recombinant bacterium for producing PHA and construction method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202410157848.9A CN117701486A (en) 2024-02-04 2024-02-04 Recombinant bacterium for producing PHA and construction method and application thereof

Publications (1)

Publication Number Publication Date
CN117701486A true CN117701486A (en) 2024-03-15

Family

ID=90162767

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202410157848.9A Pending CN117701486A (en) 2024-02-04 2024-02-04 Recombinant bacterium for producing PHA and construction method and application thereof

Country Status (1)

Country Link
CN (1) CN117701486A (en)

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101139575A (en) * 2007-08-06 2008-03-12 中国科学院微生物研究所 Extremely halophilic archaea polyhydroxy fatty acid ester synthases and encoding gene and application
CN102816729A (en) * 2012-07-24 2012-12-12 清华大学 Construction and application of polygene knockout strain of Halomonas sp. TD01
CN108060111A (en) * 2017-10-27 2018-05-22 中国科学院微生物研究所 A kind of pseudomonas aeruginosa for improving rhamnolipid yield and its construction method
CN111979163A (en) * 2019-05-24 2020-11-24 深圳蓝晶生物科技有限公司 Recombinant Roche bacterium, preparation method and application thereof
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
CN114672447A (en) * 2020-12-24 2022-06-28 中国科学院微生物研究所 Bacterial strain with self-flocculation capability and preparation method and application thereof
CN116286931A (en) * 2023-04-03 2023-06-23 北京蓝晶微生物科技有限公司 Double-plasmid system for rapid gene editing of Ralstonia eutropha and application thereof
EP4273209A1 (en) * 2022-05-04 2023-11-08 The Procter & Gamble Company Machine-cleaning compositions containing enzymes

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101139575A (en) * 2007-08-06 2008-03-12 中国科学院微生物研究所 Extremely halophilic archaea polyhydroxy fatty acid ester synthases and encoding gene and application
CN102816729A (en) * 2012-07-24 2012-12-12 清华大学 Construction and application of polygene knockout strain of Halomonas sp. TD01
CN108060111A (en) * 2017-10-27 2018-05-22 中国科学院微生物研究所 A kind of pseudomonas aeruginosa for improving rhamnolipid yield and its construction method
CN111979163A (en) * 2019-05-24 2020-11-24 深圳蓝晶生物科技有限公司 Recombinant Roche bacterium, preparation method and application thereof
CN114672447A (en) * 2020-12-24 2022-06-28 中国科学院微生物研究所 Bacterial strain with self-flocculation capability and preparation method and application thereof
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
EP4273209A1 (en) * 2022-05-04 2023-11-08 The Procter & Gamble Company Machine-cleaning compositions containing enzymes
CN116286931A (en) * 2023-04-03 2023-06-23 北京蓝晶微生物科技有限公司 Double-plasmid system for rapid gene editing of Ralstonia eutropha and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
娄秋莉等: "具有抗菌效应的聚羟基脂肪酸酯生物塑料的制备与功能表征", 生物工程学报, no. 8, 25 August 2016 (2016-08-25), pages 62 - 69 *

Similar Documents

Publication Publication Date Title
CN102102086B (en) L-lactate dehydrogenase gene-deleted engineering bacterium and construction method and application thereof
CN102212501B (en) Recombinant escherichia coli and method for applying same to produce poly(3-hydroxybutyrate-3-hydroxyvalerate) (PHBV) by utilizing single carbon source
Cui et al. Improved productivity of poly (3-hydroxybutyrate)(PHB) in thermophilic Chelatococcus daeguensis TAD1 using glycerol as the growth substrate in a fed-batch culture
CN114381415B (en) Gene recombination strain for high-yield PHA and construction method thereof
JP2017504330A (en) Recombinant microorganisms enriched with carbohydrates
CN106434510A (en) Genetically engineered bacterium for producing L-aspartic acid through fermentation
CN116376856A (en) Engineered microorganisms expressing acetoacetyl-CoA reductase variants and methods of increasing PHA production
CN114807206A (en) Bacterial strain for synthesizing poly (3-hydroxybutyrate-co-4-hydroxybutyrate) and construction method and application thereof
CN102517303B (en) Recombination blue-green alga for producing lactic acid as well as preparation method and applications thereof
CN116970659B (en) Method for producing polyhydroxyalkanoate
CN109136313B (en) Method for synthesizing 2&#39; -deoxyadenosine by using klebsiella melitensis
CN109136314B (en) Method for synthesizing 2&#39; -deoxy-2-aminoadenosine by using klebsiella melitensis
Saranya et al. Effect of nitrogen and calcium sources on growth and production of PHA of Pseudomonas sp. LDC-5 and its mutant
CN104762242A (en) Producing strain of mevalonic acid, and method used for producing mevalonic acid
JP2024515917A (en) Genetically engineered microorganisms expressing acetoacetyl-CoA reductase variants and methods for increasing the proportion of 3-hydroxyhexanoic acid in PHAs
CN117701486A (en) Recombinant bacterium for producing PHA and construction method and application thereof
CN101029316B (en) Production of succinate from colon bacillus
CN117701488A (en) Recombinant bacterium for producing PHA and method for improving PHA yield
CN104046659A (en) Poly-3-hydroxy propionic acid copolymer and production method thereof
CN106801063B (en) Construction method of engineering escherichia coli with changed form, engineering escherichia coli and application
CN117363554B (en) Engineered halophilic microorganism and construction method and application thereof
CN103436477B (en) Escherichia coli strain for producing succinic acid with glycerol as well as construction method and use
Xiuhong et al. Optimization of L-lactic acid production of Rhizopus oryzae mutant RLC41-6 by ion beam implantation at low-energy
CN117946225A (en) Recombinant engineering bacterium for improving yield of polyhydroxyalkanoate and application thereof
Son et al. Biosynthesis of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) from structurally unrelated single carbon sources by newly isolated Pseudomonas sp. EL-2

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination