CN104450594B - Produce genetic engineering bacterium and its construction method and the application of poly butyric-valerate - Google Patents

Abstract

The invention discloses a genetically engineered bacterium for producing polyhydroxybutyrate-valerate, its construction method and application. The genetically engineered bacterium of the present invention is a gene-deleted strain of Roseburia eutrophicum or the coding gene yliK of methylmalonyl-CoA mutase, the coding gene argK of GTP kinase and methylmalonyl-CoA The coding gene ygfG of decarboxylase is introduced into Roseburia eutropha or a gene-deleted strain of Roseburia eutropha; 1 and at least one of the genes encoding 2-methylcitrate synthase-2 is deleted. The production of polyhydroxybutyrate-valerate by using the recombinant bacteria constructed by the invention has the following advantages: safe bacterial strains, relatively cheap raw materials, easy control of the fermentation process, and easy large-scale production.

Description

Genetically engineered bacteria producing polyhydroxybutyrate-valerate and its construction method and application

technical field

The invention belongs to the field of biotechnology, and in particular relates to a genetically engineered bacterium for producing polyhydroxybutyrate-valerate and its construction method and application.

Background technique

Due to the dependence of the plastic industry on petroleum and the seriousness of environmental pollution caused by plastic waste, people are forced to look for environmentally friendly and sustainable materials that can replace chemical plastics. Poly-3-hydroxybutyrate (hereinafter referred to as polyhydroxybutyrate, PHB) is a biodegradable polymer material based on bio-based production, and its performance is similar to that of plastic. However, PHB is brittle, and post-processing has certain difficulties. If 3-hydroxyvaleric acid (3HV) components are mixed into PHB, the obtained poly-3-hydroxybutyric acid copolymerized 3-hydroxyvaleric acid ester (hereinafter referred to as poly Hydroxybutyrate-valerate, PHBV) can improve the performance of the material, and the hardness, strength, and melting point are all reduced, but the decomposition temperature does not drop, which is more conducive to broadening the application range of the product. PHBV can be used as a scaffold platform for tissue and organ repair, outer packaging of slow-release drugs, bone regeneration guiding membrane, construction of cardiac biological valves, medical textiles, degradable packaging materials and adsorption materials, etc.

At present, the only production strain used in large-scale production of PHBV is the mutant strain of Rosia eutropha, but propionic acid (salt) or valeric acid (salt) needs to be added during cultivation. Propionyl-CoA is synthesized from propionate (salt), and propionyl-CoA is condensed with acetyl-CoA to generate valeryl-CoA, which is the direct precursor for the synthesis of 3-hydroxyvaleric acid (3HV), and valeric acid (salt) can be directly synthesized Valeryl-CoA. However, propionic acid (salt) or valeric acid (salt) is cytotoxic and more expensive. Therefore, when producing PHBV in the prior art, it is necessary to strictly control the feeding process of propionic acid (salt) or valeric acid (salt), so as to reach a certain amount of cells and meet the needs of propionyl-CoA synthesis. The control process of producing PHBV in the prior art is complex and increases the production cost, which limits the application. In recent years, researchers have used genetic engineering techniques to transform strains, but the performance of the strains is far from meeting the requirements for commercial production, and the host bacteria may be pathogenic bacteria.

Contents of the invention

One object of the present invention is to provide a recombinant bacterium.

The recombinant bacterium provided by the present invention introduces the coding gene yliK of methylmalonyl-CoA mutase, the coding gene argK of GTP kinase, and the coding gene ygfG of methylmalonyl-CoA decarboxylase into the host. Obtained from Ralstonia eutropha.

In the above recombinant bacteria, the host R. eutropha is R. eutropha Rem-1, R. eutropha Rem-3, R. eutropha Rem-5 or R. eutropha Rem-7.

Among the above-mentioned recombinant bacteria, the Rem-3 is the bacterium obtained after the deletion of the 2-methylcitrate synthase-1 gene prpC1 in Rostella eutropha Rem-1; the Rostella eutropha Rem-1 can be obtained by homologous recombination Knock out the prpC1 gene, or inactivate the prpC1 gene by insertion, or obtain a strain with the deletion of the prpC1 gene by mutagenesis; the specific method for obtaining Rem-3 is:

(1) Insert the DNA fragment shown in sequence 3 in the sequence listing into the multiple cloning site of the pJQ200mp18Tc vector, and the resulting recombinant vector is denoted as pJQ200mp18Tc::ΔprpC1;

(2) The pJQ200mp18Tc::ΔprpC1 vector was transformed into E.coli S17-1, and the obtained recombinant bacteria were recorded as ΔprpC1pJQ200/S17-1 as the donor bacteria;

(3) Rem-1 is used as the recipient bacterium, and the donor bacterium ΔprpC1pJQ200/S17-1 is conjugated and transferred with the recipient bacterium Rem-1, so that the prpC1 gene in Rem-1 is deleted, and the recombinant bacterium obtained is The Rem-3.

Among the above-mentioned recombinant bacteria, the Rem-5 is the bacterium obtained after the deletion of the 2-methylcitrate synthase-2 gene prpC2 in Rostella eutropha Rem-1; the Rostella eutropha Rem-1 can be obtained by homologous recombination Knock out the prpC2 gene, or inactivate the prpC2 gene by insertion, or obtain a strain with the deletion of the prpC2 gene by mutagenesis; the specific method for obtaining Rem-5 is:

(1) Insert the DNA fragment shown in sequence 6 in the sequence listing into the multiple cloning site of the pJQ200mp18Tc vector, and the resulting recombinant vector is denoted as pJQ200mp18Tc::ΔprpC2;

(2) The pJQ200mp18Tc::ΔprpC2 vector was transformed into E.coli S17-1, and the obtained recombinant bacterium was recorded as ΔprpC2pJQ200/S17-1 as the donor bacterium;

(3) Rem-1 is used as the recipient bacterium, and the donor bacterium ΔprpC2pJQ200/S17-1 is conjugated and transferred to the recipient bacterium Rem-1, and the Rem-1 with prpC2 gene deletion is obtained as ΔprpC2/Rem-1, Namely the Rem-5.

In the above-mentioned recombinant bacteria, the Rem-7 is obtained after the deletion of the 2-methylcitrate synthase-1 gene prpC1 and the 2-methylcitrate synthase-2 gene prpC2 in Rostella eutropha Rem-1 bacterium; the Rostella eutrophicum Rem-1 can knock out the prpC1 and prpC2 genes by homologous recombination, or inactivate the prpC1 and prpC2 genes by insertion, and can also obtain the prpC1 and prpC2 gene deletion strains by mutagenesis; the The specific method of obtaining Rem-7 is:

(1) Insert the DNA fragment shown in sequence 6 in the sequence listing into the multiple cloning site of the pJQ200mp18Tc vector, and the resulting recombinant vector is denoted as pJQ200mp18Tc::ΔprpC2;

(2) The pJQ200mp18Tc::ΔprpC2 vector was transformed into E.coli S17-1, and the obtained recombinant bacterium was recorded as ΔprpC2pJQ200/S17-1 as the donor bacterium;

(3) Using the Rem-3 as the recipient bacterium, the donor bacterium ΔprpC2pJQ200/S17-1 was conjugated and transferred to the recipient bacterium Rem-3, and the Rem-3 with prpC2 gene deletion was obtained as ΔprpC2/Rem- 3, namely the Rem-7.

In the above-mentioned recombinant bacteria, the amino acid sequence of the 2-methylcitrate synthase-1 is shown in sequence 2 in the sequence listing; the coding gene sequence of the 2-methylcitrate synthase-1 is shown in sequence 1 in the sequence listing The 878-2035th nucleotide molecule from the 5' end in the middle; the amino acid sequence of the 2-methylcitrate synthase-2 is as shown in sequence 5 in the sequence listing; the 2-methylcitrate synthase The coding gene sequence of Enzyme-2 is shown in the 1032-2229th nucleotide molecule from the 5' end in Sequence 4 in the Sequence Listing.

In the above-mentioned recombinant bacteria, the encoding gene yliK of the methylmalonyl-CoA mutase, the encoding gene argK of the GTP kinase and the encoding gene ygfG of the methylmalonyl-CoA decarboxylase The recombinant expression vector pZMwf was introduced into the host Rostella eutrophicum.

In the above-mentioned recombinant bacteria, the construction method of the recombinant expression vector pZMwf includes the following steps: inserting the yliK-argK-ygfG gene cluster fragment shown in sequence 7 in the sequence listing into the multiple cloning site of the expression vector pLXM1, and the obtained recombinant The vector is designated as pZMwf.

In the above recombinant bacteria, the amino acid sequence of the methylmalonyl-CoA mutase is shown in sequence 8 in the sequence listing; the amino acid sequence of the GTP kinase is shown in sequence 9 in the sequence listing; the formazan The amino acid sequence of ylmalonyl-CoA decarboxylase is shown in sequence 10 in the sequence listing.

Another object of the present invention is to provide a coding gene yliK containing methylmalonyl-CoA mutase, the coding gene argK of GTP kinase and the coding gene ygfG of methylmalonyl-CoA decarboxylase recombinant expression vectors.

The recombinant expression vector containing the coding gene yliK of methylmalonyl-CoA mutase, the coding gene argK of GTP kinase and the coding gene ygfG of methylmalonyl-CoA decarboxylase provided by the present invention is to The yliK-argK-ygfG gene cluster fragment shown in sequence 7 in the sequence listing is inserted into the multiple cloning site of the expression vector pLXM1.

In the above recombinant expression vector, the amino acid sequence of the methylmalonyl-CoA mutase is shown in sequence 8 in the sequence listing; the amino acid sequence of the GTP kinase is shown in sequence 9 in the sequence listing; the The amino acid sequence of methylmalonyl-CoA decarboxylase is shown in sequence 10 in the sequence listing.

Another object of the present invention is to provide the above-mentioned Rem-3 or the above-mentioned Rem-5 or the above-mentioned Rem-7.

The application of the above-mentioned recombinant bacteria or the above-mentioned recombinant expression vector or the above-mentioned Rem-3 or Rem-5 or Rem-7 in the production of polyhydroxybutyrate-valerate also belongs to the protection scope of the present invention .

The application of the above-mentioned recombinant bacteria or the above-mentioned recombinant expression vector or the above-mentioned Rem-3 or Rem-5 or Rem-7 in improving the content of 3HV components in the synthetic PHBV of the strain also belongs to the protection scope of the present invention .

A final object of the present invention is to provide a process for the production of polyhydroxybutyrate-valerate.

The method for producing polyhydroxybutyrate-valerate provided by the present invention includes the following steps: using glucose as a substrate, fermenting and cultivating the above-mentioned recombinant bacteria or the above-mentioned Rem-3 or Rem-5 or Rem-7, The polyhydroxybutyrate-valerate was obtained.

In the above method, the fermentation medium is a basal medium; the preparation method of the basal medium: every liter of the basal medium contains 6.7g Na ₂ HPO ₄ ·2H ₂ O, 1.5g KH ₂ PO4, 1g (NH ₄ ) ₂ SO ₄ , 0.2g MgSO ₄ ·7H ₂ O, 1ml of trace elements and 20g of glucose; the preparation method of the trace elements: each liter of trace elements contains 0.3g H ₃ BO ₃ , 0.2gCoCl ₂ , 0.1g ZnSO ₄ 7H ₂ O, 0.03g MnCl ₂ 4H ₂ O, 0.02g NaMoO ₄ 2H ₂ O, 0.02g NiCl ₂ 6H ₂ O, 0.01g CuSO ₄ 5H ₂ O, 0.01g CaCl ₂ 2H ₂ O and 0.06 g Fe(NH ₄ ) ₂ (SO ₄ ) ₂ .

In the above method, the medium of the fermentation culture does not contain propionic acid or propionate or valeric acid or valerate.

The production of polyhydroxybutyrate-valerate by using the recombinant bacteria constructed by the invention has the following advantages: safe bacterial strains, relatively cheap raw materials, easy control of the fermentation process, and easy large-scale production.

Description of drawings

Figure 1 shows the verification of the knockout vectors ΔprpC1pJQ200mp18Tc and ΔprpC2pJQ200mp18Tc.

Fig. 2 is a schematic diagram of the expression vector pZMwF carrying the yliK-argK-ygfG gene cluster.

Fig. 3 is the GC measurement result of PHBV standard.

Fig. 4 is the GC assay result of Rem-1 synthesized PHBV.

Fig. 5 is the GC assay result of the PHBV synthesized by the genetically engineered bacteria.

detailed description

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Ralstonia eutropha H16 was purchased from DSM.

Ralstonia eutropha Rem-1 (formerly named 65-7) is a strain obtained by mutagenizing Ralstonia eutropha H16. The document "Chen Qi et al., Breeding of strains producing poly-β-hydroxybutyric acid by fermentation of glucose, Microbiology Bulletin, 1994, 21(6), 333-335" is published, and the public can obtain it from the Institute of Microbiology, Chinese Academy of Sciences.

E.coli S17-1 in the literature "Simon R PU, Püller A., A broad host rangemobilization system for in vivo genetic engineering: transposon mutagenesis ingram negative bacteria, Nat.Biotechnol., 1983,1(9):784-789" Publicly available from the Institute of Microbiology, Chinese Academy of Sciences.

The pJQ200mp18Tc vector is disclosed in the document "Potter M., Steinbüchel A., Poly(3-hydroxybutyrate) granule-associated proteins: impacts on poly(3-hydroxybutyrate) synthesis and degradation, Biomacromolecules, 20056(2), 552-60." However, the public can obtain it from the Institute of Microbiology, Chinese Academy of Sciences.

The pLXM1 plasmid was disclosed in the document "Lu Xuemei et al., Engineering Transformation of L-arabinose Metabolic Pathway of Ralstonia eutropha W50, Acta Microbiology, 2013, 53(12) 1147-1155.", and the public can obtain it from the Institute of Microbiology, Chinese Academy of Sciences.

Escherichia coli W3110 has been disclosed in the document "Barfknecht T.R., Smith K.C., Ultraviolet radiation-induced mutation of isogenic uvrA and uvrB strains of Escherichia coli K-12W3110. Photochemistry and photobiology, 1977 (26), 643-645." Obtained from Institute of Microbiology, Chinese Academy of Sciences.

The pMDT19-simple plasmid was purchased from Takara Bioengineering Co., Ltd., the product catalog number is TAKARA Code: D104.

The preparation method of PG medium: each liter of PG medium contains 10g of peptone, 5g of yeast powder, 3g of glucose and 3g of ammonium sulfate.

Preparation method of basal medium: each liter of basal medium contains 6.7g Na ₂ HPO ₄ ·2H ₂ O, 1.5g KH ₂ PO4, 1g(NH ₄ ) ₂ SO ₄ , 0.2g MgSO ₄ ·7H ₂ O, 1ml of Trace elements and 20g glucose.

Each liter of trace elements contains 0.3g H ₃ BO ₃ , 0.2g CoCl ₂ , 0.1g ZnSO ₄ ·7H ₂ O, 0.03gMnCl ₂ ·4H ₂ O, 0.02g NaMoO ₄ ·2H ₂ O, 0.02g NiCl ₂ · 6H ₂ O, 0.01 g CuSO ₄ .5H ₂ O, 0.01 g CaCl ₂ .2H ₂ O and 0.06 g Fe(NH ₄ ) ₂ (SO ₄ ) ₂ .

The construction of the gene knockout vector of embodiment 1, prpC1 and prpC2

1. Construction of prpC1 gene knockout vector

Using the genomic DNA of Ralstonia eutropha H16 as a template, PCR amplification was performed using prpC1df/prpC1dr primers. The primer sequences are as follows:

prpC1df: 5'-TAGATCTCAAGCGCGCCGGCTACCGGGCC-3';

prpC1dr: 5'-TTCTAGACAGGTCGAACTTCAGCACGCGCT-3'.

The amplified fragment with a size of 3222bp was connected to the plasmid pMDT19-simple, and the resulting recombinant vector was designated as pMD19-prpC1. The recombinant vector pMD19-prpC1 was transformed into E. coli DH5α, single colonies were picked, and the plasmid was extracted for sequencing. Sequencing results show that the sequence of the PCR amplification product is shown as sequence 1 in the sequence listing, which contains DNA fragments of the prpC1 gene and its two homologous arms. The nucleotide sequence of the prpC1 gene is shown in the 878-2035 nucleotide molecule from the 5' end in Sequence 1 of the sequence listing; the amino acid sequence of 2-methylcitrate synthase-1 encoded by the prpC1 gene is shown in the sequence listing Sequence 2 is shown.

The pMD19-prpC1 obtained above was digested with the restriction endonuclease SacII. Since the prpC1 gene fragment contained two SacII restriction sites, the plasmid was removed from the sequence 1 in the sequence table after digestion. The 1222bp fragment was recovered as a large fragment, and the recombinant vector obtained after self-ligation with T4 ligase was designated as pMD19-ΔprpC1, and pMD19-ΔprpC1 was transformed into Escherichia coli DH5α, a single colony was picked, and the extracted plasmid was sequenced with the prpC1df/prpC1dr primer pair. Sequencing results show that the plasmid carries the DNA molecule shown in sequence 3 in the sequence listing, which is denoted as ΔprpC1 DNA fragment, which is obtained by removing the 1001-2222 nucleotides from the 5' end of sequence 1 in the sequence listing sequence.

pMD19-ΔprpC1 was double-digested and blunted with restriction endonucleases BglII and XbaI, then ligated with the pJQ200mp18Tc vector digested with SmaI and dephosphorylated with T4 ligase, transformed into Escherichia coli DH5α, and coated on Transformants were obtained on LB plates with tetracycline. The plasmid of the transformant was extracted and digested with KpnI. The pJQ200mp18Tc vector has two Kpn I restriction sites. When there is no foreign gene, the result of restriction digestion is two 3kb fragments; when a foreign gene is inserted into the multi-cloning site, the nucleic acid will be Gel electrophoresis obtained two bands of different sizes, indicating that there was an insertion of a foreign fragment in the plasmid. The plasmid is a knockout vector containing a homologous fragment of the prpC1 gene, named pJQ200mp18Tc::ΔprpC1 (Figure 1).

2. Construction of prpC2 gene knockout vector

Using the genomic DNA of Rostella eutropha H16 as a template, the primers prpC2df/prpC2dr were used for PCR amplification. The primer sequences are as follows:

prpC2df:TGAGCTCCGTGCATCCTTCCGAGGTCTTGT;

prpC2dr: TCTCGAGTCGAAGGATGATGCGATGGCATT.

The amplified DNA fragment of 3218bp was connected to the plasmid pMDT19-simple, and the obtained recombinant vector was designated as pMD19-prpC2. The recombinant vector pMD19-prpC2 was transformed into E. coli DH5α, single colony was picked, and the plasmid was extracted for sequencing. Sequencing results show that the sequence of the PCR amplification product is shown as sequence 4 in the sequence listing, and the PCR product is a DNA fragment comprising the prpC2 gene and its two homologous arms. The nucleotide sequence of the prpC2 gene is shown in the 1032-2229th nucleotide molecule from the 5' end in sequence 4 in the sequence listing; the amino acid sequence of 2-methylcitrate synthase-2 encoded by the prpC2 gene is shown in the sequence listing Shown in Sequence 5.

Digest pMD19-prpC2 with restriction endonucleases PstI and ClaI. After digestion, the 1443bp gene fragment was removed from the inside of sequence 4 in the sequence table in the recombinant vector pMD19-prpC2, and the large fragment was recovered. The recombinant vector obtained after concatenation is designated as pMD19-ΔprpC2. Transform pMD19-ΔprpC2 into Escherichia coli DH5α, pick a single colony, extract the plasmid and sequence it with the prpC2df/prpC2dr primer pair. The plasmid carries the DNA shown in sequence 6 in the sequence table The molecule has a total of 1775 bp and is named as ΔprpC2 DNA fragment, which is the sequence obtained by removing the 922-2364th nucleotides from the 5' end of sequence 4 in the sequence listing.

pMD19-ΔprpC2 was double-digested and smoothed with restriction endonucleases SacI and XhoI, and then ligated with the fragment of pJQ200mp18Tc vector digested with SmaI and dephosphorylated with T4 ligase, transformed into Escherichia coli DH5α, and coated on Transformants were obtained on LB plates with tetracycline. The plasmid of the transformant was extracted and digested with KpnI. The pJQ200mp18Tc vector has two Kpn I restriction sites. When there is no foreign gene, the result of restriction digestion is two 3kb fragments; when a foreign gene is inserted into the multi-cloning site, the nucleic acid will be Gel electrophoresis obtained two bands of different sizes, indicating that there was an insertion of a foreign fragment in the plasmid. The plasmid is a knockout vector containing a homologous fragment of the prpC2 gene, named pJQ200mp18Tc::ΔprpC2 (Figure 2).

Example 2, Construction of prpC1 and prpC2 gene knockout strains

1. Construction of prpC1 knockout strain

Gene knockout was performed by the method of conjugal transfer. The pJQ200mp18Tc::ΔprpC1 knockout vector was transformed into E.coli S17-1, spread on the LB plate containing tetracycline, and the correct transformant was selected and named ΔprpC1pJQ200/S17-1.

ΔprpC 1pJQ200/S17-1 was used as the donor bacteria for conjugative transfer and the recipient bacteria Rem-1 for conjugative transfer. The process of conjugative transfer is as follows: first, the recipient bacteria Rem-1 was inoculated in PG medium, and cultured at 30°C until the OD value was 1.0; the donor bacteria ΔprpC 1pJQ200/S17-1 was inoculated in LB culture based on 37°C culture, and the growth When the OD value is 0.4, take 100 μl of the culture solution of the recipient bacteria and the donor bacteria, and centrifuge at 3000 rpm to remove the supernatant, suspend and mix the two strains with 100 μl of fresh PG medium, culture them at 30°C for 12 hours, and spread them Incubate at 30°C on a double-antibody LB plate containing ampicillin (50 μg/ml) and tetracycline (5 μg/ml). Since Rem-1 can grow on a 50μg/ml ampicillin plate, but E.coli S17-1 cannot grow on an ampicillin plate, after culturing at 30°C for 48 hours, only the colony that grows on the double-antibody plate is once The exchanged Rem-1 colonies were screened out of the target strain, streaked on an LB plate containing 20% sucrose, and a single colony was picked, and the correct gene knockout strain ΔprpC1/Rem-1 was screened by colony PCR using prpC1df/prpC1dr as primers , and the resulting strain was named Rem-3. Using prpC1df/prpC1dr primers to verify the colony PCR of Rem-3, the fragment size obtained is 2kb, while the fragment size obtained by colony PCR of the original colony Rem-1 with prpC1df/prpC1dr primers is 3.2kb, proving that Rem-3 prpC1 has been knocked out.

2. Construction of prpC2 gene knockout strain

In addition to replacing the pJQ200mp18Tc::ΔprpC1 knockout vector with pJQ200mp18Tc::ΔprpC2 to transform E.coli S17-1, the obtained ΔprpC2pJQ200/S17-1 was used as the donor bacteria for the transfer, and the correct one was screened by colony PCR with prpC2df/prpC2dr as primers Except for the gene knockout strain, the construction method of the gene knockout strain ΔprpC2/Rem-1 was the same as that of the above gene knockout strain ΔprpC1/Rem-1, and the obtained strain was named Rem-5. Using prpC2df/prpC2dr primers to verify the colony PCR of Rem-5, the obtained fragment size is 1.8kb, while the fragment size obtained by colony PCR of the original colony Rem-1 with prpC2df/prpC2dr primers is 3.2kb, proving that Rem- All 5 prpC2 have been knocked out.

3. Construction of simultaneous knockout strains of prpC1 and prpC2 genes

The prpC2 gene was knocked out in Rem-3. The specific operation is to replace the pJQ200mp18Tc::ΔprpC1 knockout vector with pJQ200mp18Tc::ΔprpC2 to transform E.coli S17-1 to obtain ΔprpC2pJQ200/S17-1 as the donor bacteria for binding transfer, and replace the recipient bacteria Rem-1 with Rem -3 and using prpC2df/prpC2dr as primers to screen the correct gene knockout strain by colony PCR, the construction method of ΔprpC1ΔprpC2/Rem-1 is the same as that of the above gene knockout strain ΔprpC1/Rem-1, and the obtained strain is named Rem-7. Using prpC1df/prpC1dr as primers, the colony PCR verification of Rem-7 was performed, and the obtained fragment size was 2kb. Using prpC2df/prpC2dr as primers, the colony PCR verification of Rem-7 was performed, and the obtained fragment size was 1.8kb, proving that Rem- Both prpC1 and prpC2 have been knocked out in 7.

Embodiment 3, produce the construction of PHBV genetically engineered bacteria

1. Construction of expression vector pZMwf containing exogenous gene cluster yliK-argK-ygfG

Using the genomic DNA of Escherichia coli W3110 as a template, using primers muyayf/muyayr, a yliK-argK-ygfG gene cluster with a size of 3929 bp was obtained by PCR amplification, and the yliK-argK-ygfG gene cluster was connected to pMD19-Tsimple , the obtained recombinant vector was denoted as pT-yliK-argK-ygfG, the recombinant vector pT-yliK-argK-ygfG was transformed into Escherichia coli, a single colony was picked, and the plasmid was extracted for sequencing. The sequences of the primer pairs are as follows:

Upstream primer Muyayf: 5'-TGC GAGCTC ATCGTTAATTCTTCAGAAGCGTTCGT-3';

Downstream primer Muyayr: 5'-TAT AAGCTT TTAATGACCAACGAAATTAGGTT-3'.

Digest pT-yliK-argK-ygfG with restriction endonucleases XbaI and HindIII to obtain the yliK-argK-ygfG gene cluster fragment, and use restriction endonucleases XbaI and HindIII to perform double digestion of the expression vector pLXM1, The large fragment that obtains; The yliK-argK-ygfG gene cluster fragment is connected with the large fragment, and the recombinant vector obtained is denoted as pZMwf (as shown in Figure 2); pZMwf is transformed into Escherichia coli DH5α, chloramphenicol resistance screening, picking Plasmids of positive clones.

Sequencing results show that the yliK-argK-ygfG gene cluster fragment inserted between the Xba I and Hind III restriction sites of the pLXM1 vector is shown in the nucleotide molecule of sequence 7 in the sequence table, indicating that the vector is correct. The proteins encoded by the yliK-argK-ygfG gene cluster are methylmalonyl-CoA mutase, GTP kinase and methylmalonyl-CoA decarboxylase. Wherein, the amino acid sequence of methylmalonyl-CoA mutase is shown in sequence 8 in the sequence listing, and the coding gene sequence of methylmalonyl-CoA mutase is shown in sequence 7 in the sequence listing 1 to 2143 nucleotide molecules; the amino acid sequence of GTP kinase is shown in sequence 9 in the sequence listing, and the coding gene sequence of GTP kinase is shown in nucleotide molecules 2138 to 3131 in sequence 7 in the sequence listing; The amino acid sequence of methylmalonyl-CoA decarboxylase is shown in sequence 10 in the sequence listing, and the coding gene sequence of methylmalonyl-CoA decarboxylase is shown in the 3144th to 3929th of sequence 7 in the sequence listing The nucleotide molecules are shown.

2. Construction of PHBV-producing genetically engineered bacteria

The expression vector pZMwf with the target gene cluster yliK-argK-ygfG was introduced into the competent cells of Rem-1 by electric shock transformation, positive clones were screened on the chloramphenicol plate, and the genetically engineered bacteria were obtained through colony PCR verification, named For Rem-2. Rem-2 contains the yliK-argK-ygfG gene cluster fragment shown in the nucleotide molecule of sequence 7 in the sequence listing.

Preparation of competent cells of R. eutropha Rem-1: Cultivate a single colony of R. eutropha Rem-1 on solid PG medium overnight, then inoculate in 30ml of liquid PG medium, culture at 30°C for 8h, and then The bacterial solution was cooled in an ice bath for 15 min, and the cells were collected by centrifugation. Wash the cells three times with pre-cooled 10% glycerol; finally add 100 μl sterilized cold 10% glycerol to suspend the cells, keep in ice bath for 20 min, store at -80°C after aliquoting or directly use for electric shock transformation.

Using the above method, the expression vector pZMwf carrying the target gene cluster yliK-argK-ygfG was introduced into Rem-3 competent cells by electric shock transformation, positive clones were screened on the chloramphenicol plate, and genetic engineering was obtained through colony PCR verification. bacteria, named Rem-4. Rem-4 contains the yliK-argK-ygfG gene cluster fragment shown by the nucleotide molecule of sequence 7 in the sequence listing.

Using the above method, the expression vector pZMwf carrying the target gene cluster yliK-argK-ygfG was introduced into Rem-5 competent cells by electric shock transformation, positive clones were screened on the chloramphenicol plate, and the genetic engineering was obtained through colony PCR verification. bacteria, named Rem-6. Rem-6 contains the yliK-argK-ygfG gene cluster fragment shown in the nucleotide molecule of sequence 7 in the sequence listing.

Using the above method, the expression vector pZMwf with the target gene cluster yliK-argK-ygfG was introduced into Rem-7 competent cells by electric shock transformation, positive clones were screened on the chloramphenicol plate, and the genetic engineering was obtained through colony PCR verification. bacteria, named Rem-8. Rem-8 contains the yliK-argK-ygfG gene cluster fragment shown by the nucleotide molecule of sequence 7 in the sequence listing.

Example 4. Fermentation of genetically engineered bacteria to produce polyhydroxybutyrate-valerate

Single colonies of Rem-2, Rem-3, Rem-4, Rem-5, Rem-6, Rem-7 and Rem-8 were picked respectively, streaked on solid PG medium overnight, and then inoculated with 20 μg/ mL of chloramphenicol basal medium, cultivated at 30°C for 18-20h, and transferred the culture to shake flasks with 10% inoculum size of the basal medium (cultivation of Rem-2, Rem-4, Rem -6 and Rem-8, add 0.1-15μM VB12), and continue to culture. During the fermentation process, glucose was added three times at 8 hours, 24 hours, and 32 hours, and 1ml of 20% glucose was added each time, while the pH was adjusted to 7.0, and the fermentation was carried out for 48 hours. At the same time, Rem-1 bacteria were cultured under the same conditions as a control.

After the fermentation, take 10 mL of the fermentation broth, centrifuge at 12000 rpm for 5 minutes to collect the bacteria, wash the bacteria twice with water, dry at 70°C and weigh them. Weigh 20-30g of dried cells, transfer the dried cells to an anaerobic tube, add 2 mL of benzoic acid-acidified methanol and 2 mL of chloroform, cap tightly, put in a water bath at 100°C for 4 hours, take it out and place it at room temperature, then add 2 mL of deionized H ₂ O extraction, moved to 10mL anaerobic tube, using gas chromatography to detect 3-hydroxybutyrate methyl ester (3HB methyl ester) and 3-hydroxyvalerate methyl ester (3HV methyl ester) components.

Gas chromatographic analysis uses Shimadzu GC-2010 gas chromatograph, chromatographic column is DB-5 (SHIMADZU company), column length 25 meters, 0.25um thick, internal diameter 0.25m, FID detector. Use high-purity nitrogen as the carrier gas, hydrogen as the fuel gas, and air as the supporting gas. Column temperature: 180°C, inlet temperature 250°C, using split mode with a split ratio of 2. Detector temperature 250°C. Polyhydroxybutyrate-valerate (PHBV) standard was purchased from Sigma. The experiment was repeated three times, and the results were averaged.

The gas chromatographic analysis spectrum of polyhydroxybutyrate-valerate (PHBV) standard product is shown in Figure 3, the peak time of 3HB methyl ester is 3.3min, and the peak time of 3HV methyl ester is 3.9min. The gas chromatographic analysis profile of the fermentation product of the control strain is shown in Figure 4. The gas chromatographic analysis spectrum of the genetically engineered bacteria fermentation product (Rem-2 shake flask fermentation product) is shown in Figure 5. The peak with a retention time of 3.3 min is 3HB methyl ester, and the peak with a retention time of 3.9 min is 3HV methyl ester.

Table 1 shows the fermentation experiment results of genetically engineered bacterial strains obtained by knocking out the prpC1 gene or the prpC2 gene, or knocking out both prpC1 and prpC2.

Shake flask fermentation experiment of table 1, Rem-1, Rem-3, Rem-5 and Rem-7

Note: L in the table represents the volume of fermentation broth

The results showed that: compared with Rem-1, the 3HV component content of Rem-3 increased by 57.6%, the 3HV component content of Rem-5 increased by 24.2%, and the 3HV component content of Rem-7 increased by 3 times. The above results indicate that blocking the metabolic bypass of propionyl-CoA can significantly increase the content of 3HV components in the PHBV synthesis of the strain.

The yliK-argK-ygfG gene cluster was expressed in different host bacteria, and the results of PHBV production by genetically engineered bacteria were shown in Table 2.

Shake flask fermentation experiment of table 2, Rem-2, Rem-4, Rem-6 and Rem-8

Note: L in the table represents the volume of fermentation broth

The results showed that, compared with Rem-1, the content of 3HV components in Rem-2 was increased by 24.8 times, indicating that enhancing the synthesis of propionyl-CoA could significantly increase the content of 3HV components in strain PHBV synthesis. Compared with Rem-3, the 3HV component content of Rem-4 increased by 27.9 times; compared with Rem-5, the 3HV component content of Rem-6 increased by 14.4 times; compared with Rem-7, Rem-8 The content of the 3HV component of 20.8 times increased. The above results indicate that blocking the metabolic bypass of propionyl-CoA and enhancing the synthesis of propionyl-CoA can significantly increase the content of 3HV components in the strain PHBV synthesis.

Claims (6)

1. Recombinant bacteria, the coding gene yliK of methylmalonyl-CoA mutase, the coding gene argK of GTP kinase and the coding gene ygfG of methylmalonyl-CoA decarboxylase are introduced into the host true oxygen Roche Bacteria (Ralstoniaeutropha) to obtain the recombinant bacteria; the host R. eutropha is R. eutropha Rem-1 or R. eutropha Rem-3 or R. eutropha Rem-5 or R. eutropha Rem-7 ; The amino acid sequence of the methylmalonyl-CoA mutase is shown in sequence 8 in the sequence listing; The amino acid sequence of the GTP kinase is shown in sequence 9 in the sequence listing; The amino acid sequence of the methylmalonyl-CoA decarboxylase is shown in sequence 10 in the sequence listing; The Rostella eutropha Rem-1 is Alcaligenes eutropha 65-7; The Rem-3 is a bacterium obtained by deleting the 2-methylcitrate synthase-1 gene prpC1 of Rostella eutropha Rem-1; The Rem-5 is a bacterium obtained after the deletion of the 2-methylcitrate synthase-2 gene prpC2 in Rostella eutrophica Rem-1; The Rem-7 is a bacterium obtained by deleting the 2-methylcitrate synthase-1 gene prpC1 and the 2-methylcitrate synthase-2 gene prpC2 of Rostella eutropha Rem-1; The amino acid sequence of the 2-methylcitrate synthase-1 is shown in sequence 2 in the sequence listing; The amino acid sequence of the 2-methylcitrate synthase-2 is shown as sequence 5 in the sequence listing. 2. The recombinant bacterium according to claim 1, characterized in that: the coding gene yliK of the methylmalonyl-CoA mutase, the coding gene argK of the GTP kinase and the methylmalonyl The coding gene ygfG of acid monoacyl-CoA decarboxylase is introduced into the host Roseburia eutropha through the recombinant expression vector pZMwf; The construction method of the recombinant expression vector pZMwf includes the following steps: inserting the yliK-argK-ygfG gene cluster fragment shown in sequence 7 in the sequence listing into the multiple cloning site of the expression vector pLXM1, and the obtained recombinant vector is designated as pZMwf. 3. The use of the recombinant bacterium according to claim 1 or 2 in the production of polyhydroxybutyrate-valerate. 4. The use of the recombinant bacterium according to claim 1 or 2 in improving the content of the 3-hydroxyvaleric acid component in the synthesis of poly-3-hydroxybutyric acid copolymerized 3-hydroxyvaleric acid ester. 5. A method for producing polyhydroxybutyrate-valerate, comprising the steps of: using glucose as a substrate, fermenting and culturing the recombinant bacteria described in claim 1 or 2, to obtain the polyhydroxybutyrate-valerate . 6. The method according to claim 5, characterized in that: the medium of the fermentation culture does not contain propionic acid or propionate or valeric acid or valerate.