CN112920984A - Construction is based on formic acid and CO2Method and application of growing recombinant strain - Google Patents

Construction is based on formic acid and CO2Method and application of growing recombinant strain Download PDF

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CN112920984A
CN112920984A CN202110162536.3A CN202110162536A CN112920984A CN 112920984 A CN112920984 A CN 112920984A CN 202110162536 A CN202110162536 A CN 202110162536A CN 112920984 A CN112920984 A CN 112920984A
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formic acid
glycine
growing
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recombinant strain
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王昕�
王静
陈可泉
廖杨
朱强强
王静雯
吴怡菲
欧阳平凯
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Nanjing Tech University
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Abstract

The invention discloses a method for constructing a composite material based on formic acid and CO2Methods and uses of growing recombinant strains. Exogenous genes of formate-tetrahydrofolate ligase (FtfL), methylenetetrahydrofolate dehydrogenase (MtdA) and methyltetrahydrofolate cyclohydrolase (Fch) are expressed in an escherichia coli engineering strain MG1655, and glycine cleavage system aminomethyltransferase (GcvT), glycine cleavage system H protein (GcvH) and glycine decarboxylase (GcvP) are overexpressed, so that the efficient utilization of substrates of formic acid and CO is constructed2The produced Escherichia coli. Meanwhile, glycine auxotroph strains are constructed to verify the feasibility of the path. The invention realizes the utilization of substrates formic acid and CO by escherichia coli for the first time2Production of Glycine to pyruvate into the tricarboxylic acid cycleThe biomass now increases.

Description

Construction is based on formic acid and CO2Method and application of growing recombinant strain
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a construction method based on formic acid and CO2Methods and uses of growing recombinant strains.
Background
Formic acid is an organic substance (Formic acid), the common name Formic acid, and is the simplest carboxylic acid. Colorless and pungent. It is used as an important organic-carbon resource, is one of basic organic chemical raw materials, and is widely applied to the industries of pesticides, leather, dyes, medicines, rubber and the like. In recent years, due to the influence of fluctuation of industrial demand, the formic acid production faces the dilemma of excess capacity, and a new transformation path is urgently needed to be developed to expand and extend the related industrial chain, and the biological route is one of important directions. The problems of slow growth, low formic acid metabolism efficiency, poor genetic modification caused by lack of molecular tools and the like of natural formic acid utilization microorganisms generally exist, and modification and optimization are urgently needed; the research of artificially constructing formic acid and utilizing microorganisms is still in the initial stage, and has great development space and is worth paying attention.
The CRISPR-Cas system is an immune defense system of bacteria, is formed by the evolution of bacteria in the process of long-term protection against exogenous DNA, can degrade invading exogenous DNA (viruses or phages and the like), and is widely present in bacteria and archaea. The II-type CRISPR-Cas9 system is most widely applied to the aspect of current gene editing. The system mainly comprises three parts: crRNA serving as a target sequence, tracrRNA capable of being connected with the crRNA in a pairing mode, and Cas9 protein for enzyme digestion of the target sequence. The CRISPR sequence transcribes mature crRNA and tracrRNA to form a partially double-stranded RNA structure through base complementary pairing, and then forms a complex with Cas9 protein to cut the exogenous DNA. Researchers integrate crRNA and tracrRNA into the same single strand, and design single-stranded guide RNA (sgRNA) with the length of about 20bp, wherein the sgRNA has two functions, namely complementary pairing with a target DNA sequence and simultaneously guiding Cas9 protein, so that the functions of gene knockout and the like are realized. In recent years, the CRISPR-Cas9 technology has been widely applied to genome editing and functional research of microorganisms, and particularly, great progress has been made in gene editing of bacteria.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for constructing a catalyst based on formic acid and CO2The construction realizes that the Escherichia coli can utilize formic acid and CO for the first time2The growth lays a foundation for producing compounds by using formic acid in the future, and has profound significance.
Construction is based on formic acid and CO2A method of growing a recombinant strain comprising the steps of:
step 1, constructing a pathway plasmid capable of utilizing formic acid to generate 5, 10-methylenetetrahydrofolate: pCWJ-ftfL-mtdA-fch;
step 2, constructing a glycine pathway plasmid capable of generating glycine by using 5, 10-methylenetetrahydrofolic acid: pTrc99a-rGCS;
step 3, CO-transforming pCWJ-ftfL-mtdA-fch and pTrc99a-rGCS into Escherichia coli MG1655, namely based on formic acid and CO2Growing the recombinant strain.
As an improvement, the pCWJ-ftfL-mtdA-fch construction method in the step 1 comprises the following steps: from methylotrophic bacteria (Methylobacterium extorquens Formate tetrahydrofolate synthetase (AM 1)FtfL) NADP dependent methylenetetrahydrofolate dehydrogenase (a)MtdA) And methyl tetrahydrofolate cyclohydrolase (Fch) After gene codon optimization, the gene is handed over to the Protechs Biotech Limited company of Oncology to be synthesized into the carrier pCWJEcoRI/Hind III between the enzyme cutting sites.
As an improvement, the construction method of pTrc99a-rGCS in step 2 is as follows: primers were selected and the aminomethyltransferase from E.coli MG1655 was replicated in operon form by PCRGcvT) Glycine cleavage system H protein: (GcvH) Glycine decarboxylase (c)GcvP) And then ligated with vector pTrc99 a.
In a further improvement, the upstream primer of the primer carriesNco I Cleavage site, downstream primer withBamH IAnd (4) enzyme cutting sites.
The above is based on formic acid and CO2Use of a growing recombinant strain to catalyse the degradation of formic acid. Has the advantages that:
compared with the prior art, the construction of the invention is based on formic acid and CO2The method and the application of the growing recombinant strain have the following specific advantages:
1. the method realizes that the cheaper formic acid is used as the substrate on the basis that the traditional industrial microorganism mainly uses glycosyl raw materials as fermentation substrates, and is economic and environment-friendly;
2. the method realizes the first time that formic acid and CO are used in the escherichia coli2Growth is realized for the source;
3. a glycine-related generating gene in Escherichia coli MG1655 is knocked out by using a Crispr-Cas9 technology, glycine-like defective cells are constructed, and the feasibility of a path can be accurately verified.
Drawings
FIG. 1 is a schematic diagram of the construction of plasmid pCWJ-ftfL-mtdA-fch;
FIG. 2 is a schematic diagram of the construction of plasmid pTrc99a-rGCS;
FIG. 3 is a gel diagram for verifying the construction of glycine auxotroph strain, wherein (a) isglyAThe gel map of successful gene knock-out is (b)aceA、ItaE、kblKnocking out a successful glue picture;
FIG. 4 is a diagram showing the growth of recombinant E.coli in M9 medium.
Detailed Description
The present invention is further described in the following description of the specific embodiments, which is not intended to limit the invention, but various modifications and improvements can be made by those skilled in the art according to the basic idea of the invention, within the scope of the invention, as long as they do not depart from the basic idea of the invention.
The techniques not mentioned in the examples are all conventional in the art, and in addition, use is made ofEscherichia coli MG655, methylotrophic bacterium (A)Methylobacterium extorquens AM 1), Trans1-T1, pCWJ, pTrc99a and other materials are all commercial products and can be purchased directly.
Example 1 construction of pCWJ-ftfL-mtdA-fch plasmid
Will be provided withFtfL、MtdA、FchAfter being optimized, the three genes are handed over to the Protechs Biotechnology Limited company of Oncology of department to be synthesized in carrier pCWEcoRI/Hind III(said formate tetrahydrofolate synthetase between the cleavage sitesFtfLThe nucleotide sequence after gene optimization is shown as SCN NO.1, and the methylenetetrahydrofolate dehydrogenaseMtdAThe nucleotide sequence of the optimized gene is shown as SCN NO.2, and the methyl tetrahydrofolate cyclohydrolaseFchThe nucleotide sequence of the optimized gene is shown as SCN NO. 3) is transferred into Escherichia coli Trans1-T1, a positive strain Trans 1-T1-pCWJ-ftfL-mtdA-fch is screened by PCR, DNA sequencing is carried out, and the construction of the recombinant plasmid is verified to be correct.
SCN NO.1
ATGCCGAGCGACATTGAAATTGCGCGCGCGGCGACCCTGAAACCGATTGCGCAGGTGGCGGAAAAACTGGGCATTCCGGACGAAGCGCTGCACAACTACGGCAAACACATCGCGAAAATTGATCATGATTTTATTGCGAGCCTGGAAGGCAAACCGGAAGGCAAACTGGTGCTGGTGACGGCGATTAGCCCGACCCCGGCGGGTGAAGGCAAAACCACGACGACCGTGGGTCTGGGCGATGCGCTGAACCGCATTGGCAAACGCGCGGTGATGTGCCTGCGCGAACCGAGCCTGGGCCCGTGTTTTGGTATGAAAGGCGGCGCGGCGGGCGGCGGTAAAGCACAGGTTGTGCCGATGGAACAGATCAACCTGCATTTTACCGGTGATTTTCATGCGATTACCAGCGCGCATAGCCTGGCGGCGGCGCTGATCGATAATCATATTTATTGGGCGAACGAACTGAACATTGACGTGCGCCGCATTCATTGGCGTCGCGTGGTGGATATGAACGATCGCGCACTGCGTGCGATTAACCAGAGCCTGGGTGGCGTGGCGAACGGCTTTCCGCGCGAAGATGGCTTTGACATTACCGTGGCGAGCGAAGTGATGGCCGTGTTTTGTCTGGCGAAAAACCTGGCGGATCTGGAAGAACGCCTGGGCCGCATTGTGATTGCGGAAACCCGCGATCGCAAACCGGTTACCCTGGCGGATGTGAAAGCGACCGGCGCGATGACCGTGCTGCTGAAAGATGCGCTGCAGCCGAACCTGGTGCAGACGCTGGAAGGTAACCCGGCGCTGATTCATGGCGGCCCGTTTGCGAACATTGCGCATGGCTGTAACAGCGTGATTGCGACCCGCACCGGCCTGCGTCTGGCCGACTACACCGTGACCGAAGCGGGCTTTGGCGCAGATCTGGGCGCGGAAAAATTTATCGATATTAAATGTCGCCAGACCGGCCTGAAACCGAGCGCGGTGGTGATTGTGGCGACCATTCGCGCGCTGAAAATGCATGGTGGCGTGAACAAAAAGGATCTGCAGGCGGAAAACCTGGATGCGCTGGAAAAAGGCTTTGCGAACCTGGAACGCCATGTGAATAACGTGCGTAGCTTCGGCCTGCCGGTGGTGGTGGGCGTGAATCACTTCTTTCAGGACACCGATGCGGAACACGCGCGCCTGAAAGAACTGTGCCGTGACCGCCTGCAGGTGGAAGCGATCACCTGCAAACATTGGGCCGAAGGCGGCGCGGGCGCGGAAGCACTGGCACAGGCAGTGGTGAAACTGGCCGAAGGTGAACAGAAACCGCTGACCTTTGCGTACGAAACCGAAACCAAAATTACCGACAAAATCAAAGCGATTGCGACCAAACTGTATGGTGCGGCGGATATTCAGATTGAAAGCAAAGCGGCGACCAAACTGGCCGGCTTCGAAAAAGACGGCTATGGCGGCCTGCCGGTTTGCATGGCGAAAACCCAGTATAGCTTCAGCACGGATCCGACCCTGATGGGCGCGCCGAGCGGTCATCTGGTGAGCGTGCGTGATGTGCGCCTGAGCGCGGGCGCAGGTTTCGTGGTGGTGATTTGTGGCGAAATTATGACGATGCCGGGCCTGCCGAAAGTGCCGGCGGCAGATACCATTCGCCTGGATGCGAATGGCCAGATTGATGGCCTGTTTTAA
SCN NO.2
ATGAGCAAAAAGCTGCTGTTTCAGTTTGACACCGACGCGACCCCGAGCGTGTTTGACGTGGTGGTGGGCTACGACGGCGGCGCAGATCACATTACCGGCTACGGCAACGTGACCCCGGATAACGTGGGCGCGTATGTTGATGGCACCATTTATACGCGCGGCGGCAAAGAAAAACAGAGCACCGCGATTTTTGTGGGTGGTGGCGATATGGCAGCGGGCGAACGCGTGTTCGAAGCGGTGAAAAAGCGTTTTTTTGGCCCGTTTCGCGTGAGCTGCATGCTGGATAGCAACGGTAGCAACACCACCGCGGCGGCGGGCGTTGCACTGGTTGTTAAAGCGGCCGGTGGCAGCGTTAAAGGTAAAAAGGCGGTGGTGCTGGCGGGCACCGGCCCGGTGGGTATGCGTAGCGCAGCGCTGCTGGCGGGTGAAGGCGCAGAAGTTGTGCTGTGTGGCCGTAAACTGGATAAAGCGCAGGCGGCGGCGGATAGCGTGAACAAACGCTTTAAAGTGAATGTGACCGCGGCGGAAACCGCCGATGATGCGAGCCGCGCGGAAGCGGTGAAAGGCGCACACTTTGTGTTTACCGCGGGCGCGATTGGTCTGGAACTGCTGCCGCAGGCGGCGTGGCAGAATGAAAGCAGCATTGAAATTGTGGCGGATTATAACGCGCAGCCGCCGCTGGGTATTGGTGGTATTGATGCGACCGATAAAGGCAAAGAATATGGTGGCAAACGTGCGTTCGGTGCGCTGGGCATTGGCGGCCTGAAACTGAAACTGCATCGTGCGTGCATTGCGAAACTGTTTGAAAGCAGCGAAGGCGTGTTTGATGCGGAAGAAATTTATAAACTGGCCAAAGAAATGGCATAA
SCN NO.3
ATGGCGGGTAACGAAACGATTGAAACCTTCCTGGACGGCCTGGCGAGCAGCGCGCCGACCCCGGGTGGTGGTGGTGCAGCAGCAATTAGCGGCGCGATGGGCGCGGCGCTGGTGAGCATGGTGTGCAACCTGACCATTGGCAAAAAGAAATATGTGGAAGTGGAAGCGGATCTGAAACAGGTGCTGGAAAAATCGGAAGGTCTGCGCCGTACCCTGACGGGCATGATTGCGGATGATGTGGAAGCATTTGACGCGGTGATGGGCGCGTATGGCCTGCCGAAAAACACCGATGAAGAAAAAGCGGCGCGCGCGGCGAAAATTCAGGAAGCACTGAAAACGGCGACCGATGTGCCGCTGGCGTGCTGCCGTGTGTGCCGTGAAGTTATCGATCTGGCGGAAATCGTGGCGGAAAAAGGTAACCTGAACGTGATTAGCGACGCCGGCGTGGCCGTGCTGAGCGCATATGCGGGCCTGCGCAGCGCGGCACTGAACGTTTATGTGAACGCGAAAGGCCTGGACGATCGTGCGTTCGCGGAAGAACGCCTGAAAGAACTGGAAGGCCTGCTGGCGGAAGCAGGTGCGCTGAATGAACGCATTTATGAAACCGTGAAAAGCAAAGTGAACTAA
The positive strain Trans 1-T1-pCWJ-ftfL-mtdA-fch was inoculated into 5ml LB/Cmr liquid medium consisting of: 10g/L peptone, 5g/L yeast powder and 5g/L sodium chloride, and culturing overnight at 37 deg.C and 200rpm with shaking.
After 24 hours, the plasmid pCWJ-ftfL-mtdA-fch was extracted according to the instructions of the Tiangen plasmid extraction kit. The construction schematic diagram is shown in figure 1.
Example 2 construction of pTrc99a-rGCS plasmid
Amplification of aminomethyltransferase from E.coli MG1655 in operon form by conventional PCR using E.coli MG1655 whole genome as templateGcvT) Glycine cleavage system H protein: (GcvH) Glycine decarboxylase (c)GcvP) A coding sequence;
the upstream primer used hasNco ICleavage site, sequence SCN NO. 4: CATGCCATGGCAATGGCACAACAGACTCCTTTG are provided.
The downstream primer hasBamH ICleavage site, sequence SCN No. 5: CGCGGATCCTTACTGGTATTCGCTAATCGGTACGC are provided.
The reaction conditions are as follows: 30 cycles of 95 ℃ for 2min, 95 ℃ for 20s, 55 ℃ for 20s, and 72 ℃ for 165 s; 5min at 72 ℃;
the obtained sequence is subjected to 1% agarose gel electrophoresis, and then the corresponding fragment is recovered; the sequence and expression vector pTrc99a were obtained from TakaraNco IAndBamH Ienzyme digestion, wherein the enzyme digestion reaction system is as follows: 10 Xbuffer H2. mu.l,Nco I 0.5μl，BamH I0.5. mu.l, gene fragment and pTrc99a vector 3. mu.l, H2O 14μl。
The digestion system was reacted at 37 ℃ for 2 hours. Connecting the enzyme digestion products, wherein the reaction system is as follows: 10 XLigase buffer 1. mu.l, T4 DNA Ligase (Takara) 1. mu.l, gene fragment 7. mu.l, vector 1. mu.l. The reaction was carried out at 25 ℃ for 3 hours. The ligation product was transformed into E.coli Trans 1-T1. Positive strain Trans1-T1-pTrc99a-rGCS is screened by PCR and DNA sequencing is carried out, and the construction correctness of the recombinant plasmid is verified.
The positive strain is inoculated to 5ml LB/Amp liquid culture medium, the composition of the LB/Amp liquid culture medium is 10g/L peptone, 5g/L yeast powder and 5g/L sodium chloride, and the shake culture is carried out overnight under the conditions of 37 ℃ and 200 rpm. After 24 hours, the plasmid pTrc99a-rGCS was extracted according to the instructions of the Tiangen plasmid extraction kit, and the construction scheme is shown in FIG. 2.
Example 3 verification of MG1655 (Δ glyA) -pCWJ-ftfL-mtdA-fch/pTrc99a-rGCS Strain
Firstly, the middle of Escherichia coli MG1655 is knocked out by using a Crispr-Cas9 editing technologyglyAThe gene whose knockout validation gel is shown in FIG. 3 (a). Then knock out the two simultaneouslyaceA、ItaE、kblThe gene whose knockout validation gel map is shown in FIG. 3 (b) gave the glycine auxotrophic strain MG1655 (Δ glyA).
Mu.l of pCWJ-ftfL-mtdA-fch and pTrc99a-rGCS plasmid were transferred to glycine auxotrophic strain by heat shock transformation to obtain recombinant E.coli MG1655 (Δ glyA) -pCWJ-ftfL-mtdA-fch/pTrc99 a-rGCS.
Culturing recombinant Escherichia coli MG1655 (Δ glyA) -pCWJ-ftfL-mtdA-fch/pTrc99a-rGCS until OD600 is 0.5-0.7, adding IPTG to the final concentration of 0.5-1 per mill, performing induced expression at 30 ℃ for 12h, collecting cells, washing twice with M9 culture medium, and re-suspending to obtain the cell re-suspension of the recombinant Escherichia coli. The composition of the M9 medium used was: m9 Medium (g.L)-1)：NH4Cl 10, NaCl 0.5, Na2HPO4·12H2O 17.1,KH2PO4 3, MgSO4 0.12, CaCl21.1×10-2。
Addition of the final concentration OD600The cells were resuspended at 0.1 to 10mL in a medium containing M9/AmpR/CmR, 30mM formic acid, and 50mM sodium bicarbonate, and their growth was observed, and the results were shown in FIG. 4 in which formic acid and CO were added2Under the conditions of (1), the strain realizes growth, and the judgment path is feasible.
The experiments show that the construction of the recombinant strain of the invention realizes that the Escherichia coli can utilize formic acid and CO for the first time2Growth of formic acid to produce compoundsThe foundation is determined, and the method has profound significance.
The above description is only a preferred embodiment of the present invention, and the scope of the present invention is not limited thereto, and any simple modifications or equivalent substitutions of the technical solutions that can be obviously obtained by those skilled in the art within the technical scope of the present invention are within the scope of the present invention.
Sequence listing
<110> Nanjing university of industry
<120> a method for constructing a recombinant strain based on formic acid and CO2 growth and application thereof
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1674
<212> DNA
<213> Artificial sequence (Artificial Gene)
<400> 1
atgccgagcg acattgaaat tgcgcgcgcg gcgaccctga aaccgattgc gcaggtggcg 60
gaaaaactgg gcattccgga cgaagcgctg cacaactacg gcaaacacat cgcgaaaatt 120
gatcatgatt ttattgcgag cctggaaggc aaaccggaag gcaaactggt gctggtgacg 180
gcgattagcc cgaccccggc gggtgaaggc aaaaccacga cgaccgtggg tctgggcgat 240
gcgctgaacc gcattggcaa acgcgcggtg atgtgcctgc gcgaaccgag cctgggcccg 300
tgttttggta tgaaaggcgg cgcggcgggc ggcggtaaag cacaggttgt gccgatggaa 360
cagatcaacc tgcattttac cggtgatttt catgcgatta ccagcgcgca tagcctggcg 420
gcggcgctga tcgataatca tatttattgg gcgaacgaac tgaacattga cgtgcgccgc 480
attcattggc gtcgcgtggt ggatatgaac gatcgcgcac tgcgtgcgat taaccagagc 540
ctgggtggcg tggcgaacgg ctttccgcgc gaagatggct ttgacattac cgtggcgagc 600
gaagtgatgg ccgtgttttg tctggcgaaa aacctggcgg atctggaaga acgcctgggc 660
cgcattgtga ttgcggaaac ccgcgatcgc aaaccggtta ccctggcgga tgtgaaagcg 720
accggcgcga tgaccgtgct gctgaaagat gcgctgcagc cgaacctggt gcagacgctg 780
gaaggtaacc cggcgctgat tcatggcggc ccgtttgcga acattgcgca tggctgtaac 840
agcgtgattg cgacccgcac cggcctgcgt ctggccgact acaccgtgac cgaagcgggc 900
tttggcgcag atctgggcgc ggaaaaattt atcgatatta aatgtcgcca gaccggcctg 960
aaaccgagcg cggtggtgat tgtggcgacc attcgcgcgc tgaaaatgca tggtggcgtg 1020
aacaaaaagg atctgcaggc ggaaaacctg gatgcgctgg aaaaaggctt tgcgaacctg 1080
gaacgccatg tgaataacgt gcgtagcttc ggcctgccgg tggtggtggg cgtgaatcac 1140
ttctttcagg acaccgatgc ggaacacgcg cgcctgaaag aactgtgccg tgaccgcctg 1200
caggtggaag cgatcacctg caaacattgg gccgaaggcg gcgcgggcgc ggaagcactg 1260
gcacaggcag tggtgaaact ggccgaaggt gaacagaaac cgctgacctt tgcgtacgaa 1320
accgaaacca aaattaccga caaaatcaaa gcgattgcga ccaaactgta tggtgcggcg 1380
gatattcaga ttgaaagcaa agcggcgacc aaactggccg gcttcgaaaa agacggctat 1440
ggcggcctgc cggtttgcat ggcgaaaacc cagtatagct tcagcacgga tccgaccctg 1500
atgggcgcgc cgagcggtca tctggtgagc gtgcgtgatg tgcgcctgag cgcgggcgca 1560
ggtttcgtgg tggtgatttg tggcgaaatt atgacgatgc cgggcctgcc gaaagtgccg 1620
gcggcagata ccattcgcct ggatgcgaat ggccagattg atggcctgtt ttaa 1674
<210> 2
<211> 867
<212> DNA
<213> Artificial sequence (Artificial Gene)
<400> 2
atgagcaaaa agctgctgtt tcagtttgac accgacgcga ccccgagcgt gtttgacgtg 60
gtggtgggct acgacggcgg cgcagatcac attaccggct acggcaacgt gaccccggat 120
aacgtgggcg cgtatgttga tggcaccatt tatacgcgcg gcggcaaaga aaaacagagc 180
accgcgattt ttgtgggtgg tggcgatatg gcagcgggcg aacgcgtgtt cgaagcggtg 240
aaaaagcgtt tttttggccc gtttcgcgtg agctgcatgc tggatagcaa cggtagcaac 300
accaccgcgg cggcgggcgt tgcactggtt gttaaagcgg ccggtggcag cgttaaaggt 360
aaaaaggcgg tggtgctggc gggcaccggc ccggtgggta tgcgtagcgc agcgctgctg 420
gcgggtgaag gcgcagaagt tgtgctgtgt ggccgtaaac tggataaagc gcaggcggcg 480
gcggatagcg tgaacaaacg ctttaaagtg aatgtgaccg cggcggaaac cgccgatgat 540
gcgagccgcg cggaagcggt gaaaggcgca cactttgtgt ttaccgcggg cgcgattggt 600
ctggaactgc tgccgcaggc ggcgtggcag aatgaaagca gcattgaaat tgtggcggat 660
tataacgcgc agccgccgct gggtattggt ggtattgatg cgaccgataa aggcaaagaa 720
tatggtggca aacgtgcgtt cggtgcgctg ggcattggcg gcctgaaact gaaactgcat 780
cgtgcgtgca ttgcgaaact gtttgaaagc agcgaaggcg tgtttgatgc ggaagaaatt 840
tataaactgg ccaaagaaat ggcataa 867
<210> 3
<211> 627
<212> DNA
<213> Artificial sequence (Artificial Gene)
<400> 3
atggcgggta acgaaacgat tgaaaccttc ctggacggcc tggcgagcag cgcgccgacc 60
ccgggtggtg gtggtgcagc agcaattagc ggcgcgatgg gcgcggcgct ggtgagcatg 120
gtgtgcaacc tgaccattgg caaaaagaaa tatgtggaag tggaagcgga tctgaaacag 180
gtgctggaaa aatcggaagg tctgcgccgt accctgacgg gcatgattgc ggatgatgtg 240
gaagcatttg acgcggtgat gggcgcgtat ggcctgccga aaaacaccga tgaagaaaaa 300
gcggcgcgcg cggcgaaaat tcaggaagca ctgaaaacgg cgaccgatgt gccgctggcg 360
tgctgccgtg tgtgccgtga agttatcgat ctggcggaaa tcgtggcgga aaaaggtaac 420
ctgaacgtga ttagcgacgc cggcgtggcc gtgctgagcg catatgcggg cctgcgcagc 480
gcggcactga acgtttatgt gaacgcgaaa ggcctggacg atcgtgcgtt cgcggaagaa 540
cgcctgaaag aactggaagg cctgctggcg gaagcaggtg cgctgaatga acgcatttat 600
gaaaccgtga aaagcaaagt gaactaa 627
<210> 4
<211> 33
<212> DNA
<213> Artificial sequence (Artificial Gene)
<400> 4
catgccatgg caatggcaca acagactcct ttg 33
<210> 5
<211> 35
<212> DNA
<213> Artificial sequence (Artificial Gene)
<400> 5
cgcggatcct tactggtatt cgctaatcgg tacgc 35

Claims (5)

1. Construction is based on formic acid and CO2A method of growing a recombinant strain comprising the steps of:
step 1, constructing a pathway plasmid capable of utilizing formic acid to generate 5, 10-methylenetetrahydrofolate: pCWJ-ftfL-mtdA-fch;
step 2, constructing a glycine pathway plasmid capable of generating glycine by using 5, 10-methylenetetrahydrofolic acid: pTrc99a-rGCS;
step 3, CO-transforming pCWJ-ftfL-mtdA-fch and pTrc99a-rGCS into Escherichia coli MG1655, namely based on formic acid and CO2Growing the recombinant strain.
2. A process according to claim 1 based on formic acid and CO2The method for growing the recombinant strain is characterized in that the pCWJ-ftfL-mtdA-fch in the step 1 is constructed by the following steps: synthesizing formate tetrahydrofolate synthetase, NADP dependent methylenetetrahydrofolate dehydrogenase and methyltetrahydrofolate cyclohydrolase from methylotrophic bacteria into vector pCWJ by gene codon optimization and then transferring the vector pCWJ to Onck Biotechnology LimitedEcoRI/Hind III between the enzyme cutting sites.
3. A process according to claim 1 based on formic acid and CO2The method for growing the recombinant strain is characterized in that the pTrc99a-rGCS construction method in the step 2 comprises the following steps: primers were selected, and the aminomethyltransferase, glycine cleavage system H protein, glycine decarboxylase on E.coli MG1655, and the like were replicated in operon form by PCR, and then ligated with vector pTrc99 a.
4. A construction according to claim 3 based on formic acid and CO2Method for growing recombinant strains, characterized in that the primer upstream of said primer carriesNco I Cleavage site, downstream primer withBamH IAnd (4) enzyme cutting sites.
5. Based on formic acid and CO as per one of claims 1 to 42Use of a growing recombinant strain to catalyse the degradation of formic acid.
CN202110162536.3A 2021-02-05 2021-02-05 Construction is based on formic acid and CO2Method and application of growing recombinant strain Pending CN112920984A (en)

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CN114480239A (en) * 2022-02-26 2022-05-13 南京工业大学 Reconstructed methylotrophus bacterium for synergistically assimilating methanol by utilizing WLP (white Spot protein) pathway and reductive glycine pathway and application thereof

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* Cited by examiner, † Cited by third party
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CN114015620A (en) * 2021-12-13 2022-02-08 青岛农业大学 Demethylating bacillus evolved strain capable of efficiently utilizing formic acid and application thereof
CN114480239A (en) * 2022-02-26 2022-05-13 南京工业大学 Reconstructed methylotrophus bacterium for synergistically assimilating methanol by utilizing WLP (white Spot protein) pathway and reductive glycine pathway and application thereof
CN114480239B (en) * 2022-02-26 2024-02-13 南京工业大学 Restructuring bacillus methyl butyrate for synergetically assimilating methanol by utilizing WLP (wlP) pathway and reductive glycine pathway and application thereof

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