CN113913448A - Method for increasing yield of methylotrophic bacteria pyrroloquinoline quinone and application - Google Patents
Method for increasing yield of methylotrophic bacteria pyrroloquinoline quinone and application Download PDFInfo
- Publication number
- CN113913448A CN113913448A CN202110838234.3A CN202110838234A CN113913448A CN 113913448 A CN113913448 A CN 113913448A CN 202110838234 A CN202110838234 A CN 202110838234A CN 113913448 A CN113913448 A CN 113913448A
- Authority
- CN
- China
- Prior art keywords
- sequence
- bacteria
- pyrroloquinoline quinone
- methylotrophic
- homology arm
- 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.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/182—Heterocyclic compounds containing nitrogen atoms as the only ring heteroatoms in the condensed system
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Mycology (AREA)
- Medicinal Chemistry (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for preparing engineering bacteria for producing pyrroloquinoline quinone, which is characterized by comprising the following steps: TCS3 in the genome of methylotrophic bacteria is replaced by a screening marker gene, so that the engineering bacteria for producing pyrroloquinoline quinone are obtained. The growth rate of the knockdown bacterium J1-1. DELTA. mpq-0782-0783 of the present invention is faster than that of the wild bacterium J1-1The time reaching the plateau period is short, and the maximum OD after reaching the plateau period600The value is 7.7 percent higher than that of the wild strain J1-1. The production rate and growth condition of PQQ are consistent and higher than those of wild bacteria J1-1, and the highest PQQ yield of the knockout bacteria is increased by 11.3 percent compared with that of wild bacteria J1-1.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for improving the yield of methylotrophic bacteria pyrroloquinoline quinone and application thereof.
Background
Pyrroloquinoline quinone (PQQ) is a quinone compound synthesized by methylotrophic bacteria, and is a reddish brown small molecule compound, namely a third class of oxidoreductase coenzymes except NAD (P) and FAD. Has important physiological functions such as scavenging oxygen free radicals, enhancing mitochondrial function, regulating immunity, promoting cognition, preventing and treating radiation injury and liver injury, promoting wound healing, etc. The chemical synthesis process of PQQ is complex, serious in pollution and high in cost, and microbial fermentation is the current industrial preparation process of PQQ. Gram-negative bacteria are the largest group of organisms reported to be able to synthesize PQQ, with methylotrophic bacteria producing the highest levels. And (3) screening a plurality of methylotrophic bacteria capable of producing PQQ from soil in the early stage, and selecting one strain (MP688) with the highest yield to perform continuous mutagenesis by methods such as ultraviolet, nitrosoguanidine and the like to obtain the PQQ high-producing bacteria J1-1. At present, the further improvement of PQQ yield by physical and chemical mutagenesis is difficult, and another effective means is urgently required to be searched.
Disclosure of Invention
The invention aims to obtain a PQQ high-producing strain through metabolic engineering modification, and further obtain a method for improving the yield of methylotrophic bacteria pyrroloquinoline quinone by utilizing the PQQ high-producing strain and application thereof.
The invention provides a method for preparing engineering bacteria for producing pyrroloquinoline quinone, which comprises the following steps: replacing TCS3 in the genome of methylotrophic bacteria with a screening marker gene to obtain engineering bacteria for producing pyrroloquinoline quinone; the sequence of the TCS3 is shown as a sequence 1 in a sequence table.
Further, the selection marker gene is a Gm gene; the sequence of the Gm gene is a sequence 2 in a sequence table.
Further, the methylotrophic bacterium is methylotrophic bacterium J1-1.
Furthermore, the TCS3 in the methylotrophic bacterium J1-1 is replaced by a Gm gene in a homologous recombination mode, the sequence of an upstream homology arm is shown as a sequence 3 in a sequence table, and the sequence of a downstream homology arm is shown as a sequence 4 in the sequence table.
Further, the method comprises the steps of: introducing a recombinant vector into methylotrophic bacteria J1-1, wherein the recombinant vector contains the upstream homology arm, the Gm gene and the downstream homology arm which are sequentially connected from 5 'end to 3' end, and the upstream homology arm and the downstream homology arm are subjected to homologous recombination with a homologous fragment on the genome of methylotrophic bacteria J1-1 to obtain the engineering bacteria for producing pyrroloquinoline quinone.
Further, the recombinant vector is obtained by inserting the upstream homology arm, the Gm gene and the downstream homology arm which are sequentially connected from the 5 'end to the 3' end between the enzyme cutting sites BamH I and Apa I of the pWM91 vector.
The engineering bacteria prepared by the method for producing pyrroloquinoline quinone also falls into the protection scope of the invention.
The application of the method or the engineering bacteria in the production of pyrroloquinoline quinone also falls within the protection scope of the present invention.
The invention also provides a method for producing pyrroloquinoline quinone, which comprises the step of carrying out fermentation culture on the engineering bacteria and preparing pyrroloquinoline quinone from a fermentation product.
The fermentation product refers to fermentation liquor obtained after the engineering bacteria are subjected to fermentation culture.
The growth rate of the knockdown bacterium J1-1 delta mpq-0782-0783 is faster than that of the wild bacterium J1-1, the time for reaching the plateau phase is short, and the maximum OD is reached after the plateau phase is reached600The value is 7.7 percent higher than that of the wild strain J1-1. The production rate and growth condition of PQQ are consistent and higher than those of wild bacteria J1-1, and the highest PQQ yield of the knockout bacteria is increased by 11.3 percent compared with that of wild bacteria J1-1.
Drawings
FIG. 1 is a graph of linearized plasmid and knock-out fragment recovery, where M: marker; 1: linearized particles pWM 91; 2: knocking out upstream homology arms of target fragments of the genes, wherein the size of the upstream homology arms is 1500 bp; 3: knocking out a resistance fragment of a target fragment of the gene, wherein the size of the resistance fragment is 750 bp; 4: the downstream homology arm of the target fragment of the knocked-out gene is 1500bp in size.
FIG. 2 is a knockout plasmid colony PCR plot wherein: m: marker; 1: single colonies were used as templates.
FIG. 3 is a restriction map of a knockout plasmid, wherein: m: marker; 1: knock-out plasmid double-enzyme cutting map.
FIG. 4 shows the electrotransformation plate of J1-1. DELTA. mpq-0782-0783.
FIG. 5 is a knockout strain PCR validation in which: m: marker; 1-3: 3 single colonies were used as templates.
FIG. 6 shows the results of resistance screening of knockout strains.
FIG. 7 is a J1-1. DELTA. mpq-0782-0783 anchor validation in which: m: marker; 1: wild-type J1-1 anchored PCR control; 2: the TCS3 knock-out strain was anchored for PCR validation.
FIG. 8 is a phenotype assay for wild type strain J1-1 and knockdown bacterium J1-1. DELTA. mpq-0782-0783, wherein: (a) a growth curve; (b) the method comprises the following steps PQQ yield curve.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Methylotrophic bacteria J1-1 (China general microbiological culture Collection center CGMCC4096), Escherichia coli lambda competence (Shanghai Weidi Biotech, Inc.), pWM91 plasmid, and pBBR1MCS-5 plasmid (Wuhan vast Ling Biotech, Inc.) in the examples described below.
Solvent CaCl in the following examples2The formulation of the glycerol solution is: 60mM CaCl215% glycerol, 10mmol/L PIPES, pH 7.0.
Trans 2K Plus II DNA Marker in the following examples,
The MultiS recombinant cloning kit, the agarose gel DNA recovery kit and the plasmid miniprep kit are purchased from Davidae; kanamycin (Kan) 40mg/mL, gentamicin (Gm) 40mg/mL, Trans Taq HiFi DNA Polymerase (all-type gold), restriction enzymes BamH I, Apa I, Xba I and Kpn I,T4 DNA ligase (TaKaRa);
the formulation of LB liquid medium in the following examples is: weighing 10g NaCl, 5g yeast extract and 10g peptone, dissolving in 800mL ddH2And in O, keeping the volume to 1L.
The formulation of methylotrophic bacteria seed Medium (MP) in the following examples is: MgSO (MgSO)4·7H2O 0.2g/L，(NH4)2SO43 g/L，KH2PO41.4 g/L，Na2HPO4·12H2O3 g/L, ferric citrate 30mg/L, MnCl2·4H2O 5mg/L，ZnSO4·7H2O 5mg/L，CuSO4·5H2O 0.5mg/L。
The formulation of methylotrophic fermentation medium (HM) in the following examples was: KH (Perkin Elmer)2PO42.8 g/L、Na2HPO46g/L ferric citrate 60mg/L, MnCl2·4H2O 5mg/L，ZnSO4·7H2O 5mg/L，CuSO4·5H2O 0.5mg/L、MgSO4·7H2O 0.2g/L，(NH4)2SO43g/L, no CaCl2。
Example 1 construction of an engineering bacterium for producing pyrroloquinoline quinone
1. Knock-out plasmid construction
Amplifying an upstream (Up) homologous arm fragment of TCS3 by using a primer 1F/1R by taking a wild type (methylotrophic bacterium J1-1) as a template; wherein, the sequence of the primer 1F is shown as the sequence 5 in the sequence table, the sequence of the primer 1R is shown as the sequence 6 in the sequence table, the sequence of the upstream (Up) homologous arm is shown as the sequence 3 in the sequence table, and the sequence of the TCS3 fragment is shown as the sequence 1.
Amplifying a downstream (Down) homologous arm fragment of the TCS3 fragment by using a primer 3F/3R by using a wild type (methylotrophic bacterium J1-1) as a template; wherein, the sequence of the primer 3F is shown as the sequence 9 in the sequence table, the sequence of the primer 3R is shown as the sequence 10 in the sequence table, and the sequence of the downstream (Down) homologous arm fragment is shown as the sequence 4 in the sequence table.
And (2) amplifying a middle Gm resistant fragment by using a pBBR1MCS-5 plasmid as a template and using a corresponding primer 2F/2R, wherein the sequence of the primer 2F is shown as a sequence 7 in a sequence table, the sequence of the primer 2R is shown as a sequence 8 in the sequence table, and the sequence of the middle Gm resistant fragment is shown as a sequence 2 in the sequence table.
Primer sequence table:
1-F 38bp
TCCGGTAAGGAGGAATTCTAACATGGCCTGTATCGCGG
1-R 35bp
TTCCACGGTGTGCGTCGCCTGACATCAGTGCAGAA
2-F 39bp
TTTTTCTGCACTGATGTCAGGCGACGCACACCGTGGAAA
2-R 39bp
TCTCCAGGCAGCGACAGCCGCTTAGGTGGCGGTACTTGG
3-F 33bp
CCCAAGTACCGCCACCTAAGCGGCTGTCGCTGC
3-R 38bp
TGGTGGTGGTGGTGCTCGAG TACTTTACCCAGCGCGCC
the above amplification was performed using Trans Taq HiFi DNA Polymerase, the PCR system is shown in Table 1, and the reaction conditions are shown in Table 2.
TABLE 1 PCR amplification System
TABLE 2 PCR reaction conditions
After the fragment is amplified, the required plasmid pWM91 is cut by enzyme, the pWM91 plasmid is cut by restriction enzymes BamH I and Apa I, the cut plasmid system is shown in Table 3, and the reaction conditions are as follows: reacting at 37 ℃ for 30min, and inactivating at 65 ℃ for 10min to obtain the linearized plasmid pWM 91.
TABLE 3 linearized plasmid System
And recombining the recovered linearized plasmid pWM91 with a recovered fragment (consisting of an upstream homology arm fragment, a Gm fragment and a downstream homology arm fragment) to obtain a recombinant plasmid pWM91-Up-Gm-Down, wherein the recombinant plasmid pWM91-Up-Gm-Down is obtained by replacing a sequence between enzyme cutting sites of BamH I and Apa I of pWM91 with a recovered fragment sequence and keeping other sequences unchanged. The above recombination system is shown in Table 4. The recombination reaction condition is 1h at 37 ℃ and then ice bath is carried out for 5 min.
Recovering the PCR amplified knockout fragment by 1% agarose gel electrophoresis; the linearized plasmid pWM91 was recovered by 1% agarose gel electrophoresis. The results are shown in FIG. 1.
TABLE 4 recombination reaction System
The constructed recombinant plasmid is transformed into the escherichia coli lambda competence by heat shock, and the steps are as follows:
(1) adding 50 μ L of lambda competence to the recombinant reaction product;
(2) carrying out ice bath for 30min, and then carrying out heat shock for 45s at 42 ℃;
(3) ice-bath for 2min, adding 1mL LB basic culture medium, and culturing at 37 deg.C and 100rpm for 1 h;
(4) centrifugation was carried out at 3000rpm for 10min, the supernatant was discarded, and 200. mu.L of LB was used for resuspension and plating onto Kan-resistant plates (Kan-resistant first to verify the seamless cloning results).
(5) Culturing at 37 deg.C for 12 h.
2 validation of Gene knockout plasmids
And (3) selecting positive transformants on the Kan resistant plate to be cultured in a Gm resistant LB culture medium for 24h at 37 ℃, carrying out PCR verification on strains growing in the Kan resistant plate, extracting plasmids, carrying out double enzyme digestion verification by using Apa I and BamH I (the double enzyme digestion system is the same as the table 2-3, and the system size is 10 mu L), carrying out electrophoresis detection, confirming the absence of errors, preserving and using for subsequent operation.
Constructing knock-out plasmid by a seamless cloning method, carrying out chemical transformation by using escherichia coli lambda competence, and screening transformants by using Gm resistant LB. And (3) carrying out electrophoresis detection after the construction of the PCR verification plasmid, wherein the size of the fragment is consistent with the expected result (the result is shown in figure 2), extracting the knockout plasmid, carrying out double-enzyme digestion verification, and the electrophoresis result shows that the double-enzyme digestion is successful (the result is shown in figure 3), which indicates that the construction of the knockout plasmid is successful.
3 construction and identification of engineering bacteria for producing pyrroloquinoline quinone
And (3) preparing electrotransformation competence by using methylotrophic bacteria J1-1, and electrotransfering the correct knockout plasmid verified in the step (2) into J1-1 competent cells to construct knockout bacteria so as to obtain knockout bacteria J1-1 delta mpq-0782-0783. The method comprises the following specific steps:
(1) add 8. mu.L of knock-out plasmid to the 50. mu. L J1-1 electroporation competence and add to the electroporation cuvette;
(2) after the voltage is changed under 2.5kV, 1mL of MP culture medium is immediately added and moved to a 1mL of EP tube;
(3) culturing at 30 deg.C and 100rpm/min for 4 hr;
(4) centrifuging at 3000rpm/min for 10min, discarding the supernatant, and coating on Gm resistant plate with 200 μ L MP culture medium by weight;
(5) culturing for 72h in a constant temperature incubator at 30 ℃;
and performing PCR verification, resistance verification and anchoring PCR on the constructed knockout bacteria for knocking out bacteria.
Making electrotransformation competence by using methylotrophic bacteria J1-1, electrically transducing correct knockout plasmid constructed in the step 2 into methylotrophic bacteria J1-1 competence to obtain an electrotransfer plate (as shown in figure 4), selecting a single colony Gm resistance for screening, performing preliminary PCR verification on knockout strains, wherein the electrophoresis detection result is consistent with the expectation, the PCR size is 3789bp (as shown in figure 5), screening knockout strains by using the Gm resistance plate, screening out mutant strains in a Gm resistance MP culture medium, inoculating the knockout strains into a Kan resistance culture medium, not growing in the Kan resistance culture medium (as shown in figure 6), and performing anchoring PCR verification on the preliminarily obtained knockout bacteria (as shown in figure 7), wherein the results show that the knockout bacteria are constructed successfully.
Example 2 determination of growth and PQQ Synthesis Capacity of engineering bacteria for production of pyrroloquinoline quinone
Respectively activating wild bacteria J1-1 and knockdown bacteria J1-1 delta mpq-0782-0783, culturing in MP culture medium at 30 ℃ and 200rpm/min to a logarithmic phase, inoculating into HM culture medium added with 1% methanol according to the inoculum size of 1%, sampling at certain intervals to determine the cell growth and PQQ yield change curve, and the result is shown in FIG. 8.
The method for calculating the yield of PQQ comprises the following steps: the culture supernatant was measured for OD326 and OD400 by an ultraviolet spectrophotometer, and the PQQ content in the supernatant was calculated by the formula (OD326-OD 400). times. 47.619+ 0.3333.
The results show that in the determination process, the growth rate of the knockdown bacterium J1-1 delta mpq-0782-0783 is faster than that of the wild bacterium J1-1, the time for reaching the plateau phase is shorter, and the strain is cultured for 120h to reach the maximum OD600The value is 3.22, while the maximum OD of the wild-type strain J1-1 is reached at 120h600The value was 2.99. Knockdown bacteria J1-1. DELTA. mpq-0782-0783 maximum OD after plateau600The value was 7.7% higher than that of wild strain J1-1 (shown as a in FIG. 8). The production rate and growth condition of the PQQ of the knock-out bacterium J1-1 delta mpq-0782-0783 are consistent and higher than those of the wild strain J1-1, the yield is highest at 132h and the highest yield is 108.93mg/L, while the wild strain J1-1 achieves the highest yield of 97.87mg/L at 120h, and the highest PQQ yield of the knock-out bacterium is 11.3 percent higher than that of the wild strain J1-1 (shown as b in FIG. 8).
The above experimental results show that the growth rate of the knockdown bacterium J1-1 delta mpq-0782-0783 is faster than that of the wild bacterium J1-1, the time for reaching the plateau phase is short, and the maximum OD is reached after reaching the plateau phase600The value is 7.7 percent higher than that of the wild strain J1-1. The production rate and growth condition of PQQ are consistent and higher than those of wild bacteria J1-1, and the highest PQQ yield of the knockout bacteria is increased by 11.3 percent compared with that of wild bacteria J1-1.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Sequence listing
<110> military medical research institute of military science institute of people's liberation force of China
<120> method for improving methylotrophic bacterium pyrroloquinoline quinone yield and application
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2084
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atgtcggatg aagcctatct ggtggatgct aataacatgc aactggcctg cgtcagcagt 60
caggcgctgg tcagtcatca atgcacgctg acgcaattgc atgcaatgcc attgcacgcg 120
gtgctcggca tgcccgagac cgagcttgtg cagtggcagg cccagtgcgt gaaacagcct 180
tcaggcgcac cgttgacgac gctgttatta cccaaggccg atggcgaaaa agggcggcgt 240
gcaggcgaca gcaccaaagt gagctttttt gaggccgaag ggcggtcctg gatgctggtt 300
atgaaaaaca gcgtcagccg ctcagccatg caggcgctgg atgacagcga gtcgcgcttt 360
caggcgattg tggccaacac gccgggtctg gtattccagt tcagattgct ggggccgggc 420
cggctgggtt ttatctatct gagcgaggcc tgttatgccg tgctgggcat taccgaagcc 480
gagttgacca agacgccatc gcagttttac cggctgatgg cagaagaaga ccggccatcg 540
ttccgtgaag ccttgctgac ctcggccaat gaaatgaagg tattgaactg ggagggccgc 600
atctggatca aggcctggca ggataccaag tggatcaacc tgcgtggcag tcccatcaag 660
ctgcacgatg gcgaagtgca atggggcggc atcattacca acattacgca gagcaaaaac 720
gaaaagctcg agattgaacg ctcacgccag cgcatggccg agctgtctgc ccacctgaca 780
caggtgaaag agcaggagcg tcggcgcatt gcgcgcgaga tacatgatga tctcggtggc 840
aacctgaccg cgatcaagat cggccttacc tccattatca agcgtatcaa tcccgatgaa 900
cccaagctgg tggagaaggc acacaatctg gaaagcattg tcgatcgcac ctttgatatc 960
gtgcatcgca ttgccagtga cttgcggccg gatgtgctgg aactgggcat cgtcgcggcg 1020
ctggagtggc aatgcgggca atttgaaaag cagttttccc tgccgtgccg gttttacagc 1080
tcgcagaccg agctggctgt cagtgccgat gaggcgatca cgctgttccg catctgccag 1140
gaggcgatgt ccaatatcgc caagcatgcc agggccaggc aggtggaagt tgagcttctg 1200
gtcgaagacc gcgatatcat gctgcgtatc gtcgatgacg gtcagggcat acgcatgcag 1260
gaccagcata aatccaactc ttttggtctg cggggcatgc gggagcgcgt cgctgcgctg 1320
gatggcagtc ttgaagtcga aaaacgtgta cgtgggggta cagcggtgca ggttaaaatt 1380
ccacgcagtc tcaagtaaga ttgcgacata acgaaggtaa tacggggaca agcatttata 1440
tgcctgcaga taaaaaaata aaggtcatca tcgccgatga ccatgcgatt cttcgcgaag 1500
ggctgaagca gatattgtcg gaaaccgagg atatcgaagt catcgccgag gcgcaaaatg 1560
ccagtgaggc catccactac gcctgcaaac tcgaggccga tgtgcttctg ctggatattt 1620
cattgccgga ccgcagtggt atcgatgcgc tgcgctatat caaaaaggaa aacaccaaga 1680
tcgccatcct catgctgtcc atgcatcgtg aggatcaata cgccatccgc tcgctcaagg 1740
ccggcgcagc tggctatctg ggcaagcagg gtgcctcttc cgagctggtg aatgcgatcc 1800
gggtgatcgc caagggcaag aaatacatta cgccggaagt ggccgacgcc ctggccaatc 1860
aggtgatgga taccgagaag cccttgcatg agtcgctgtc cgaccgcgaa ttccagacgc 1920
tgacgctgat tgcctccggc ctgacggtgg gtgagattgc cctgcggctg tcgctatccg 1980
tcaaaaccgt gagcatgtac cgcacccggc ttctggaaaa aatgcagctg aagcacaacg 2040
ctgaactcac gcactacgcg ataaaaaaca atctggtcga ttag 2084
<210> 2
<211> 789
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gacgcacacc gtggaaacgg atgaaggcac gaacccagtt gacataagcc tgttcggttc 60
gtaaactgta atgcaagtag cgtatgcgct cacgcaactg gtccagaacc ttgaccgaac 120
gcagcggtgg taacggcgca gtggcggttt tcatggcttg ttatgactgt ttttttgtac 180
agtctatgcc tcgggcatcc aagcagcaag cgcgttacgc cgtgggtcga tgtttgatgt 240
tatggagcag caacgatgtt acgcagcagc aacgatgtta cgcagcaggg cagtcgccct 300
aaaacaaagt taggtggctc aagtatgggc atcattcgca catgtaggct cggccctgac 360
caagtcaaat ccatgcgggc tgctcttgat cttttcggtc gtgagttcgg agacgtagcc 420
acctactccc aacatcagcc ggactccgat tacctcggga acttgctccg tagtaagaca 480
ttcatcgcgc ttgctgcctt cgaccaagaa gcggttgttg gcgctctcgc ggcttacgtt 540
ctgcccaagt ttgagcagcc gcgtagtgag atctatatct atgatctcgc agtctccgga 600
gagcaccgga ggcagggcat tgccaccgcg ctcatcaatc tcctcaagca tgaggccaac 660
gcgcttggtg cttatgtgat ctacgtgcaa gcagattacg gtgacgatcc cgcagtggct 720
ctctatacaa agttgggcat acgggaagaa gtgatgcact ttgatatcga cccaagtacc 780
gccacctaa 789
<210> 3
<211> 1500
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
catggcctgt atcgcggtat tgaagaaacc cgcaagctca agctcgatac cctggtgttt 60
ctgaccgatg gtcagcaggc accgcccgcc aatccgcggt acatgccggt gtttgatggc 120
aaaccgggcg aagtaggcgg catcattgcc ggggtcggcg ggctgcaacg gcaagccatc 180
cccaagctcg atgaaaacga caacattacc ggctactggc tgccggaaga tgccatgcaa 240
tttgccacct tcggcatgtc ccacgtgcag tctgtgctgg aaatggaagg ttaccatgga 300
cgcaatgcgc cgcatggcgc acatcccgaa ggctcgtata ccgagcatat gtcggccctg 360
gatgaaaaag gcctgcagga actcgccaag ataaccggtt tgcattatct gcatctgcag 420
gacaaggccc agctggccga tgccctgcgt cagaacacct tgcaccggca gtgggttgcc 480
agcgatatcc gtcccgtatt tgccgggctg gccttgctcc tgctgctggc ctcgtatttc 540
cccgctttta tttcatcatt gttcaagaaa ggaaaaccat gaaatcatgg attgcagcac 600
tgttattggc attacccctg gcggcgtggg cgcatggccc caccccgcag aaagccgagg 660
aaaccgtaga gatcaatgcg gcacccgctg ccgtctgggc caagatcaag gattttggcg 720
cgattgccag ctggcaacct gccgtggaaa aagtggcggc tgaaggcggc aatggcaatg 780
gcgccaagcg tacgctgaca ttcaagagcg gcggtgtgct ggaagaagaa ctggatgatt 840
actcggatgc cgagatgcgc tatgcctacc gcatgggccg tgacatggat atcaaggcct 900
tgccggtcag ctcgctgaat ggtcgtctgg aagtgaccgc aggcaaggac ggcaagaccg 960
tggtgacctg gaaggctcgt ttctaccgtg cctttaccgg caatgagccg ccagccgggc 1020
aggatgatgc cacggcagtg gaaggcgtga ctgcctattt ccgtcagggt ctggataatc 1080
tgaagcagct ggtggaaagc ggcaagtaac tttcgggaaa aattcccgag gtttttcggg 1140
atggccagtg tagtggcaac agacggcgac ctttagtatg caacacatgc tctagtgcaa 1200
ttgaaatgtc aggaatgacg gttcgcgtgt cgcacaatgt tcaggcatac tccatagaaa 1260
ttcaaccaga cttgctctcc aggtctggtt tttttttgcc ggttatcggc cggtctgatc 1320
ctccccgcaa ttaacgcttg tgaaattatg tggatgcgat tatcttaaaa tcgcttgcac 1380
gccttgtgct gactgcctgc aagcggcttg atctccataa aaacttatcc atgaacaaca 1440
ggtgatgtct tgctagaggc aacgatagcc aatccatttt tttctgcact gatgtcaggc 1500
<210> 4
<211> 1500
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
gcggctgtcg ctgcctggag acagcgcaga tgtgagtcca cgtctctggt ctgaccctac 60
tctggcaaag ctcagccgtg ggttttgccg atttaagcgg tttttacctc cctgttaggc 120
tgtttgcatt gtcgtctgtt tgattagcat ggcggaatca aactatagac ggagcctttg 180
cccgagcggc aaaggagatg cagccatggc agaccctact ctccatatct atcagcagaa 240
acccgccttg ctcggccagg aaatcctggc acgactggag gcgcgtgaca ttgcgcctac 300
gcctgaagcc tatcaggcct tgtatctgga attgactgga caacaggccg ccgcgccgac 360
gagcgcccca caacagggca gcccgtcgcc tgccgctccc agcatgctgg agcagcgctt 420
tcagaaagtg gcccgccagc tggcgaaaaa ctccaatgaa gcggggatga agctggccta 480
tgccattgag aatgaagact gggatgcatt gctggacggg attttcaagc tggtgaaggc 540
ctcggtggcc atccagtcgc aacggtcccg gtttgccgcc tttggtgatt taagcaatgc 600
cgcagatgat ggccgaccct ggcaggatca gcttgccaag atgttggcac atactttgcc 660
tgcattgttg cagaacgaat ccgagctggc catgaagtca cgtcagctgg ctgaacaggt 720
gaagacaaca caatcttacc gtgccatgca aaaggtcatg cgcgaggtca atgagctcag 780
ttatcgcatc agcatggtga ccgaagatga gtacggcaag cagaaagcgt tcatccgttt 840
gctcaatttg acactggata gtctcaccga agtgatccaa agcgacgatc aagtcctgca 900
tgaagtgcgg aattttcaga aaatcgttaa tgagccatgg gaccaaacca agctaaacaa 960
ggcagcaaag cagttgacca cgcgagtttc ggctcagcgt acataagttt aagtaattga 1020
tttttatagt tcttaataaa aatatatgag aaaggtttaa agatttgtta attacggccg 1080
tcatcaaaca caagattgct tgattcggaa tgcttgaaaa tttaaatagc caaccccgct 1140
aaattttcac taaaagtaac cgataaacaa tttaacagcg gcgcgatgaa cgcaatgctg 1200
aaatgcaaag taagtgtttg aatcaattaa caaaggagaa ttaccatggc tgctgtaatt 1260
aacaccaaca tcgcctcgct gaatgctcaa cgtaacctgt ctgcttctca gtcgagcctg 1320
aatacttcgc tgcaacgttt gtcttctggt ctgcgcgtaa acagcgctaa ggatgacgcc 1380
gctggtatgt ccatcgcaac gcgtatggac tcccaggttc gcggtcaaac tgttgcgatc 1440
cgtaacgcta acgatgcgat ctctttcgca caaactgctg aaggcgcgct gggtaaagta 1500
<210> 5
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
tccggtaagg aggaattcta acatggcctg tatcgcgg 38
<210> 6
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ttccacggtg tgcgtcgcct gacatcagtg cagaa 35
<210> 7
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
tttttctgca ctgatgtcag gcgacgcaca ccgtggaaa 39
<210> 8
<211> 39
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
tctccaggca gcgacagccg cttaggtggc ggtacttgg 39
<210> 9
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
cccaagtacc gccacctaag cggctgtcgc tgc 33
<210> 10
<211> 38
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
tggtggtggt ggtgctcgag tactttaccc agcgcgcc 38
Claims (9)
1. The method for preparing the engineering bacteria for producing the pyrroloquinoline quinone is characterized by comprising the following steps of: replacing TCS3 in the genome of methylotrophic bacteria with a screening marker gene to obtain engineering bacteria for producing pyrroloquinoline quinone; the sequence of the TCS3 is shown as a sequence 1 in a sequence table.
2. The method of claim 1, wherein: the screening marker gene is a Gm gene; the sequence of the Gm gene is a sequence 2 in a sequence table.
3. The method according to claim 1 or 2, characterized in that: the methylotrophic bacterium is methylotrophic bacterium J1-1.
4. The method of claim 3, wherein the TCS3 in the methylotrophic bacterium J1-1 is replaced by a Gm gene by homologous recombination, the sequence of the upstream homology arm is shown as sequence 3 in the sequence table, and the sequence of the downstream homology arm is shown as sequence 4 in the sequence table.
5. Method according to claim 4, characterized in that it comprises the following steps: introducing a recombinant vector into methylotrophic bacteria J1-1, wherein the recombinant vector contains the upstream homology arm, the Gm gene and the downstream homology arm which are sequentially connected from 5 'end to 3' end, and the upstream homology arm and the downstream homology arm are subjected to homologous recombination with a homologous fragment on the genome of methylotrophic bacteria J1-1 to obtain the engineering bacteria for producing pyrroloquinoline quinone.
6. The method of claim 4, wherein the recombinant vector is a recombinant plasmid obtained by inserting the upstream homology arm, the Gm gene and the downstream homology arm, which are sequentially connected from the 5 'end to the 3' end, between the BamHI and ApaI cleavage sites of the pWM91 vector.
7. The engineering bacteria for producing pyrroloquinoline quinone prepared by the method of any one of claims 1 to 6.
8. Use of the method according to any one of claims 1 to 6 or the engineered bacterium according to claim 7 for the production of pyrroloquinoline quinone.
9. A method for producing pyrroloquinoline quinone, characterized in that the engineered bacterium of claim 8 is subjected to fermentation culture to produce pyrroloquinoline quinone from the fermentation product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110838234.3A CN113913448B (en) | 2021-07-23 | 2021-07-23 | Method for improving yield of pyrroloquinoline quinone of methylotrophic bacteria and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110838234.3A CN113913448B (en) | 2021-07-23 | 2021-07-23 | Method for improving yield of pyrroloquinoline quinone of methylotrophic bacteria and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113913448A true CN113913448A (en) | 2022-01-11 |
| CN113913448B CN113913448B (en) | 2023-10-20 |
Family
ID=79233052
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110838234.3A Active CN113913448B (en) | 2021-07-23 | 2021-07-23 | Method for improving yield of pyrroloquinoline quinone of methylotrophic bacteria and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113913448B (en) |
Citations (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012118225A1 (en) * | 2011-03-03 | 2012-09-07 | Ajinomoto Co.,Inc. | A method for producing pyrroloquinoline quinone using a bacterium of the genus methylobacterium or hyphomicrobium |
| CN103224965A (en) * | 2013-03-27 | 2013-07-31 | 中国人民解放军军事医学科学院生物工程研究所 | Method for producing pyrroloquinoline quinine through microbial fermentation and fermentation medium used in same |
| CN103361390A (en) * | 2013-03-27 | 2013-10-23 | 中国人民解放军军事医学科学院生物工程研究所 | Method for eliminating side product polysaccharide in fermentation production of pyrroloquinoline quinone, and application of method |
| CN103898011A (en) * | 2014-03-11 | 2014-07-02 | 江南大学 | Methylotrophic bacteria and method for producing pyrroloquinoline quinine (PQQ) by fermentation thereof |
| CN105378077A (en) * | 2013-06-18 | 2016-03-02 | 凯利斯塔公司 | Compositions and methods for biological production of lactate from c1 compounds using lactate dehydrogenase transformants |
| CN105624084A (en) * | 2016-01-28 | 2016-06-01 | 福建师范大学 | Oriented domestication and breeding of methylotrophic bacterium capable of producing pyrroloquinoline quinone at high yield |
| CA2998981A1 (en) * | 2015-10-06 | 2017-04-13 | Ioannis PAPAPETRIDIS | Eukaryotic cell with increased production of fermentation product |
| WO2017123775A1 (en) * | 2016-01-12 | 2017-07-20 | The Regents Of The University Of California | Methanol dehydrogenases |
| WO2019068011A2 (en) * | 2017-09-28 | 2019-04-04 | Lanzatech, Inc. | Genetic knockouts in wood-ljungdahl microorganisms |
| CN111057660A (en) * | 2014-04-15 | 2020-04-24 | Ao生物医学有限责任公司 | Ammonia oxidizing nitrosomonas D23 |
| CN111139210A (en) * | 2020-01-15 | 2020-05-12 | 青岛农业大学 | Recombinant methylotrophic bacterium and oral vaccine |
| CN111378680A (en) * | 2020-04-07 | 2020-07-07 | 陕西师范大学 | CRISPR-Cas9 double-vector system suitable for Serratia gene modification |
| CN113337432A (en) * | 2021-06-04 | 2021-09-03 | 华熙生物科技股份有限公司 | Methylophagus for producing pyrroloquinoline quinone and application thereof |
| JP2022523540A (en) * | 2019-03-01 | 2022-04-25 | ブラスケム エス．エー． | Methods for In vivo Synthesis of 4-Hydroxymethylfurfural and its Derivatives |
| CN115747275A (en) * | 2022-07-29 | 2023-03-07 | 郑州尼采生物科技有限公司 | Method for increasing yield of pyrroloquinoline quinone fermented by methylobacterium |
-
2021
- 2021-07-23 CN CN202110838234.3A patent/CN113913448B/en active Active
Patent Citations (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012118225A1 (en) * | 2011-03-03 | 2012-09-07 | Ajinomoto Co.,Inc. | A method for producing pyrroloquinoline quinone using a bacterium of the genus methylobacterium or hyphomicrobium |
| CN103224965A (en) * | 2013-03-27 | 2013-07-31 | 中国人民解放军军事医学科学院生物工程研究所 | Method for producing pyrroloquinoline quinine through microbial fermentation and fermentation medium used in same |
| CN103361390A (en) * | 2013-03-27 | 2013-10-23 | 中国人民解放军军事医学科学院生物工程研究所 | Method for eliminating side product polysaccharide in fermentation production of pyrroloquinoline quinone, and application of method |
| CN105378077A (en) * | 2013-06-18 | 2016-03-02 | 凯利斯塔公司 | Compositions and methods for biological production of lactate from c1 compounds using lactate dehydrogenase transformants |
| CN103898011A (en) * | 2014-03-11 | 2014-07-02 | 江南大学 | Methylotrophic bacteria and method for producing pyrroloquinoline quinine (PQQ) by fermentation thereof |
| CN111057660A (en) * | 2014-04-15 | 2020-04-24 | Ao生物医学有限责任公司 | Ammonia oxidizing nitrosomonas D23 |
| CA2998981A1 (en) * | 2015-10-06 | 2017-04-13 | Ioannis PAPAPETRIDIS | Eukaryotic cell with increased production of fermentation product |
| WO2017123775A1 (en) * | 2016-01-12 | 2017-07-20 | The Regents Of The University Of California | Methanol dehydrogenases |
| CN105624084A (en) * | 2016-01-28 | 2016-06-01 | 福建师范大学 | Oriented domestication and breeding of methylotrophic bacterium capable of producing pyrroloquinoline quinone at high yield |
| WO2019068011A2 (en) * | 2017-09-28 | 2019-04-04 | Lanzatech, Inc. | Genetic knockouts in wood-ljungdahl microorganisms |
| JP2022523540A (en) * | 2019-03-01 | 2022-04-25 | ブラスケム エス．エー． | Methods for In vivo Synthesis of 4-Hydroxymethylfurfural and its Derivatives |
| CN111139210A (en) * | 2020-01-15 | 2020-05-12 | 青岛农业大学 | Recombinant methylotrophic bacterium and oral vaccine |
| CN111378680A (en) * | 2020-04-07 | 2020-07-07 | 陕西师范大学 | CRISPR-Cas9 double-vector system suitable for Serratia gene modification |
| CN113337432A (en) * | 2021-06-04 | 2021-09-03 | 华熙生物科技股份有限公司 | Methylophagus for producing pyrroloquinoline quinone and application thereof |
| CN115747275A (en) * | 2022-07-29 | 2023-03-07 | 郑州尼采生物科技有限公司 | Method for increasing yield of pyrroloquinoline quinone fermented by methylobacterium |
Non-Patent Citations (7)
| Title |
|---|
| NCBI: "Methylovorus sp. MP688, complete genome", 《GENBANK DATABASE》 * |
| XIANG-HUA XIONG 等: "Complete Genome Sequence of the Bacterium Methylovorus sp. Strain MP688, a High-Level Producer of Pyrroloquinolone Quinone", 《JOURNAL OF BACTERIOLOGY》 * |
| 孙晓宇;薄明井;杨亚欣;张惟材;葛欣;熊向华;: "通过突变甲醇脱氢酶启动子提高甲基营养菌吡咯喹啉醌产量", 生物技术通讯 * |
| 寇航 等: "基于Methylovorus sp. J1-1 基因组尺度代谢网络优化吡 咯喹啉醌合成", 《生物技术通报》 * |
| 李彤;景爱霞;杨亚欣;刘晓阳;汪建华;刘党生;熊向华;张惟材;: "食甲基杆菌J1-1吡咯喹啉醌生物合成突变株的筛选和鉴定", 生物技术通讯 * |
| 杨靖;陈文静;张敏;袁肖杰;杨益明;朱丽萍;杨松;: "甲基营养菌代谢网络途径和代谢工程改造的研究进展", 生物加工过程 * |
| 熊向华;杨璐;韩晓东;汪建华;张惟材;: "大肠杆菌pqqL基因敲除与功能分析", 微生物学报 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113913448B (en) | 2023-10-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107916283B (en) | A kind of production technology of niacinamide | |
| CN110157654B (en) | Bacillus natto recombinant strain and construction method and application thereof | |
| CN101983240B (en) | Flocculent yeast and method for production thereof | |
| CN111073902A (en) | CRISPR/dCas9 vector for improving expression level of gliotoxin biosynthesis gene and construction method and application thereof | |
| CN111849852A (en) | Construction method of high-optical-purity L-lactic acid engineering bacteria | |
| CN116023449A (en) | Fusarium fumonisin synthesis and pathogenicity related gene FpFUM21 and application thereof | |
| CN106119180B (en) | Mycobacterium recombinant genetic engineering bacterium and application thereof | |
| CN114606149A (en) | Saccharomyces cerevisiae engineering strain for producing ergosterol and application thereof | |
| CN111733179A (en) | Method for synthesizing resveratrol by oleaginous microorganism yarrowia lipolytica | |
| CN111690549A (en) | Recombinant yarrowia lipolytica strain for producing protopanoxadiol by using xylose and construction method and application thereof | |
| CN118103513A (en) | Engineering bacterium for efficiently expressing D-psicose 3-epimerase and application | |
| CN113913448B (en) | Method for improving yield of pyrroloquinoline quinone of methylotrophic bacteria and application | |
| CN108588108B (en) | Preparation method and application of bacillus for efficiently metabolizing glycerol | |
| CN115927234A (en) | application of amt gene and mutant M107 in expression of azotobacter siderophin | |
| CN113667650A (en) | 11 alpha-hydroxylase mutant and application thereof | |
| CN105505933B (en) | Novel arthrobacterium promoter sequence and application thereof | |
| CN113584023B (en) | Metal ion induced aspergillus oryzae gene promoter, preparation method and application thereof | |
| CN113151262B (en) | Yeast promoter with weakened regulation strength and application thereof in metabolic flux regulation | |
| CN114634883B (en) | Recombinant engineering bacterium for producing 2' -fucosyllactose and construction method and application thereof | |
| CN107574174B (en) | Construction method of plasmid expression vector for improving yield of rhodobacter sphaeroides coenzyme Q10 | |
| CN106967627A (en) | A kind of fermenting and producing ALA recombinant bacterial strain and preparation method thereof | |
| CN118291405A (en) | Camptothecine 10-hydroxylase and preparation method and application thereof | |
| CN116676328A (en) | Method for improving fusarium oxysporum gene knockout efficiency and application | |
| CN116179373A (en) | Cordyceps militaris strain without producing ustilaginoidea virens and preparation method thereof | |
| CN117247882A (en) | High-yield lipopeptide engineering strain modified by constitutive promoter and construction method thereof |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |