CN103361390A - Method for eliminating side product polysaccharide in fermentation production of pyrroloquinoline quinone, and application of method - Google Patents
Method for eliminating side product polysaccharide in fermentation production of pyrroloquinoline quinone, and application of method Download PDFInfo
- Publication number
- CN103361390A CN103361390A CN2013101029549A CN201310102954A CN103361390A CN 103361390 A CN103361390 A CN 103361390A CN 2013101029549 A CN2013101029549 A CN 2013101029549A CN 201310102954 A CN201310102954 A CN 201310102954A CN 103361390 A CN103361390 A CN 103361390A
- Authority
- CN
- China
- Prior art keywords
- gene
- pyrroloquinoline quinone
- polysaccharide
- bacterial strain
- production
- 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
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a method for eliminating a side product polysaccharide in fermentation production of pyrroloquinoline quinone. According to the method, in microorganism fermentation production of pyrroloquinoline quinone, a gene knockout method is used for knockout of an important gene in a polysaccharide synthetic gene cluster of a pyrroloquinoline quinone production strain so that the production strain loses the capability of exopolysaccharide synthesis without reducing the capability of producing pyrroloquinoline quinone. According to the method disclosed by the invention, the important gene mpq_1844 in the polysaccharide synthetic gene cluster of the PQQ (pyrroloquinoline quinone) production strain Methylovorussp.MP688 is successfully knocked out by a double-exchange homologous recombination method, the gene knockout strain of the original production strain no longer synthesizes exopolysaccharide, thus the metabolism side product polysaccharide generated in microorganism fermentation production of the pyrroloquinoline quinone is eliminated, the aim of simplifying the purification technique is achieved and the foundation is laid for industrial and efficient preparation of pyrroloquinoline quinone.
Description
Technical field
The present invention relates to microbial fermentation technology and gene engineering technology field, be specifically related to eliminate in a kind of fermentative production pyrroloquinoline quinone process method and the application thereof of by product polysaccharide.
Background technology
Pyrroloquinoline quinone (Pyrroloquinoline quinone, PQQ) is the coenzyme by the 3rd class oxydo-reductase, has different physiological roles and potential pharmaceutical use.Bibliographical information, the synthetic level of methylotrophic bacteria PQQ is higher, and has cleaning low-carbon (LC), the gentle advantage such as controlled, with low cost, is a kind of production bacterial strain that has potentiality.Yet most methylotrophic bacterias have the ability of synthetic exocellular polysaccharide, limit microbial fermentation and produced the PQQ large-scale application, the main unfavorable factor of polysaccharide has: (1) bacterium secretion soluble polysaccharide not only causes purification column congestion affects purification efficiency, and can shorten the work-ing life of purification media; (2) viscosity of fermented liquid increases the oxygen transfer efficiency that affects in the fermenting process; (3) insoluble polysaccharide is attached to bacterium surface and makes the separating effect of fermented liquid extracellular fraction and thalline not good, increases power cost.
Polysaccharide synthetic comprises a series of biochemical reactions in the bacterium, the gene of enzyme of these reactions of encoding generally exists with the form of gene cluster, therefore one of method of blocking the bacterial polysaccharides route of synthesis is exactly that the synthetic important gene of polysaccharide is knocked out, reach not affecting under the synthetic prerequisite of bacterial growth and target meta-bolites, in the purpose of certain level reduction or Polysaccharide removing.So far, the methods and applications of eliminating the polysaccharide that produces in the fermentation using bacteria by this genetic modification have no report.
Summary of the invention
In order to address the above problem, the purpose of this invention is to provide the method for eliminating the by product polysaccharide in a kind of fermentative production pyrroloquinoline quinone process, for utilizing the purifying process of microorganism scale operation PQQ, simplification lays a good foundation.
Another object of the present invention provides the application of aforesaid method in microbial fermentation production coenzyme or VITAMIN process.
In order to realize the foregoing invention purpose, technical scheme of the present invention is as follows:
Eliminate the method for by product polysaccharide in a kind of fermentative production pyrroloquinoline quinone process, the method is in the process of microbial fermentation production pyrroloquinoline quinone, knock out pyrroloquinoline quinone by the gene knockout means and produce important gene in the bacterial strain polysaccharide synthetic gene bunch, make this production bacterial strain lose the ability of transposon mutagenesis, and do not reduce the ability of this production bacterial strain production pyrroloquinoline quinone.
Preferably, described production bacterial strain is methylotrophic bacteria Methylovorus sp.MP688.This bacterium is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on August 20th, 2010, and its preserving number is CGMCCNo.4096.
Preferably, the important gene in the described polysaccharide synthetic gene bunch is the gene of phosphotransferase class, such as mpq_1844.
The preferred concrete steps of above-mentioned method are:
(1) the screening pyrroloquinoline quinone is produced the important gene in the bacterial strain polysaccharide synthetic gene bunch;
(2) extract the genomic dna that described PQQ produces bacterial strain, and the design primer, take this genomic dna as template, by the flanking sequence of the described important gene 5 ' end of PCR method amplification step (1) and 3 ' end;
(3) flanking sequence of step (2) gained the 5 ' distolateral wing sequence and 3 ' end is made respectively double digestion and process, be connected on the gene knockout carrier;
(4) the described gene knockout carrier of step (3) imports in the described PQQ production bacterial strain by the mode of conventional electricity conversion or conjugal transfer, cultivate at the MP solid medium that contains penbritin, the single bacterium colony of picking resistance is going down to posterity 2-4 time without under the screening conditions, contain the positive colony on the culture medium flat plate of sucrose by anti-sieve label screening, and then identify by bacterium colony PCR whether important gene lacks;
(5) in shaking flask, carry out the PQQ fermenting experiment of genetically deficient bacterium and original production bacterial strain, get respectively fermented liquid centrifugal, observe the thalline configuration of surface, identify having or not of bacterium surface insoluble polysaccharide, measure the content of soluble polysaccharide in the supernatant liquor.
Preferably, to produce the method for the important gene in the bacterial strain polysaccharide synthetic gene bunch be to download important gene sequence in the polysaccharide route of synthesis in NCBI gene bank database (concrete network address is: http://www.ncbi.nlm.nih.gov/nuccore/NC_014733.1 from=1899875﹠amp to the described screening pyrroloquinoline quinone of step (1); To=1901296﹠amp; Report=fasta), more preferably, phosphotransferase class mpq_1844 and flanking sequence thereof, the nucleotide sequence of described mpq_1844 is shown in SEQ.ID No.1, the nucleotide sequence of described flanking sequence is shown in SEQ.ID No.2 and SEQ.ID No.3, described flanking sequence length, preferred 1500-2500bp.
Wherein, the primer of design described in the step (2) comprises forward and reverse two primers, and be specially: the 5 ' distolateral wing sequence forward primer UpF is shown in SEQ.ID No.4, and reverse primer UpR is shown in SEQ.ID No.5; The flanking sequence forward primer DwF of 3 ' end is shown in SEQ.ID No.6, and reverse primer DwR is shown in SEQ.ID No.7.
Preferably, double digestion described in the step (3) is processed, and is specially: the 5 ' distolateral wing sequence is processed (37 ℃, 4-10 hour) with ApaI and BamHI double digestion; The flanking sequence of 3 ' end is processed (37 ℃, 4-10 hour) with SpeI and NotI double digestion; Described double digestion reaction solution system is as shown in table 3; Described gene knockout carrier is preferably plasmid pWM91, and connecting temperature is 16-25 ℃, 1-6 hour; Described linked system is as shown in table 4.
Preferably, the final concentration of penbritin is 50 μ g/ml in the step (4); Described anti-sieve is labeled as sacB; Employed sucrose concentration is specially 0.01～0.05g/mL in the described substratum; The used primer of bacterium colony PCR of described identified gene disappearance is UpF and the DwR in the step (2); Described genetically deficient bacterium amplification is 4000bp, and specifically shown in SEQ.ID No.8, producing the bacterial strain amplification is 5422bp, specifically shown in SEQ.ID No.9;
Mode and the importing condition of described conventional electricity conversion or conjugal transfer, with reference to people the document Magnetosome vesicles are present before magnetite formation in 2004 such as Komeili A, andMamA is required for their activation.
Preferably, soluble polysaccharide measuring method described in the step (5) is the phenolsulfuric acid method, the method steps of specifically reporting according to people document Colorimetric in 1956 method for determination of sugarsand related substancede such as Dubois M.; Described centrifugal rotational speed and time, be specially 12000-12000 rev/min, 5-10 minute.
Further, the invention provides the application of aforesaid method in microbial fermentation production coenzyme or VITAMIN process.Described coenzyme is preferably pyrroloquinoline quinone.
Beneficial effect of the present invention is as follows:
The present invention is by the method for double exchange homologous recombination, important gene mpq_1844 in the polysaccharide synthetic gene of PQQ being produced bacterial strain Methylovorussp.MP688 bunch of success knocks out, the no longer synthetic exocellular polysaccharide of the clpp gene degerming of original production bacterial strain, under the prerequisite that the production to bacterial growth and PQQ does not impact, greatly reduce the synthetic level of soluble polysaccharide and insoluble polysaccharide, realized the removal of metabolic by-prods polysaccharide in the microbial fermentation production pyrroloquinoline quinone process, thereby reached simplify purifying process purpose, lay a good foundation for industrial high efficiency prepares pyrroloquinoline quinone.
Description of drawings
The PCR proof diagram of the mpq_1844 gene knock-out bacterial strain that Fig. 1 shows.From left to right be respectively dna molecular amount marker, the amplification take original production strain gene group DNA as template, be the amplification take mpq_1844 gene knock-out bacterial strain genomic dna as template.
What Fig. 2 showed is mpq_1844 gene knock-out bacterial strain and original production bacterial strain difference aspect the soluble polysaccharide outside growth and born of the same parents.▲ be the synthetic situation of original production bacterial strain soluble polysaccharide, ■ is the synthetic situation of gene knock-out bacterial strain soluble polysaccharide.
What Fig. 3 showed is mpq_1844 gene knock-out bacterial strain and the synthetic difference of original production bacterial strain capsular polysaccharide.Left figure is the synthetic situation of original production bacterial strain capsular polysaccharide, and right figure is the synthetic situation of gene knock-out bacterial strain capsular polysaccharide.
What Fig. 4 showed is the comparison of mpq_1844 gene knock-out bacterial strain and original production bacterial strain PQQ throughput.▲ be the synthetic situation of original production bacterial strain PQQ, ■ is the synthetic situation of gene knock-out bacterial strain PQQ.
Embodiment
Introduce in detail the present invention below in conjunction with accompanying drawing and embodiment thereof.But protection of the present invention orientation is not limited to following instance, should comprise the full content in claims.
Experiment material related in following examples all can prepare by the commercial sources acquisition or by normal experiment; Used restriction endonuclease, ligase enzyme, pcr amplification enzyme are all available from the Beijing Quanshijin Biotechnology Co., Ltd; Experiment is the biology laboratory routine with instrument and equipment.
The acquisition of embodiment 1mpq_1844 gene and 5 ' end and the 3 ' flanking fragment of holding and knock out the structure of plasmid
(concrete network address is: http://www.ncbi.nlm.nih.gov/nuccore/NC_014733.1 from=1899875﹠amp from NCBI gene bank database; To=1901296﹠amp; Report=fasta) input mpq_1844 in downloads the flanking sequence of preserving gene order and 5 ' end and 3 ' end; The nucleotide sequence of described mpq_1844 shown in SEQ.ID No.1, the nucleotide sequence of described flanking sequence such as SEQ.ID No.2 and SEQ.ID No.3.Shown in use genome to extract test kit (Tiangen company bacterial genomes is extracted test kit) extracting PQQ to produce the genomic dna of bacterial strain Methylovorus sp.MP688, use the 5 ' distolateral wing sequence forward primer UpF (ATT GGG CCT GCC GAT GGG CGC TGA CC) and reverse primer UpR (CG GGA TCC GAGGCC CGT GGA GTC TGC) shown in SEQ.ID No.5 shown in SEQ.ID No.4, the flanking sequence forward primer DwF of 3 ' end is (GG ACT AGT AGT AAA GGA GTC GTT CAT) and reverse primer DwR (GAA AAA AGC GGC CGC CCC GGA TGA CTGGGT TGG) amplification shown in SEQ.ID No.7 shown in SEQ.ID No.6, the flanking sequence of amplification respectively is 2000bp, and is consistent with the flanking sequence SEQ.ID No.2 and the SEQ.ID No.3 that download respectively.Pcr amplification system and condition be as shown in Table 1 and Table 2:
Each substances content in the table 1PCR50 μ l reaction solution
Table 2PCR reaction conditions
The 5 ' distolateral wing sequence PCR product is processed with ApaI and BamHI double digestion, and described double digestion treatment condition are 37 ℃, 4-10 hour; The 3 ' distolateral wing sequence PCR product is processed with SpeI and NotI double digestion, and described double digestion treatment condition are 37 ℃, 4-10 hour; Described double digestion reaction solution system is as shown in table 3.After carrier pWM91 processes successively equally, enzyme is cut rear PCR product is connected with carrier after enzyme is cut in turn, the connection temperature is 16-25 ℃, 1-6 hour; Described linked system is as shown in table 4.Final must build knock out plasmid (being gene knockout carrier).
Table 3 double digestion reaction solution system
Table 4 linked system
Embodiment 2 knocks out mpq_1844 gene and evaluation
By conventional electricity conversion or conjugal transfer the plasmid that knocks out that embodiment 1 obtains is imported among the PQQ production bacterial strain Methylovorus sp.MP688, the importing condition is with reference to people document Magnetosome vesicles are present before magnetite formation in 2004 such as Komeili A, and MamA isrequired for their activation, reacted bacterium liquid is coated on the penbritin MP solid medium upper flat plate that contains 50 μ g/ml, and described MP solid culture based component is to contain MgSO in 1 liter
47H
2O:0.2g, (NH
4)
2SO
4: 3g, KH
2PO
4: 1.4g, Na
2HPO
412H
2O:3g, ironic citrate: 30mg, CaCl
2: 30mg; MnCl
24H
2O:5mg, ZnSO
47H
2O:5mg, CuSO
45H
2O:0.5mg, 1% agar powder.Cultivate 2 days afterwards random single bacterium colonies of picking for 30 ℃, be inoculated in the liquid nutrient medium without any screening pressure, described liquid culture based formulas is the same, do not add agar powder, go down to posterity 2-4 time, draw the flat board that 100 μ l coating contains 0.02g/mL sucrose, culture medium prescription is the same, cultivate 2 days afterwards random 100 single bacterium colonies of picking for 30 ℃, use the 5 ' distolateral wing sequence forward primer UpF(ATT GGG CCTGCC GAT GGG CGC TGA CC) and the flanking sequence reverse primer DwR(GAA AAAAGC GGC CGC CCC GGA TGA CTG GGT TGG of 3 ' end) do bacterium colony PCR evaluation, amplification system and program see Table 5 and table 6:
Each substances content in the table 550 μ l bacterium colony PCR reaction solution
Table 6PCR reaction conditions
Genetically deficient bacterium amplification is 4000bp, and its nucleotide sequence sees SEQ.ID No.8 for details; Producing the bacterial strain amplification is 5422bp, and its nucleotide sequence sees SEQ.ID No.9 for details, and concrete amplification sees Fig. 1 for details.
The mensuration of embodiment 3 exocellular polysaccharides and pyrroloquinoline quinone content
The knock-out bacterial strain that screens and original production bacterial strain are inoculated into the MP liquid nutrient medium that contains 100ml simultaneously, and (culture medium prescription is with embodiment 3, do not add agar powder) the 250ml triangular flask in, 30 ℃, 200rpm cultivates, phenolsulfuric acid method according to people the document Colorimetric method fordetermination of sugars and related substancede in 1956 such as Dubois M. report is measured soluble polysaccharide content, the polysaccharide content of measuring in the genetically deficient fermented liquid is below 10mg/l, can think does not have polysaccharide, and producing and detecting polysaccharide content in 60 hours the fermented liquid of bacterial strain is that 700mg/l(sees Fig. 2 for details); Original production bacterial strain has floss on centrifugal rear thalline surface, and the genetically deficient bacterium without, illustrate that the transposon mutagenesis ability loses (seeing Fig. 3 for details).Get respectively the fermented liquid of knock-out bacterial strain and original production bacterial strain, measure the content of pyrroloquinoline quinone according to the Hexose phosphate dehydrogenase method of bibliographical information, concrete document is with reference to the people such as GeigerO. document Enzymatic determination of pyrroloquinoline quinoneusing crude membranes from Escherichia coli in 1987, and the result shows that the content of two strain bacterium pyrroloquinoline quinone in cultivating 70 hours secondary fermentation liquid is respectively 41.5mg/l and 39.5mg/l(sees Fig. 4 for details).Do not affect the synthetic of pyrroloquinoline quinone behind this explanation Polysaccharide removing.
The above only is preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. eliminate the method for by product polysaccharide in the fermentative production pyrroloquinoline quinone process, it is characterized in that, the method is in the process of microbial fermentation production pyrroloquinoline quinone, knock out pyrroloquinoline quinone by the gene knockout means and produce important gene in the bacterial strain polysaccharide synthetic gene bunch, make this production bacterial strain lose the ability of transposon mutagenesis, and do not reduce the ability of this production bacterial strain production pyrroloquinoline quinone.
2. method according to claim 1 is characterized in that, described production bacterial strain is methylotrophic bacteria Methylovorus sp.MP688; Important gene in the described polysaccharide synthetic gene bunch is the phosphotransferase class.
3. method according to claim 1 is characterized in that, described phosphotransferase class is mpq_1844.
4. the described method of any one according to claim 1～3, its feature is used for, and the concrete steps of described method are:
(1) the screening pyrroloquinoline quinone is produced the important gene in the bacterial strain polysaccharide synthetic gene bunch;
(2) extract the genomic dna that described PQQ produces bacterial strain, and the design primer, take this genomic dna as template, by the flanking sequence of the described important gene 5 ' end of PCR method amplification step (1) and 3 ' end;
(3) flanking sequence of step (2) gained the 5 ' distolateral wing sequence and 3 ' end is made respectively double digestion and process, be connected on the gene knockout carrier;
(4) the described gene knockout carrier of step (3) imports in the described PQQ production bacterial strain by the mode of conventional electricity conversion or conjugal transfer, cultivate at the MP solid medium that contains penbritin, the single bacterium colony of picking resistance is going down to posterity 2-4 time without under the screening conditions, contain the positive colony on the culture medium flat plate of sucrose by anti-sieve label screening, and then identify by bacterium colony PCR whether important gene lacks;
(5) in shaking flask, carry out the PQQ fermenting experiment of genetically deficient bacterium and original production bacterial strain, get respectively fermented liquid centrifugal, observe the thalline configuration of surface, identify having or not of bacterium surface insoluble polysaccharide, measure the content of extracellular soluble exo polysaccharides in the supernatant liquor.
5. method according to claim 4, it is characterized in that, the method that the described screening pyrroloquinoline quinone of step (1) is produced the important gene in the bacterial strain polysaccharide synthetic gene bunch is the important gene sequence of downloading in NCBI gene bank database in the polysaccharide route of synthesis, wherein, described important gene is phosphotransferase class mpq_1844 and flanking sequence thereof, the nucleotide sequence of described mpq_1844 is shown in SEQ.ID No.1, and the nucleotide sequence of described flanking sequence is shown in SEQ.ID No.2 and SEQ.ID No.3.
6. method according to claim 4 is characterized in that, described design primer comprises forward and reverse two primers, is specially: the 5 ' distolateral wing sequence forward primer UpF is shown in SEQ.ID No.4, and reverse primer UpR is shown in SEQ.ID No.5; The flanking sequence forward primer DwF of 3 ' end is shown in SEQ.IDNo.6, and reverse primer DwR is shown in SEQ.ID No.7.
7. method according to claim 4 is characterized in that, double digestion described in the step (3) is processed, and is specially: the 5 ' distolateral wing sequence is processed with ApaI and BamHI double digestion; The flanking sequence of 3 ' end is processed with SpeI and NotI double digestion; Described gene knockout carrier is plasmid pWM91.
8. method according to claim 4 is characterized in that, the final concentration of the penbritin described in the step (4) is 50 μ g/mL; Described anti-sieve is labeled as sacB; Employed sucrose concentration is 0.01～0.05g/mL in the described substratum.
9. method according to claim 4 is characterized in that, the soluble polysaccharide measuring method is the phenolsulfuric acid method described in the step (5); Described centrifugal rotational speed and time, be specially 10000-12000 rev/min, 5-10 minute.
10. the application of the described method of claim 1～3 any one in microbial fermentation production coenzyme or VITAMIN process.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310102954.9A CN103361390B (en) | 2013-03-27 | 2013-03-27 | Method for eliminating side product polysaccharide in fermentation production of pyrroloquinoline quinone, and application of method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310102954.9A CN103361390B (en) | 2013-03-27 | 2013-03-27 | Method for eliminating side product polysaccharide in fermentation production of pyrroloquinoline quinone, and application of method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103361390A true CN103361390A (en) | 2013-10-23 |
| CN103361390B CN103361390B (en) | 2015-01-07 |
Family
ID=49363646
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310102954.9A Active CN103361390B (en) | 2013-03-27 | 2013-03-27 | Method for eliminating side product polysaccharide in fermentation production of pyrroloquinoline quinone, and application of method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103361390B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113913448A (en) * | 2021-07-23 | 2022-01-11 | 中国人民解放军军事科学院军事医学研究院 | Method for increasing yield of methylotrophic bacteria pyrroloquinoline quinone and application |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004033671A1 (en) * | 2002-10-11 | 2004-04-22 | Cj Corp. | The microorganism of which the fadr gene was knocked out on the chromosome and a process for producing l-threonine by fermentation with the same mutant |
| CN102061278A (en) * | 2010-11-17 | 2011-05-18 | 中国人民解放军军事医学科学院生物工程研究所 | Methylovorus sp. MP688 and application thereof |
| CN102653755A (en) * | 2011-03-01 | 2012-09-05 | 中国农业科学院作物科学研究所 | Knockout method for verticillium dahliae gene |
-
2013
- 2013-03-27 CN CN201310102954.9A patent/CN103361390B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004033671A1 (en) * | 2002-10-11 | 2004-04-22 | Cj Corp. | The microorganism of which the fadr gene was knocked out on the chromosome and a process for producing l-threonine by fermentation with the same mutant |
| CN102061278A (en) * | 2010-11-17 | 2011-05-18 | 中国人民解放军军事医学科学院生物工程研究所 | Methylovorus sp. MP688 and application thereof |
| CN102653755A (en) * | 2011-03-01 | 2012-09-05 | 中国农业科学院作物科学研究所 | Knockout method for verticillium dahliae gene |
Non-Patent Citations (2)
| Title |
|---|
| DAHE ZHAO ET AL.: "Improving polyhydroxyalkanoate production by knocking out the genes involved in exopolysaccharide biosynthesis in Haloferax mediterranei", 《APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY》 * |
| 蒋建东等: "同源重组法构建多功能农药降解基因工程菌研究", 《生物工程学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113913448A (en) * | 2021-07-23 | 2022-01-11 | 中国人民解放军军事科学院军事医学研究院 | Method for increasing yield of methylotrophic bacteria pyrroloquinoline quinone and application |
| CN113913448B (en) * | 2021-07-23 | 2023-10-20 | 中国人民解放军军事科学院军事医学研究院 | Method for improving yield of pyrroloquinoline quinone of methylotrophic bacteria and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103361390B (en) | 2015-01-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107354188B (en) | Process for producing N-acetylglucosamine by fermentation of Escherichia coli JL-GlcN | |
| CN102102086B (en) | L-lactate dehydrogenase gene-deleted engineering bacterium and construction method and application thereof | |
| CN105420154A (en) | Double knockout recombinant rhodococcus as well as construction method and application thereof | |
| CN101663389A (en) | Nitrile hydratase producing engineering bacteria with amidase gene knocked out and construction method and application thereof | |
| Choi et al. | Bioethanol production by a flocculent hybrid, CHFY0321 obtained by protoplast fusion between Saccharomyces cerevisiae and Saccharomyces bayanus | |
| US10612007B2 (en) | Method for increasing citric acid production by Aspergillus niger fermentation | |
| CN102994439A (en) | Escherichia coli recombinant strain producing shikimic acid, and construction method and application thereof | |
| CN103468581A (en) | Mortierella alpine uracil auxotroph with ura5 gene knocked out through homologous recombination, and construction method thereof | |
| CN105624080A (en) | Bacillus licheniformis gene engineering bacterium capable of producing polysaccharide flocculant at high yield and establishment method thereof | |
| CN105154381A (en) | Novel succinic acid-producing mutant microorganism simultaneously using sucrose and glycerol and method for producing succinic acid using the same | |
| CN102191203B (en) | Bacillus amyloliquefaciens and method for producing chymosin by fermenting using same | |
| CN110055204A (en) | A kind of method and application for knocking out II Q and pcf gene of spo and improving the lichen bacillus ferments producing enzyme | |
| CN103865863B (en) | One strain leukonid mutant strain and construction process and methods for using them thereof | |
| Zhong et al. | Application of T-DNA insertional mutagenesis for improving cellulase production in the filamentous fungus Trichoderma reesei | |
| CN109554386A (en) | A kind of utilizing works Escherichia coli are using Corncob hydrolysate as the method for substrate high yield D- xylonic | |
| CN101979587A (en) | Phytoene desaturase gene of sphingomonas sp. and application thereof | |
| CN105624176A (en) | Engineering bacterium with overexpressed uridine diphosphoglucose pyrophosphorylase gene and establishment thereof | |
| CN104845926B (en) | Gene knockout escherichia coli beneficial to recombinant protein extracellular secretion and application thereof | |
| CN104152483A (en) | Application of argJ gene in fermentation production of L-citrulline | |
| CN103233034B (en) | Produce the construction method of the engineering strain of tetracycline one-component | |
| CN103361390B (en) | Method for eliminating side product polysaccharide in fermentation production of pyrroloquinoline quinone, and application of method | |
| CN103820362B (en) | A kind of structure of biosynthesizing gentamicinX 2 engineering bacteria and application thereof | |
| CN109055417A (en) | A kind of recombinant microorganism, preparation method and its application in production Co-Q10 | |
| CN108949784A (en) | Application of the sporulation related gene sigmaF in producing enzyme | |
| CN112239742B (en) | Bacillus licheniformis for producing alkaline protease and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |