CN103361390B - 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
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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 method and the application thereof of eliminating by product polysaccharide in a kind of fermentative production pyrroloquinoline quinone process.
Background technology
Pyrroloquinoline quinone (Pyrroloquinoline quinone, PQQ) is by the coenzyme of the 3rd class oxydo-reductase, has different physiological roles and potential pharmaceutical use.Bibliographical information, it is higher that methylotrophic bacteria PQQ synthesizes level, and have clean low-carbon (LC), the gentle advantage such as controlled, with low cost, is a kind of production bacterial strain of great potential.But most methylotrophic bacteria has the ability of synthesis exocellular polysaccharide, limit fermentable and produce PQQ large-scale application, the main unfavorable factor of polysaccharide has: (1) bacterial secretory soluble polysaccharide not only causes purification column to block affects purification efficiency, and can shorten the work-ing life of purification media; (2) viscosity of fermented liquid increases the oxygen transfer efficiency affected in 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.
In bacterium, the synthesis of polysaccharide comprises a series of biochemical reaction, the gene of enzymes of these reactions of encoding generally exists with the form of gene cluster, therefore one of method blocking bacterial polysaccharides route of synthesis is exactly knock out the important gene of polysaccharide synthesis, reach under the prerequisite not affecting bacterial growth and target metabolic Product formation, the object of reduction or Polysaccharide removing on certain level.So far, the methods and applications being eliminated the polysaccharide produced in fermentation using bacteria by this genetic modification have no report.
Summary of the invention
In order to solve the problem, the object of this invention is to provide a kind of method eliminating by product polysaccharide in fermentative production pyrroloquinoline quinone process, utilize the purifying process of microorganism scale operation PQQ to lay a good foundation for simplifying.
Another object of the present invention provides aforesaid method to produce the application in coenzyme or VITAMIN process at fermentable.
In order to realize foregoing invention object, technical scheme of the present invention is as follows:
The method of by product polysaccharide is eliminated in a kind of fermentative production pyrroloquinoline quinone process, the method is in the process of fermentable production pyrroloquinoline quinone, knock out pyrroloquinoline quinone by gene knockout means and produce important gene in 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's common micro-organisms center on August 20th, 2010, and its preserving number is CGMCCNo.4096.
Preferably, the important gene in described polysaccharide synthetic gene bunch is the gene of Phospholipase C γ1, as mpq_1844.
The preferred concrete steps of above-mentioned method are:
(1) screen pyrroloquinoline quinone and produce important gene in bacterial strain polysaccharide synthetic gene bunch;
(2) extract the genomic dna that described PQQ produces bacterial strain, and design primer, with this genomic dna for template, held and the 3 ' flanking sequence held by the described important gene 5 ' of PCR method amplification step (1);
(3) flanking sequence that step (2) gained 5 ' end flanking sequence and 3 ' is held is done double digestion process respectively, be connected on gene knockout carrier;
(4) step (3) described gene knockout carrier imports in described PQQ production bacterial strain by the mode of conventional electricity conversion or conjugal transfer, MP solid medium containing penbritin is cultivated, picking resistance list bacterium colony is going down to posterity 2-4 time without under screening conditions, contain positive colony on the culture medium flat plate of sucrose by the anti-label screening that sieves, and then identify whether important gene lacks by bacterium colony PCR;
(5) in shaking flask, carry out the PQQ fermenting experiment of genetically deficient bacterium and original production bacterial strain, get fermented liquid respectively centrifugal, observe phage surface form, the presence or absence of qualification bacterium surface insoluble polysaccharide, measures the content of soluble polysaccharide in supernatant liquor.
Preferably, the method of the important gene that step (1) described screening pyrroloquinoline quinone is produced in bacterial strain polysaccharide synthetic gene bunch is the important gene sequence (concrete network address is: http://www.ncbi.nlm.nih.gov/nuccore/NC_014733.1 from=1899875 & to=1901296 & report=fasta) downloaded in NCBI gene bank database in polysaccharide route of synthesis, be more preferably, Phospholipase C γ1 mpq_1844 and flanking sequence thereof, the nucleotide sequence of described mpq_1844 is as shown in SEQ.ID No.1, the nucleotide sequence of described flanking sequence is as shown in SEQ.ID No.2 and SEQ.ID No.3, described flanking sequence length, preferred 1500-2500bp.
Wherein, design primer described in step (2) and comprise forward and reverse two primers, be specially: 5 ' end flanking sequence forward primer UpF is as shown in SEQ.ID No.4, and reverse primer UpR is as shown in SEQ.ID No.5; 3 ' the flanking sequence forward primer DwF held is as shown in SEQ.ID No.6, and reverse primer DwR is as shown in SEQ.ID No.7.
Preferably, double digestion process described in step (3), is specially: 5 ' end flanking sequence ApaI and BamHI double digestion process (37 DEG C, 4-10 hour); 3 ' the flanking sequence SpeI held and NotI double digestion process (37 DEG C, 4-10 hour); 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 DEG C, 1-6 hour; Described linked system is as shown in table 4.
Preferably, in step (4), the final concentration of penbritin is 50 μ g/ml; Described anti-sieve is labeled as sacB; The sucrose concentration used in described substratum is specially 0.01 ~ 0.05g/mL; The bacterium colony PCR of described identified gene disappearance primer used is UpF and DwR in step (2); Described genetically deficient bacterium amplification is 4000bp, and specifically as shown in SEQ.ID No.8, producing bacterial strain amplification is 5422bp, specifically as shown in SEQ.ID No.9;
The mode of described conventional electricity conversion or conjugal transfer and the condition of importing, with reference to the people document Magnetosome vesicles are present before magnetite formation of 2004, andMamA is required for their activation such as Komeili A.
Preferably, soluble polysaccharide measuring method described in step (5) is phend-sulphuric acid, specifically according to the method steps of the people such as Dubois M. document Colorimetric method for determination of sugarsand related substancede report in 1956; 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 fermentable 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, the important gene mpq_1844 successfully produced by PQQ in the polysaccharide synthetic gene bunch of bacterial strain Methylovorussp.MP688 knocks out, the clpp gene of original production bacterial strain is degerming no longer synthesizes exocellular polysaccharide, under the prerequisite that the production of bacterial growth and PQQ is not impacted, greatly reduce the synthesis level of soluble polysaccharide and insoluble polysaccharide, achieve the removal of metabolic by-prods polysaccharide in fermentable production pyrroloquinoline quinone process, thus reach simplify purifying process object, prepare pyrroloquinoline quinone for industrial high efficiency to lay a good foundation.
Accompanying drawing explanation
The PCR proof diagram of the mpq_1844 gene knock-out bacterial strain of Fig. 1 display.From left to right DNA molecular amount marker respectively, with the original production strain gene group DNA amplification that is template, for the amplification that is template with mpq_1844 gene knock-out bacterial strain genomic dna.
What Fig. 2 showed is mpq_1844 gene knock-out bacterial strain and the difference of original production bacterial strain in growth and the outer soluble polysaccharide of born of the same parents.▲ be original production bacterial strain soluble polysaccharide synthesis situation, ■ is gene knock-out bacterial strain soluble polysaccharide synthesis situation.
What Fig. 3 showed is the difference that mpq_1844 gene knock-out bacterial strain and original production bacterial strain capsular polysaccharide synthesize.Left figure is original production bacterial strain capsular polysaccharide synthesis situation, and right figure is gene knock-out bacterial strain capsular polysaccharide synthesis situation.
Fig. 4 display be the comparison of mpq_1844 gene knock-out bacterial strain and original production bacterial strain PQQ throughput.▲ be that original production bacterial strain PQQ synthesizes situation, ■ is that gene knock-out bacterial strain PQQ synthesizes situation.
Embodiment
The present invention is introduced in detail below in conjunction with accompanying drawing and embodiment thereof.But protection orientation of the present invention is not limited to following instance, should comprise the full content in claims.
Experiment material involved in following examples is all obtained by commercial sources or is prepared by normal experiment; Restriction endonuclease used, ligase enzyme, pcr amplification enzyme are all purchased from Beijing Quanshijin Biotechnology Co., Ltd; Experiment instrument and equipment is biology laboratory routine.
The acquisition of embodiment 1mpq_1844 gene and 5 ' end and the 3 ' flanking fragment held and knock out the structure of plasmid
From NCBI gene bank database (concrete network address is: http://www.ncbi.nlm.nih.gov/nuccore/NC_014733.1 from=1899875 & to=1901296 & report=fasta), input mpq_1844, download and preserve gene order and 5 ' end and the 3 ' flanking sequence held; The nucleotide sequence of described mpq_1844 is as shown in SEQ.ID No.1, and the nucleotide sequence of described flanking sequence is as SEQ.ID No.2 and SEQ.ID No.3.The shown genomic dna using genome to extract test kit (Tiangen company bacterial genomes extracts test kit) extracting PQQ production bacterial strain Methylovorus sp.MP688, use 5 ' end flanking sequence forward primer UpF (ATT GGG CCT GCC GAT GGG CGC TGA CC) and reverse primer UpR as shown in SEQ.ID No.5 (CG GGA TCC GAGGCC CGT GGA GTC TGC) as shown in SEQ.ID No.4, 3 ' the flanking sequence forward primer DwF held 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 as shown in SEQ.ID No.7 as shown in SEQ.ID No.6, the flanking sequence of amplification is respectively 2000bp, consistent with the flanking sequence SEQ.ID No.2 downloaded and SEQ.ID No.3 respectively.PCR amplification system and condition be as shown in Table 1 and Table 2:
Each substances content in table 1PCR50 μ l reaction solution
Table 2PCR reaction conditions
By 5 ' end flanking sequence PCR primer ApaI and the process of BamHI double digestion, described double digestion treatment condition are 37 DEG C, 4-10 hour; 3 ' end flanking sequence PCR primer SpeI and the process of NotI double digestion, described double digestion treatment condition are 37 DEG C, 4-10 hour; Described double digestion reaction solution system is as shown in table 3.Carrier pWM91 successively equally process after, enzyme is cut rear PCR primer cut with enzyme in turn after carrier is connected, connection temperature be 16-25 DEG C, 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 qualification
The plasmid that knocks out embodiment 1 obtained by conventional electricity conversion or conjugal transfer imports in PQQ production bacterial strain Methylovorus sp.MP688, importing condition is with reference to people document Magnetosome vesicles are present before magnetite formation of 2004 such as Komeili A, and MamA isrequired for their activation, be coated on by reacted bacterium liquid on the penbritin MP solid medium upper flat plate containing 50 μ g/ml, described MP solid culture based component is containing 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 random picking single bacterium colonies afterwards for 30 DEG C, 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 100 μ l and be coated with the flat board containing 0.02g/mL sucrose, culture medium prescription is the same, cultivate 2 days random picking 100 single bacterium colonies afterwards for 30 DEG C, use 5 ' end flanking sequence forward primer UpF(ATT GGG CCTGCC GAT GGG CGC TGA CC) and 3 ' the flanking sequence reverse primer DwR(GAA AAAAGC GGC CGC CCC GGA TGA CTG GGT TGG held) be bacterium colony PCR and identify, amplification system and program are in table 5 and table 6:
Each substances content in table 550 μ l bacterium colony PCR reaction solution
Table 6PCR reaction conditions
Genetically deficient bacterium amplification is 4000bp, and its nucleotide sequence refers to SEQ.ID No.8; Producing bacterial strain amplification is 5422bp, and its nucleotide sequence refers to SEQ.ID No.9, and concrete amplification refers to Fig. 1.
The mensuration of embodiment 3 exocellular polysaccharide and pyrroloquinoline quinone content
By the knock-out bacterial strain that screens and original production bacterial strain Simultaneous vaccination, to the MP liquid nutrient medium containing 100ml, (culture medium prescription is with embodiment 3, do not add agar powder) 250ml triangular flask in, 30 DEG C, 200rpm cultivates, the phend-sulphuric acid reported according to people document Colorimetric method fordetermination of sugars and related substancede of 1956 such as Dubois M. measures soluble polysaccharide content, the polysaccharide content measured in genetically deficient fermented liquid is at below 10mg/l, can think there is no polysaccharide, produce in the bacterial strain fermented liquid of 60 hours and detect that polysaccharide content is that 700mg/l(refers to Fig. 2), original production bacterial strain after centrifugation phage surface has floss, and genetically deficient bacterium without, illustrate that transposon mutagenesis ability is lost (referring to Fig. 3).Get the fermented liquid of knock-out bacterial strain and original production bacterial strain respectively, the content of pyrroloquinoline quinone is measured according to the glucose dehydro enzyme method of bibliographical information, concrete document is with reference to people document Enzymatic determination of pyrroloquinoline quinoneusing crude membranes from Escherichia coli of 1987 such as GeigerO., and result shows two strain bacterium content of pyrroloquinoline quinone in cultivation 70 hours secondary fermentation liquid and is respectively 41.5mg/l and 39.5mg/l(and refers to Fig. 4).This does not affect the synthesis of pyrroloquinoline quinone after Polysaccharide removing is described.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the prerequisite not departing 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 (8)
1. in a fermentative production pyrroloquinoline quinone process, eliminate the method for by product polysaccharide, it is characterized in that, the method is in the process of fermentable production pyrroloquinoline quinone, knock out pyrroloquinoline quinone by gene knockout means and produce important gene in 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; Described production bacterial strain is methylotrophic bacteria Methylovorus sp.MP688; Important gene in described polysaccharide synthetic gene bunch is the mpq_1844 in Phospholipase C γ1.
2. method according to claim 1, its feature is used for, and the concrete steps of described method are:
(1) screen pyrroloquinoline quinone and produce important gene in bacterial strain polysaccharide synthetic gene bunch;
(2) extract the genomic dna that described pyrroloquinoline quinone produces bacterial strain, and design primer, with this genomic dna for template, held and the 3 ' flanking sequence held by the described important gene 5 ' of PCR method amplification step (1);
(3) flanking sequence that step (2) gained 5 ' end flanking sequence and 3 ' is held is done double digestion process respectively, be connected on gene knockout carrier;
(4) step (3) described gene knockout carrier imports in described pyrroloquinoline quinone production bacterial strain by the mode of conventional electricity conversion or conjugal transfer, MP solid medium containing penbritin is cultivated, picking resistance list bacterium colony is going down to posterity 2-4 time without under screening conditions, contain positive colony on the culture medium flat plate of sucrose by the anti-label screening that sieves, and then identify whether important gene lacks by bacterium colony PCR;
(5) in shaking flask, carry out the pyrroloquinoline quinone fermenting experiment of genetically deficient bacterium and original production bacterial strain, get fermented liquid respectively centrifugal, observe phage surface form, the presence or absence of qualification bacterium surface insoluble polysaccharide, measures the content of extracellular soluble exo polysaccharides in supernatant liquor.
3. method according to claim 2, it is characterized in that, the method of the important gene that the described screening pyrroloquinoline quinone of step (1) is produced in bacterial strain polysaccharide synthetic gene bunch is the important gene sequence downloaded in NCBI gene bank database in polysaccharide route of synthesis, wherein, described important gene is Phospholipase C γ1 mpq_1844 and flanking sequence thereof, the nucleotide sequence of described mpq_1844 is as shown in SEQ.ID No.1, and the nucleotide sequence of described flanking sequence is as shown in SEQ.ID No.2 and SEQ.ID No.3.
4. method according to claim 2, is characterized in that, described design primer comprises forward and reverse two primers, is specially: 5 ' end flanking sequence forward primer UpF is as shown in SEQ.ID No.4, and reverse primer UpR is as shown in SEQ.ID No.5; 3 ' the flanking sequence forward primer DwF held is as shown in SEQ.ID No.6, and reverse primer DwR is as shown in SEQ.ID No.7.
5. method according to claim 2, is characterized in that, double digestion process described in step (3), is specially: 5 ' end flanking sequence ApaI and the process of BamHI double digestion; 3 ' the flanking sequence SpeI held and the process of NotI double digestion; Described gene knockout carrier is plasmid pWM91.
6. method according to claim 2, is characterized in that, the final concentration of the penbritin described in step (4) is 50 μ g/mL; Described anti-sieve is labeled as sacB; The sucrose concentration used in described substratum is 0.01 ~ 0.05g/mL.
7. method according to claim 2, is characterized in that, described in step (5), soluble polysaccharide measuring method is phend-sulphuric acid; Described centrifugal rotational speed and time, be specially 10000-12000 rev/min, 5-10 minute.
8. the application of method described in claim 1 in fermentable production pyrroloquinoline quinone process.
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