CN105543154A - L-tryptophan production genetically engineered bacterium and construction method and application thereof - Google Patents
L-tryptophan production genetically engineered bacterium and construction method and application thereof Download PDFInfo
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Abstract
The invention relates to the field of microorganisms, and discloses an L-tryptophan production genetically engineered bacterium and a construction method and application thereof. A common pathway of an Escherichia coli central carbon metabolic pathway is modified through a genetic engineering method, a phosphoric enol pyruvic acid-phosphotransferase system with phosphoric enol pyruvic acid as a substrate of escherichia coli is knocked out, a glucose transfer system with non-PEP as a substrate is enhanced, a glucose transfer endocytosis pathway is changed, the L-tryptophan production genetically engineered bacterium is obtained, and consumption of L-tryptophan synthesis precursor phosphoric enol pyruvic acid in the glucose transferring process is avoided, so that the intracellular PEP level is improved, and then the capability of L-tryptophan produced through strain fermentation is improved. An experiment shows that the constructed L-tryptophan production genetically engineered bacterium is an escherichia coli L-tryptophan high-producing strain, L-tryptophan can be effectively accumulated, the yield of the L-tryptophan is improved, and a foundation is laid for industrial production of the L-tryptophan.
Description
Technical field
The invention belongs to microorganism field, relate to and a kind of produce L-Trp genetic engineering bacterium and construction process thereof and purposes.
Background technology
Tryptophane, formal name used at school tryptophane (β-indolylalanine), it is one of essential amino acid in human body and animal life activity, the growing of humans and animals, metabolism are played an important role, be called as the second indispensable amino acid, be widely used in the aspect such as medicine, food and feed.At field of medicaments, tryptophane is the important component of amino acid transfusion and important medicine intermediate.In food applications field, tryptophane can be used for nutrient fortified food, improves local flavor, also can be used for bread and promotes fermentation.Field is added at feed, after Methionin and methionine(Met) are met, tryptophane becomes the important limiting amino acid of daily ration, supplemented with exogenous tryptophane can improve the content of tryptophane in poultry, fowl, fish daily ration, improve daily ration amino acid composition and ratio, improve value and the utilising efficiency of dietary protein.
The production method of current L-Trp mainly contains four kinds, comprises albumen hydrolysis, chemical synthesis, enzymatic synthesis and biological fermentation process.Albumen hydrolysis and chemical synthesis also exist that raw material sources is limited respectively, production technique and step is many, optical resolution is difficult and yield is low, the production cycle is long etc. shortcoming, are extremely restricted in the industrial production.Enzymatic synthesis has the advantages such as end product accumulation volume is high, reaction time is short, separating-purifying is easy, is the effective ways producing L-Trp, is once widely used in the suitability for industrialized production of L-Trp by countries in the world.But enzymatic synthesis produces the substrate Serine of L-Trp and the expensive of indoles, and indoles is insoluble in water, tryptophan synthetic enzyme suppresses strong, affects the raising of transformation efficiency, and the cost causing enzymatic synthesis to produce L-Trp remains high.Therefore biological fermentation process is the main method being widely used in industrial production L-Trp at present.
The research that biological fermentation process produces L-Trp starts from the twentieth century initial stage sixties, but within one quite long afterwards period, do not reach the requirement of suitability for industrialized production, major cause is that the biosynthetic pathway from glucose to L-Trp is very long, its metabolic fluxes is also more weak, and L-Trp synthesis needs multiple precursor (as Serine, glutamine, PRPP etc.).On the other hand, the metabolic regulation mechanism also more complicated in L-Trp biosynthetic pathway.
1979, Tribe and Pittard utilizes DNA recombinant technology first trpE gene to be introduced intestinal bacteria (Escherichiacoli), L-Trp output reaches 1g/L (TribeDE, PittardJ.HyperproductionoftryptophanbyEscherichiacoli:ge neticmanipulationofthepathwaysleadingtotryptophanformati on.ApplEnvironMicrobiol, 1979,38 (2): 181 – 190.).
After this, along with the widespread use of gene recombination technology in Microbial Breeding, the triage techniques that L-Trp produces bacterial strain achieves important breakthrough, and L-Trp acid yield have also been obtained huge raising.
1996, Berry removed the aroG of feedback inhibition (feed-backresistance, fbr) at expression in E. coli
fbrand trpE
fbrdCBA gene, ferment and produce the nearly 45g/L of L-Trp in 52 hours, the highest glucose acid invert ratio of process is about 22% (BerryA.ImprovingproductionofaromaticcompoundsinEscherich iacolibymetabolicengineering.TrendsBiotechnol, 1996,14 (7): 250 – 256.).
1999, Ikeda etc. are high expression level tktA gene in Corynebacterium glutamicum (Corynebacteriumglutamicum) pIK9960 producing L-Trp, increase the level of L-Trp synthesis precursor E4P, thus improve the combined coefficient of L-Trp, the L-Trp output of fermenting 80 hours reaches 58g/L (IkedaM, KatsumataR.HyperproductionoftryptophanbyCorynebacteriumg lutamicumwiththemodifiedpentosephosphatepathway.ApplEnvi ronMicrobiol, 1999, 65 (6): 2497 – 2502.).
In addition, be that Host Strains is produced the granted patent of L-Trp and also had WO9408031A1, EP0293207, US4371614, US4588687, CN1289676C, CN100582220C and CN101622338B etc. with recombination bacillus coli.The site of its new structure of these patent protections, also illustrates the ability that their different fermentations produce acid.
Intestinal bacteria are by number of ways transhipment and phosphorylation glucose generates G6P (Fig. 1), then imported glycolytic pathway.
Wild-type e. coli utilizes phosphoenolpyruvic acid-sugar phosphotransferase system (being called for short PTS system) transhipment and phosphorylation glucose (Fig. 1).PTS system is the abbreviation of phosphoenolpyruvic acid-sugar phosphotransferase system, and be made up of E I, HPr and E II s, wherein E I and HPr is respectively by ptsI and ptsH genes encoding, is kytoplasm soluble proteins; E II s comprises E II
man, E II
fru, E II
bgl, E II A
glcwith E II CB
glcdeng, mostly be protein complexes, to carbohydrate, there is specificity, wherein E II A
glcwith E II CB
glcrespectively by crr and ptsG genes encoding.PTS system plays an important role in glucose transport and Phosphorylation events, is the core of intestinal bacteria glucose metabolism genes expression regulation.PTS system transports 1 mol Glucose need consume 1 mole of phosphoric acid enol pyruvic acid (PEP) (1molGlucose+1molPEP → 1molGlucose-6-P+1molPyruvate) (NeidhardtFC, PostmaPW, LengelerJW, etal.EscherichiacoliandSalmonella.2nded.WashingtonD.C.:A SMPress, 1996:1824 – 1866.; Hern á ndez-MontalvoV, Mart í nezA, Hern á ndez-ChavezG, etal.ExpressionofgalPandglkinaEscherichiacoliPTSmutantre storesglucosetransportandincreasesglycolyticfluxtofermen tationproducts.BiotechnolBioeng, 2003,83 (6): 687 – 694.).When intestinal bacteria grow in the restricted substratum taking glucose as carbon source, the PEP of PTS system consumption about 50% is used for transhipment and the phosphorylation of glucose, direct impact take PEP as synthesis (the GossetG.ImprovementofEscherichiacoliproductionstrainsbym odificationofthephosphoenolpyruvate:sugarphosphotransfer asesystem.MicrobCellFact of the compound (as shikimic acid, die aromatischen Aminosaeuren and aspartic acid race amino acid etc.) of precursor, 2005,4 (1): 14.).Same directly impact take PEP as the synthesis of the L-Trp of precursor.
PTS system defect type intestinal bacteria can pass through semi-lactosi/hydrogen ion cotransporter (D-Galactose/H+symporter, and glucokinase (Glucokinase GalP), Glk) synergy transhipment glucose enters born of the same parents (Fig. 1), take ATP as phosphate group donor (1molGlucose+1molATP → 1molGlucose-6-P+1molADP), but the lower (NeidhardtFC of efficiency of the phosphorylation transhipment glucose that acts synergistically with GalP and Glk, PostmaPW, LengelerJW, etal.EscherichiacoliandSalmonella.2nded.WashingtonD.C.:A SMPress, 1996:1824 – 1866., Hern á ndez-MontalvoV, Mart í nezA, Hern á ndez-ChavezG, etal.ExpressionofgalPandglkinaEscherichiacoliPTSmutantre storesglucosetransportandincreasesglycolyticfluxtofermen tationproducts.BiotechnolBioeng, 2003,83 (6): 687 – 694., GossetG.ImprovementofEscherichiacoliproductionstrainsbym odificationofthephosphoenolpyruvate:sugarphosphotransfer asesystem.MicrobCellFact, 2005,4 (1): 14.).
Summary of the invention
In view of this, the defect that the object of the invention is to exist for prior art provides the genetic engineering bacterium and construction process thereof and purposes that a kind of L-Trp output is high.
For realizing object of the present invention, the present invention adopts following technical scheme:
The invention provides and a kind of produce L-Trp genetic engineering bacterium, in its PTS system, one or more gene is knocked or inactivation, and is one or morely over-expressed with the non-PEP glucose transporter that is substrate or enzyme genes involved.
Preferably, to knock out described in or the gene of inactivation is ptsH and/or ptsI and/or crr.
Preferably, the gene be over-expressed described in is glf and glk gene.
In one embodiment, the invention provides a kind of product L-Trp genetic engineering bacterium MHZ-0811, in its PTS system, ptsH, ptsI and crr gene knocks out completely from genome, glf and glk gene is over-expressed.Described genetic engineering bacterium MHZ-0811 is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCCNo.11940.
The invention provides a kind of construction process of described product L-Trp genetic engineering bacterium, comprising:
Steps A, L-Trp are produced in bacterium PTS system one or more to glucose phosphorylation with transport relevant gene and be knocked or inactivation, acquisition mutant strain;
Step B, L-Trp are produced and one or morely in bacterium to be over-expressed with the non-PEP glucose transporter that is substrate or the relevant gene of enzyme, obtain recombinant vectors;
Step C, by step B recombinant vectors step of converting A mutant strain and get final product.
Preferably, to knock out described in or the gene of inactivation is ptsH and/or ptsI and/or crr.
Preferably, the gene be over-expressed described in is glf and glk gene.
Present invention also offers the application of described product L-Trp genetic engineering bacterium in L-Trp is produced.
Further, present invention also offers a kind of production method of L-Trp, it is numerous by deposit number to be that seed culture medium that the product L-Trp genetic engineering bacterium of CGMCCNo.11940 is inoculated in tetracyclin resistance carries out expansion, then by expand numerous after the fermention medium that proceeds to containing tetracyclin resistance of culture ferment.
Wherein, described fermention medium is preferably by 60g/L glucose, 1.0g/L yeast extract, 5g/LKH
2pO
4, 2.0g/L citric acid, 2g/LMgSO
47H
2o, 5g/L (NH
4)
2sO
4, 0.1g/LMnSO
4, 0.1g/LFeSO
47H
2o, 0.1g/LZnSO
4h
2o, 0.1g/LCoCl
26H
2o, 0.03g/LCuSO
45H
2o, 20g/LCaCO
3composition, pH value is 7.0.
The present invention is by the total approach of the colibacillary central carbon metablism approach of engineered method transformation, what knock out intestinal bacteria self take PEP as the PTS system of substrate, and that introduce motion pseudomonas source take ATP as Glf and Glk of substrate, change the path that glucose transport enters born of the same parents, avoid phosphoenolpyruvic acid (PEP) consumption in glucose transport process of L-Trp synthesis precursor, thus improve the level of PEP in born of the same parents, and then improve the ability that strain fermentation produces L-Trp.Experiment shows that the engineering strain that the present invention builds is intestinal bacteria L-Trp superior strain, effectively can accumulate L-Trp, improves the output of L-Trp, for the suitability for industrialized production of L-Trp is laid a good foundation.
Biological deposits information:
Bacterial strain MHZ-0811: Classification And Nomenclature: colon bacillus, Escherichiacoli. China Committee for Culture Collection of Microorganisms's common micro-organisms center is deposited on December 25th, 2015, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number is CGMCCNo.11940.
Accompanying drawing explanation
Fig. 1 shows the schematic diagram of recombination bacillus coli PTS system and other glucose transport systems;
Fig. 2 shows recombinant plasmid pMG43 schematic diagram, process LAN gene serA, aroG
fbrand trpE
fbrdCBA;
Fig. 3 shows recombinant plasmid pMG56 schematic diagram, process LAN gene serA, aroG
fbr, trpE
fbrdCBA, glf and glk.
Embodiment
The embodiment of the invention discloses a kind of method utilizing fermentable high yield tryptophane.Those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Method of the present invention is described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope method as herein described is changed or suitably change with combination, realize and apply the technology of the present invention.
For realizing object of the present invention, the present invention adopts following technical scheme:
In the process of intestinal bacteria synthesis L-Trp, often synthesizing 1 mole of L-Trp needs consumption 1 mole of phosphoric acid enol pyruvic acid (PEP) as precursor, and in born of the same parents, PEP level directly determines the ability that bacterial strain produces L-Trp.And intestinal bacteria utilize the PTS system of self to transport 1 mol Glucose need consume 1 mole of PEP, consume the PEP of about 50% in the transhipment and Phosphorylation events of glucose.
The present invention is according to the colibacillary central carbon metablism approach of product L-Trp, first one or more gene of PTS system in genome are knocked out, blocking-up need consume glucose phosphorylation and the transporting pathway of PEP, L-Trp biosynthetic important as precursors thing PEP is effectively avoided to enter loss in born of the same parents' process at glucose transport, improve the level of PEP in born of the same parents, thus be conducive to the accumulation of L-Trp.Secondly on plasmid vector, process LAN is one or more with non-PEP be substrate glucose transporter or enzyme genes involved, rebuild an allos glucose transport approach, take ATP as phosphate group donor, do not consume extra PEP, improve glucose transport and enter born of the same parents' efficiency, be conducive to the growth of bacterial strain and the accumulation of L-Trp, thus build a kind of engineering strain of high yield L-Trp.I.e. described product L-Trp genetic engineering bacterium, in its PTS system, one or more gene is knocked or inactivation, and is one or morely over-expressed with the non-PEP glucose transporter that is substrate or enzyme genes involved.
Preferably, to knock out described in or the gene of inactivation is ptsH and/or ptsI and/or crr.
Preferably, the gene be over-expressed described in is glf and glk gene.Preferably, described glf and glk gene source in motion pseudomonas (Zymomonasmobilis), glf genes encoding glucose transporter (Glucosefacilitator, Glf), glk gene encodes glucokinase (Glucokinase, Glk).Preferred, described glf and glk gene is positioned at the downstream of Ptac promotor and/or Pglf promotor.Namely described glf and glk gene is positioned at the downstream of Pglf promotor successively; Or glf and glk gene is positioned at the downstream of Ptac promotor successively; Or as shown in Figure 3, glf and glk gene is positioned at the downstream of Ptac promotor and Pglf promotor successively.
In one embodiment, the invention provides a kind of product L-Trp genetic engineering bacterium MHZ-0811, the basis of bacterial strain MHZ-0800 builds mutant, and in its PTS system, ptsH, ptsI and crr gene knocks out completely from genome, glf and glk gene is over-expressed.Described genetic engineering bacterium MHZ-0811 is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on December 25th, 2015, and deposit number is CGMCCNo.11940.
The invention provides a kind of construction process of described product L-Trp genetic engineering bacterium, comprising:
Steps A, L-Trp are produced in bacterium PTS system one or more to glucose phosphorylation with transport relevant gene and be knocked or inactivation, acquisition mutant strain;
Step B, L-Trp are produced and one or morely in bacterium to be over-expressed with the non-PEP glucose transporter that is substrate or the relevant gene of enzyme, obtain recombinant vectors;
Step C, by step B recombinant vectors step of converting A mutant strain and get final product.
Preferably, described steps A knocks out or the gene of inactivation is ptsH and/or ptsI and/or crr, knocks out or the gene of inactivation is one or more in ptsH, ptsI and crr namely.
Preferably, the gene of described step B process LAN is glf and glk.
In one embodiment, the present invention builds and produces L-Trp genetic engineering bacterium MHZ-0811, comprising:
Steps A: knocked out from genome completely by ptsH, ptsI and crr gene in the PTS system of bacterial strain MHZ-0800, obtains PTS system defect type bacterial strain SA01 △ ptsHIcrr;
Step B: insert glf and glk gene and obtain recombinant plasmid pMG56 between the Spe I and Sph I restriction enzyme site of plasmid vector pMG43;
Step C: recombinant plasmid pMG56 is transformed PTS system defect type bacterial strain SA01 △ ptsHIcrr, build and obtain gene recombination bacterial strain MHZ-0811.
Preferably, plasmid vector pMG43 is low copy plasmid, and copy number is 15-22; Preferred, the replicon of plasmid vector pMG43 is the replicon in pBR322 source or the replicon in p15A source.
The L-Trp utilizing the present invention to obtain is produced bacterial strain and is carried out fermentative production, can obtain effective accumulation of L-Trp, for the suitability for industrialized production of L-Trp is laid a good foundation.Therefore present invention also offers the application of described product L-Trp genetic engineering bacterium in L-Trp is produced.Preferably, described product L-Trp genetic engineering bacterium is bacterial strain MHZ-0811.
Further, present invention also offers a kind of production method of L-Trp, it is numerous by deposit number to be that seed culture medium that the product L-Trp genetic engineering bacterium of CGMCCNo.11940 is inoculated in tetracyclin resistance carries out expansion, then by expand numerous after the fermention medium that proceeds to containing tetracyclin resistance of culture ferment.
Wherein, described seed culture medium is by 20g/L glucose, 10g/L yeast extract, 9.5g/LKH
2pO
4, 5g/L (NH4)
2sO
4, 2g/LMgSO
47H
2o forms, and pH value is 7.0.
Preferably, the condition optimization that described expansion is numerous is 37 DEG C, 240rpm cultivates 5-10 hour.
Preferably, described fermention medium is preferably by 60g/L glucose, 1.0g/L yeast extract, 5g/LKH
2pO
4, 2.0g/L citric acid, 2g/LMgSO
47H
2o, 5g/L (NH
4)
2sO
4, 0.1g/LMnSO
4, 0.1g/LFeSO
47H
2o, 0.1g/LZnSO
4h
2o, 0.1g/LCoCl
26H
2o, 0.03g/LCuSO
45H
2o, 20g/LCaCO
3composition, pH value is 7.0.
Preferably, described fermentation condition optimization for for 37 DEG C, 240rpm cultivates 20-30 hour.
In order to understand the present invention further, below in conjunction with embodiment, the present invention is described in detail.
Wherein, bacterial strain MHZ-0800 (CGMCCNO.6863) is that the method described according to patent WO8701130A1 and Mascarenhas etc. builds (MascarenhasD.Tryptophanproducingmicroorganism:WO, 8701130A1.1985-08-15.; MascarenhasD, AshworthDJ, ChenCS.Deletionofpgialterstryptophanbiosynthesisinagenet icallyengineeredstrainofEscherichiacoli.ApplEnvironMicro biol, 1991,57 (10): 2995 – 2999.), its Host Strains SA01 is the E.coliK-12CICC10303 △ tnaA △ serA that E.coliK-12CICC10303 derives, contained plasmid pMG43 is pBR322 source, includes serA, aroG
fbrand trpE
fbrdCBA gene.
The structure of bacterial strain MHZ-0811, with reference to the document (DatsenkoKA of Datsenko etc., WannerBL.One-stepinactivationofchromosomalgenesinEscheri chiacoliK-12usingPCRproducts.ProcNatlAcadSciUSA, 2000, 97 (12): 6640 – 6645.) and Molecular Cloning: A Laboratory guide (SambrookJ, RussellDW.MolecularCloning:ALaboratoryManual.3rded.NewYo rk:ColdSpringHarborLaboratoryPress, 2001:2 – 110.), Red methods of homologous recombination is utilized to carry out ptsH in genome of E.coli, knocking out of ptsI and crr gene, and adopt pcr amplification, enzyme is cut, the conventional molecule clone technologies such as connection and conversion, construction of recombinant plasmid vector process LAN glf and glk gene.
Embodiment 1: the preparation knocking out the bacterial strain SA01 △ ptsHIcrr of ptsH, ptsI and crr gene
1. the preparation of mutant strain SA01 △ ptsHIcrr::kan
With plasmid pKD4 (CGSCNO.7632) for template, utilize primer pts1 and pts2, pcr amplification ptsHIcrr mutated gene segment, primer sequence is as follows:
Forward primer pts1:
5′-CTAGACTTTAGTTCCACAACACTAAACCTATAAGTTGGGGAAATACAATGGTGTAGGCTGGAGCTGCTTC-3′(SEQIDNO.1)
Reverse primer pts2:
5′-AAATGGCGCCGATGGGCGCCATTTTTCACTGCGGCAAGAATTACTTCTTGCATATGAATATCCTCCTTAG-3′(SEQIDNO.2)
Pcr amplification reaction system is:
hF damping fluid is not (containing MgSO
4) 10 μ L, dNTP (10mM) 1 μ L,
high-FidelityDNA polysaccharase 0.5 μ L, forward primer (10 μMs) 2.5 μ L, reverse primer (10 μMs) 2.5 μ L, template 0.5 μ L, adds ddH
2o to 50 μ L (the present invention's High fidelity PCR reaction reagent used is all purchased from NEB company).
Pcr amplification reaction condition is: 98 DEG C of denaturation 30sec; 98 DEG C of sex change 10sec, 60 DEG C of renaturation 20sec, 72 DEG C extend 40sec, 30 circulations; 72 DEG C extend 10min.
Glue reclaims the PCR primer (glue reclaims and reclaims test kit recovery method with reference to Tiangen glue) of about 1.5kbptsHIcrr mutated gene segment, electroporated for PCR primer (intestinal bacteria Electroporation-competent cells preparation method and electric method for transformation are with reference to 99 pages, " Molecular Cloning: A Laboratory guide III " the 1st chapter) 100 μ L are contained SA01 competent escherichia coli cell (the i.e. intestinal bacteria SA01/pKD46 competent cell of plasmid pKD46 (CGSCNO.7739), competent escherichia coli cell preparation method and method for transformation are with reference to 96 pages, " Molecular Cloning: A Laboratory guide III " the 1st chapter), at 30 DEG C, SOC substratum hatches 1-2 hour, the solid LB be coated on by bacterium liquid containing kantlex (25 μ g/mL) is dull and stereotyped, 30 DEG C of quiescent culture 20-24 hour, bacterium colony PCR checking is carried out to the transformant grown, PCR fragment size is that 1.5kb shows that corresponding transformant is the mutant strain SA01 △ ptsHIcrr::kan of ptsHIcrr gene.
By obtain positive transformant with pts3 and pts4 for primer, carry out bacterium colony PCR qualification, positive fragment size should be 1.5kb, and primer sequence is as follows.
Forward primer pts3:5 '-GCTAAAGTCGAACCGCCAGG-3 ' (SEQIDNO.3)
Reverse primer pts4:5 '-CCAGCAGCATGAGAGCGATG-3 ' (SEQIDNO.4)
PCR reaction system is: 10 × TransFastTaq damping fluid 5 μ L, dNTP (2.5mM) 4 μ L, TransFastTaqDNA polysaccharase 0.5 μ L, forward primer (10 μMs) 1.5 μ L, reverse primer (10 μMs) 1.5 μ L, template 0.5 μ L, adds ddH
2o to 50 μ L (the present invention identifies that PCR reaction reagent used is all purchased from Beijing Quan Shijin biotech company).
PCR reaction conditions is: 94 DEG C of denaturation 3min; 94 DEG C of sex change 5sec, 60 DEG C of renaturation 15sec, 72 DEG C extend 20sec, 25 circulations; 72 DEG C extend 10min.
2. the preparation (rejecting of kan resistant gene) of recombinant bacterial strain SA01 △ ptsHIcrr
Plasmid pCP20 (CGSCNO.7629) (CGSC:ColiGeneticStockCenter (YaleUniversity that can identify the recombinase FLP gene of FLP recognition site (FRT) of high temperature induction will be carried, NewHaven, CT) intestinal bacteria heredity stock center (Yale University)) electroporated (intestinal bacteria Electroporation-competent cells preparation method and electric method for transformation are with reference to 99 pages, " Molecular Cloning: A Laboratory guide III " the 1st chapter) mutant strain SA01 △ ptsHIcrr::kan, the solid LB that bacterium liquid is coated containing paraxin (25 μ g/mL) is dull and stereotyped, 30 DEG C of quiescent culture 20-24 hour, the single bacterium colony obtained is forwarded to not dull and stereotyped containing antibiotic solid LB, 42 DEG C of quiescent culture 12 hours, utilize high temperature induction to express recombinase FLP the box gene between FRT site is removed, thus obtain the recombinant bacterial strain SA01 △ ptsHIcrr that ptsHIcrr gene knocks out completely.
The gene knock-out bacterial strain obtained is with pts3 and pts6 for primer, and carry out bacterium colony PCR checking, positive fragment size should be 0.5kb, and primer sequence is as follows.
Forward primer pts3:5 '-GCTAAAGTCGAACCGCCAGG-3 ' (SEQIDNO.3)
Reverse primer pts6:5 '-TTGCCGCGATCTCGACAGTG-3 ' (SEQIDNO.5)
PCR reaction system is: 10 × TransFastTaq damping fluid 5 μ L, dNTP (2.5mM) 4 μ L, TransFastTaqDNA polysaccharase 0.5 μ L, forward primer (10 μMs) 1.5 μ L, reverse primer (10 μMs) 1.5 μ L, template 0.5 μ L, adds ddH
2o to 50 μ L.
PCR reaction conditions is: 94 DEG C of denaturation 3min; 94 DEG C of sex change 5sec, 60 DEG C of renaturation 15sec, 72 DEG C extend 10sec, 25 circulations; 72 DEG C extend 10min.
Embodiment 2: the structure of recombinant plasmid pMG56
The structure of 1.pET28-glfglk
With plasmid pSC6.090B (ATCCNo.PTA-2578) for template, increase with primer glfglk1 and glfglk2 the FK1 fragment (upstream sequence of glf-glk gene) of about 2.3kb, the FK2 fragment (downstream sequence of glf-glk gene) of the about 1.0kb that increases with primer glfglk3 and glfglk4.Above-mentioned PCR primer is carried out respectively agarose gel electrophoresis and glue recovery, obtain the DNA fragmentation of FK1 and FK2.With the DNA fragmentation of FK1 and FK2 altogether for template, the glf-glk gene fragment of the about 3.3kb that increases with primer lfglk1 and glfglk4.Introduce Xba I and Sph I restriction enzyme site at the 5 ' end of primer glfglk1 and glfglk4 respectively, primer sequence is as follows:
Forward primer glfglk1:
5′-GC
TCTAGACGACATCATAACGGTTCTG-3′(SEQIDNO.6)
Reverse primer glfglk2:
5′-ACGCGCATGGGTTCCACCGATGTCAATCG-3′(SEQIDNO.7)
Forward primer glfglk3:
5′-CGGTGGAACCCATGCGCGTTTCTCTATTG-3′(SEQIDNO.8)
Reverse primer glfglk4:
5′-ACAT
GCATGCGACTAGTCAGCCTCTTAAATTCAGTTC-3′(SEQIDNO.9)
PCR reaction system and reaction conditions are with embodiment 1 the 1st step.
Plasmid pET28a (being purchased from Merck company) Xba I, Sph I double digestion, reclaim the carrier segments of 5.1kb, connect with the glf-glk cut through same enzyme, connect product conversion intestinal bacteria Top10 competent cell (being purchased from Beijing Quan Shijin biotech company), picking transformant, with glfglk1 and glfglk4 for primer PCR is identified, get positive transformant extracting plasmid, and identify with Xba I, Sph I double digestion, gained positive recombinant plasmid pET28-glfglk size should be 8.4kb.
Double digestion reaction system: 10 × endonuclease reaction damping fluid 2 μ L, Xba I and each 1 μ L of Sph I restriction enzyme, treats digested plasmid carrier or PCR fragment, adds water and supply 20 μ L; 37 DEG C of enzymes cut 15-30min.
Ligation system: 10 × T4DNA connects damping fluid 1 μ L, and the mol ratio of carrier segments and target fragment is 1:3-7, T4DNA ligase enzyme 0.5 μ L, adds water and supplies 10 μ L; 22 DEG C connect 15-30min.
PCR reaction system and reaction conditions are with embodiment 1 the 2nd step.
2. the structure of recombinant plasmid pMG56
Plasmid pET28-glfglk Xba I, Sph I double digestion, reclaim the glf-glk gene fragment of 3.3kb; Plasmid pMG43 (CGMCCNo.6864) Spe I, Sph I double digestion, reclaim the carrier segments of about 12.5kb, be connected with the glf-glk gene fragment through Xba I, Sph I double digestion, connect product conversion intestinal bacteria Top10 competent cell, picking transformant, with glfglk1 and glfglk4 for primer PCR is identified, gets positive transformant extracting plasmid, and identify with Spe I, Sph I double digestion, gained positive recombinant plasmid pMG56 size should be 15.8kb.
Enzyme is cut, ligation system and reaction conditions be with embodiment 2 the 1st step; PCR reaction system and reaction conditions are with embodiment 1 the 2nd step.
The enzyme that in embodiment 2, pcr amplification reaction is used and reagent are all purchased from Beijing Quan Shijin biotech company; The enzyme that enzyme is cut, ligation is used and reagent are all purchased from Fermentas company; Plasmid extraction and DNA sepharose reclaim and reclaim test kit with reference to Tiangen company plasmid extraction kit and DNA; The preparation of intestinal bacteria Top10 competent cell and method for transformation are with reference to 96 pages, " Molecular Cloning: A Laboratory guide III " the 1st chapter.
Embodiment 3: the acquisition of producing L-Trp recombinant bacterial strain
The mutant E. coli SA01 △ ptsHIcrr of ptsHIcrr gene knockout embodiment 1 obtained makes competent cell (preparation of competent cell and method for transformation are with reference to 96 pages, " Molecular Cloning: A Laboratory guide III " the 1st chapter), recombinant plasmid pMG56 embodiment 2 obtained transforms Host Strains SA01 △ ptsHIcrr, obtain L-Trp fermentative production engineering strain MHZ-0811 (pMG56/SA01 △ ptsHIcrr, preserving number: 11940).
Described engineering strain MHZ-0811 is carried out biological deposits, and its relevant information is as follows:
Bacterial strain MHZ-0811: Classification And Nomenclature: colon bacillus, Escherichiacoli. common micro-7 Bio-Centers of China Committee for Culture Collection of Microorganisms are deposited on December 25th, 2015, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number is CGMCCNo.11940.
Embodiment 4: utilize L-Trp engineering strain fermentative production L-Trp
Coli strain MHZ-0800 and MHZ-0811 is got respectively at the flat lining out of LB containing tetracyclin resistance (10 μ g/mL), 37 DEG C of quiescent culture 18-24 hour from cryopreservation tube; Thalline is scraped next ring from flat board, is inoculated into and 50mL is housed containing in the shaking flask of tetracyclin resistance (10 μ g/mL) seed culture medium substratum, 37 DEG C, 240rpm cultivates 5-10 hour, OD
600control at 6-10; Get 2mL seed liquor be forwarded to be equipped with 20mL containing tetracyclin resistance (10 μ g/mL) fermention medium shaking flask in, 37 DEG C, 240rpm cultivates 20-30 hour, in culturing process, stream adds weak ammonia, controls fermented liquid pH value to 6.5-7.0, until residual sugar exhausts, fermentation ends.
After fermentation ends, adopt the L-Trp content in HPLC method mensuration fermented liquid supernatant, HPLC condition determination is as follows: HPLC (Agilenttechnologies, 1200), chromatographic column: XDB-C18 (4.6 × 250mm5 μm); Moving phase is: 8.5mmol/LNaAC (HAC adjusts pH to 4.0): methyl alcohol=95:5 (V/V), and flow velocity is 1.5mL/min; Sample size 15 μ L; Detector: VWD, determined wavelength 280nm; Testing environment: temperature 16.3 DEG C, humidity 20%RH.
Seed culture medium component:
Fermention medium component:
Carry out 3 shake flask fermentation parallel laboratory tests, shake flask fermentation L-Trp yield result is as shown in table 1:
Table 1. engineering strain L-Trp Yield compari@:
Can learn that genetic engineering bacterium MHZ-0811 shake flask fermentation L-Trp output is 5.5g/L from table 1, glucose acid invert ratio is 10.3%, improves 22% compared with the product acid 4.5g/L of starting strain MHZ-0800.Result shows that carrying out transformation to colibacillary PTS system serves obvious effect.
In sum, the L-Trp genetic engineering bacterium constructed by the present invention can realize effective accumulation of L-Trp in fermented liquid in fermenting process, and L-Trp output significantly improves, thus for L-Trp produce industrialization lay a good foundation.
The explanation of above embodiment just understands method of the present invention and core concept thereof for helping.It should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention, can also carry out some improvement and modification to the present invention, these improve and modify and also fall in the protection domain of the claims in the present invention.
Claims (10)
1. one kind is produced L-Trp genetic engineering bacterium, it is characterized in that, in phosphoenolpyruvic acid-sugar phosphotransferase system, one or more gene is knocked or inactivation, and is one or morely over-expressed with the non-PEP glucose transporter that is substrate or enzyme genes involved.
2. genetic engineering bacterium as claimed in claim 1, is characterized in that, described in be knocked or the gene of inactivation is ptsH and/or ptsI and/or crr.
3. genetic engineering bacterium as claimed in claim 1, is characterized in that, described in the gene that is over-expressed be glf and glk gene.
4. genetic engineering bacterium as claimed in claim 1, it is characterized in that, described genetic engineering bacterium is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCCNo.11940.
5. produce the construction process of L-Trp genetic engineering bacterium described in claim 1, it is characterized in that, comprising:
Steps A, L-Trp are produced in bacterium PTS system one or more to glucose phosphorylation with transport relevant gene and be knocked or inactivation, acquisition mutant strain;
Step B, L-Trp are produced and one or morely in bacterium to be over-expressed with the non-PEP glucose transporter that is substrate or the relevant gene of enzyme, obtain recombinant vectors;
Step C, by step B recombinant vectors step of converting A mutant strain and get final product.
6. construction process as claimed in claim 5, is characterized in that, described in be knocked or the gene of inactivation is ptsH and/or ptsI and/or crr.
7. construction process as claimed in claim 5, is characterized in that, described in the gene that is over-expressed be glf and glk gene.
8. described in claim 1, produce the application of L-Trp genetic engineering bacterium in L-Trp is produced.
9. the production method of a L-Trp, it is characterized in that, it is numerous by deposit number to be that seed culture medium that the product L-Trp genetic engineering bacterium of CGMCCNo.11940 is inoculated in tetracyclin resistance carries out expansion, then by expand numerous after the fermention medium that proceeds to containing tetracyclin resistance of culture ferment.
10. production method according to claim 9, it is characterized in that, described fermention medium is by 60g/L glucose, 1.0g/L yeast extract, 5g/LKH
2pO
4, 2.0g/L citric acid, 2g/LMgSO
47H
2o, 5g/L (NH
4)
2sO
4, 0.1g/LMnSO
4, 0.1g/LFeSO
47H
2o, 0.1g/LZnSO
4h
2o, 0.1g/LCoCl
26H
2o, 0.03g/LCuSO
45H
2o, 20g/LCaCO
3composition, pH value is 7.0.
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| CN109852572A (en) * | 2019-01-28 | 2019-06-07 | 江南大学 | A method of it knocking out Escherichia coli PTS system and improves L-threonine yield |
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| CN111778200B (en) * | 2019-04-04 | 2022-11-01 | 中国科学院微生物研究所 | Platform bacterium for producing L-aspartic acid, recombinant bacterium for producing beta-alanine constructed based on platform bacterium and construction method thereof |
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