CN102041284A - Method for improving acid production rate of L-phenylalanine genetic engineering bacterial - Google Patents

Abstract

The invention relates to a method for improving acid production rate of L-phenylalanine genetic engineering bacterial. The method comprises the steps of: constructing the original engineering plasmid MDphe-2 of L-phenylalanine into a new generation of engineering plasmid MDphe-3 by adopting a PCR (polymerase chain reaction)technology; carrying out deletion constructing on a whole metabolism network regulatory gene csrA of the engineering host bacterial of the engineering plasmid MDphe-3 through a Red system; then implanting the engineering plasmid MDphe-3 in the engineering host bacterial which is in deletion of the whole metabolism network regulatory gene csrA to construct a new L-phenylalanine genetic engineering bacterial; and finally verifying and reserving the L-phenylalanine gene engineering bacterial with high hereditary stability and high acid production rate. The method has the advantages that the PEP accumulation of one of precursors of aromatic amino acid is strengthened, simultaneously the carbon metabolism flux can be converted heavily to a shikimic acid way, the large flowing of the metabolism flux of the aromatic amino acid to the synthesis of the L-phenylalanine is ensured, and the ability of the engineering bacterial to ferment the L- phenylalanine is further improved.

Description

A kind of method that improves L-phenylalanine gene engineering bacteria acid production rate

[technical field]

The present invention relates to bioengineering field, be specifically related to a kind ofly adopt the Red system to mediate the method that improves L-phenylalanine acid production rate by round pcr improvement project plasmid and to the host bacterium of transforming the back plasmid.

[background technology]

L-phenylalanine (L-phenylalanine) is called for short L-Phe, be white crystalline powder, be human body and animal can not be in vivo one of necessary amino acid of synthetic voluntarily, be widely used in fields such as food, feed, medicine and makeup, the dipeptide sweetener aspartame (Aspartame) of especially low in calories, high sugariness is used increasingly extensive, increases sharply as the market demand of the L-phenylalanine of one of synthetic two kinds of raw materials of aspartame.The L-phenylalanine also is the essential raw material of antitumor drug and amino acid transfusion preparation in addition, along with its continually developing of field of medicaments, demand also constantly increases.Therefore, the research of L-phenylalanine biosynthetic pathway, L-phenylalanine are produced the improvement of bacterium and the research of its suitability for industrialized production more and more is subject to people's attention.

The production method of L-Phe mainly contains hydrolysis extraction process, chemical synthesis, enzyme process and fermentation method etc. both at home and abroad.Because the L-phenylalanine content is lower in the native protein, the hydrolysis extraction process seldom uses; The complex process of chemical synthesis, cost is higher, is replaced by enzyme process and fermentation method basically abroad.But because the price of substrate and enzyme is higher and originate limitedly, enzyme process is used and also is restricted; Because the microorganism direct fermentation can utilize raw material cheap and easy to get, can carry out at normal temperatures and pressures again, be the main stream approach of producing L-Phe at present both at home and abroad, have bigger competitive edge.Characteristics such as wherein intestinal bacteria are rapid with its breeding, cultivation is simple, easy to operate, genetic background is clear are extensively transform as the genetically engineered receptor biological of the safety that is adapted to exogenous gene expression.But, make its cell can not accumulate phenylalanine in a large number because end products such as L-Phe have the intensive feedback inhibition to the key enzyme on the intravital die aromatischen Aminosaeuren metabolic pathway of synthesizing of wild strain.Simultaneously, many competition branch roads have also been shunted the energy flow direction of L-Phe relatively, thereby make that the yield of L-Phe is limited.Therefore if will carry out real attenuation production, just must transform wild strain, remove the shunt metabolism regulation and control, make excessive the accumulating of its purpose metabolic end product energy.

Traditional strain selection is to produce the bacterial strain that acid can be strong from the occurring in nature high flux screening, but amino acid kind and output that this method obtains are limited, are establishing mutating technology and are illustrating the seed selection that develops into the auxotrophy variant on the basis of amino acid synthesis system regulation mechanism afterwards.Utilize the method for conventional selection by mutation, its blindness is big, and workload is heavy, and can not increase the number of copies of gene, and mutant strain generally all carries the multiple nutrients defective, and further raising output is restricted.

In recent years, along with deepening continuously of molecular biology research, the increasing biosynthetic pathway of organism is familiar with by people, the universality research of the mensuration of some microbial genome complete sequences and the synergy between the polygene and metabolism network, the rise of metabolic engineering and information biology research, gene, host's organism, the development of carrier system and biology tool, modification by pathways metabolism not only can improve oneself chemical substance output of having of cell or produce host cell itself can not the synthetic novel substance, and can expand the substrate use range of cell, improve some primary metabolite that important economic worth is arranged and secondary metabolites.Wherein has most the model of success to improve amino acid whose output in these class methods.

The intestinal bacteria direct fermentation synthesizes the L-phenylalanine, and biosynthetic pathway is divided into common pathway and branch's approach, and common pathway provides precursor substance for branch's approach.With glucose is that initiator is through E4P (Erythrose-4-phosphate, E4P, the intermediate product of five-carbon sugar phosphoric acid approach) and the intermediate product of glycolytic cycle---phosphoenolpyruvic acid (Phophoenol pyruvate, PEP) the two condensation forms one seven carbon ketose open chain phosphate cpd, be called 3-deoxidation-α-Arabic heptanone saccharic acid-7-phosphoric acid (3-deoxy-α-arobinoheptulosonate-7-phosphate, DAHP).This is the biosynthetic common metabolic approach of die aromatischen Aminosaeuren, also is the committed step of its productive rate of decision and output.Therefore, in the biosynthesizing of intestinal bacteria L-phenylalanine, DAHP is that first also is most important metabolic intermediate in the L-phenylalanine biosynthetic process, and can flow the synthetic of the DAHP that leads effectively to the center metabolism be the determinative of the fragrant L-phenylalanine biosynthesizing output height of decision.The enzyme of this committed step of catalysis is referred to as deoxy-arabinose type ketoheptose phosphate synthase (DS), be respectively by three isozyme of aroF, aroG and three genes encodings of aroH, be subjected to the feedback inhibition of dead drunk propylhomoserin, phenylalanine and tryptophane respectively, the DAHP synthetic enzyme also is subjected to the collaborative inhibition of phenylalanine and tyrosine simultaneously.

After the common pathway, chorismic acid is the tapping point of die aromatischen Aminosaeuren route of synthesis, is divided into two approach later on again at chorismic acid, and wherein one forms phenylalanine and tyrosine, and another bar shaped becomes tryptophane.

Chorismic acid changes prephenic acid under the chorismate mutase effect, form phenyl-pyruvic acid after dewatering, taking off shuttle, and the latter changes ammonia with L-glutamic acid and forms phenylalanine under the transaminase effect.The chorismic acid approach is that people early improve the important step of carrying out genetic manipulation to the L-phenylalanine.Chorismate mutase (CM) is the bifunctional enzyme that forms with prephenate dehydratase (PD), and its activated state is that the size of two pheA genes encodings is the homodimer of the subunit formation of 43000D; The CM/PD enzyme forms dimer and tetrameric mixture when Phe concentration is higher, enzymic activity is reduced or completely lose, so it is subjected to the feedback inhibition of product P he, Tyr also has collaborative the inhibition to this enzyme.

The stoichiometry analysis revealed: the absorption of glucose needs sugar-phosphate transfer system, a part glucose needs a part PEP, the branched acid of synthetic a part needs bimolecular PEP, so PEP becomes the restricted substrate of DAHP theoretical yield, how many supplys of PEP has determined the biosynthesizing of Phe.And PEP is the competitive substrate of PEP carboxylase, pyruvate kinase, sugar-phosphate transferring enzyme and DAHP synthetic enzyme.1mol glucose enters cell will be transformed into pyruvic acid with the PEP of 1mol, and PEP can generate pyruvic acid and careless phthalein acetate respectively again under the effect of pyruvate kinase and PEP shuttle enzyme simultaneously.Pyruvic acid can not spontaneously transform back into PEP because of high energy barrier in vivo, makes a large amount of carbon stream just generate the content of organic acid, carbonic acid gas and cell through pyruvic acid.In order to make metabolism stream flow to the direction that Phe generates, often take following several strategy: the 1) gene of clonal expression PEP synthetic enzyme (PpsA) and transketolase (tktA) and PEP carboxylation kinases (pckA) in Phe production bacterium; 2) the consumption enzyme of passivation PEP such as pyruvate kinase (Pyk); 3) transformation of phosphotransferase system (PTS).

The biosynthesizing of L-phenylalanine is a complicated system engineering, improve the L-phenylalanine and produce the synthetic output of bacterial classification only to clone several genes still be not enough, also should pay attention to the aspects such as whole metabolic regulation strategy, technology controlling and process of bacterial classification itself and carry out many-sided retrofit work.Wherein utilizing the gene knockout method is a very important technology in the middle of the host of L-phenylalanine biosynthesizing bacterial strain transforms.

Gene knockout also claims gene targeting, gene substitution technique, is the technology that the eighties grows up latter half, mainly is to utilize the character of homologous sequence generation homologous recombination of viable cell chromosomal DNA and foreign DNA to reach the purpose that fixed point knocks out modification.

The principle of Red homologous recombination technique is one section to be carried with the target gene both wings respectively have the PCR fragment of 40-60bp homologous sequence to import host's mycetocyte, utilize the effect of lambda particles phage Red recombinase, make the linear DNA fragment of transfered cell and the particular target sequence of karyomit(e) (or carrier) carry out homologous recombination, target gene is labeled gene substitution and gets off, thus the purpose of the gene knockout that achieves the goal.This recombinant technology is the new technology that can carry out genetic manipulation on the karyomit(e) level that latest developments are got up, only need short homologous sequence, and do not need specific restriction enzyme site, and can finish regrouping process in vivo, saved steps such as external DNA enzyme is cut, connection.Experiment showed, that this gene targeting is the strong instrument of gene functional research and new strain construction.

Utilize the microbial metabolism engineering method to comprise and remove the enzyme that generates by product, the key enzyme of removing feedback regulation, inhibition or the high expression level relational approach of allosteric enzyme, DNA reorganization or additive method importing heterologous gene etc.But these methods require to overcome intracellular regulatory mechanism.Bacterium under the free living state is also regulated and control metabolism by specific step on single pathways metabolism, and utilizes the many pathways metabolisms of whole metabolism network regulation, thereby coordinates the distribution of metabolism stream.Whole metabolism network makes the physiology of cell integral body, metabolism make a response rapidly to external environment by the more genomic expression of gene regulation and control of pair cell.Yet the effective regulator control system of this brute force is but seldom used in the improvement of strain system.

In the die aromatischen Aminosaeuren biosynthesizing, PEP and E4P are the restricted substrates of synthetic key intermediate product DAHP.Because intracellular central metabolic pathway is inter-related, the many generations that may influence these precursors of many metabolic reactions.Intestinal bacteria csrA gene, promptly carbon is stored regulatory factor A, and its 61 amino acid whose small molecular proteins of encoding have comprised the special combination albumen of a RNA binding domains KH.CsrA knocks out the biosynthesizing that can increase glyconeogenesis and glycogen, reduces glycolysis-.There are some researches show that the csrA mutant strain can be than the glycogen concentration of wild strain 20 times of poly collection in cell.CsrA also regulates and control to participate in directly the activity level of metabolic three enzymes of PEP: just regulating and control pyruvate kinase (PykF), negative regulation PEP shuttle enzyme (pckA) and PEP synthetic enzyme.Meanwhile, the enzyme of some non-direct PEP of influence is also regulated and control by CsrA.The change of term single gene (csrA) can make carbon metabolism flow transform to shikimic acid pathway greatly.Because the activity of a plurality of enzymes of the whole metabolism network coordination and the metabolism of pathways metabolism stream, and easy handling simply effectively again, be that ideal is selected on the genetic manipulation of strain system improvement.And reaction that the whole metabolic regulation factor is regulated and control and pathways metabolism might be carried out different even opposite effect, and this just makes not only to have strengthened desirable pathways metabolism but also suppressed the competition pathways metabolism becomes possibility.The transformation of engineering plasmid and engineering host's transformation itself are combined, when both the speed limit functional enzyme of key being carried out the synthetic relevant enzyme of accelerated reaction and high expression level precursor, again by the coordination of plurality of enzymes is carried out in the modification of whole metabolism network gene, thereby further strengthen the expression and the accumulation of purpose product, realize further improving the possibility of L-phenylalanine productive rate.

[summary of the invention]

Technical problem to be solved by this invention is to provide a kind of method of L-phenylalanine gene engineering bacteria acid production rate, not only strengthened the accumulation of the PEP of one of die aromatischen Aminosaeuren precursor, and can make a large amount of the transforming of carbon metabolism flow to shikimic acid pathway, guarantee die aromatischen Aminosaeuren bigizationner of metabolism stream flow to the synthetic of L-phenylalanine, further improve the ability of engineering bacterium fermentation L-phenylalanine.

The present invention solves the problems of the technologies described above by the following technical programs: a kind of method that improves L-phenylalanine gene engineering bacteria acid production rate, adopt round pcr that the original engineering plasmid MDphe-2 of L-phenylalanine is built into engineering plasmid MDphe-3 of new generation; And the whole metabolism network regulation gene csrA of the engineering host bacterium of this project plasmid MDphe-3 is lacked structure by the Red system; Then engineering plasmid MDphe-3 is implanted in the engineering host bacterium of whole metabolism network regulation gene csrA disappearance to make up new L-phenylalanine gene engineering bacteria; At last the genetic stability and the product acid of this new L-phenylalanine gene engineering bacteria are verified.

Further, this method comprises following step:

Step 1: the enzyme that has that will design has the pckA gene employing round pcr of restriction enzyme site to increase;

Step 2: the pckA gene that step 1 amplification back is obtained carries out electrophoresis and reclaims, and the fragment that reclaims is adopted T-A cloning clone, and the positive colony carrier after will connecting carries out the closure screening, with acquisition PMDpckA carrier;

Step 3:, finally obtain the expressed intact element of pckA gene by in the PMDpckA carrier, merging temperature control promotor Pl sequence and the strong terminator sequence of T7;

Step 4: the original engineering plasmid MDphe-2 of L-phenylalanine is carried out sequential analysis, the flat end of the pckA gene complete Expression element after the flat endization of insertion of then connecting again after the 1293bp site of this project plasmid MDphe-2 is equalled endization is to make up engineering plasmid MDphe-3 of new generation;

Step 5: the homologous recombination function of employing Red system lacks the whole metabolism network regulation of the engineering host bacterium gene csrA of engineering plasmid MDphe-3;

Step 6: express in the middle of the engineering host bacterium with engineering plasmid MDphe-3 implantation csrA genetically deficient, to finish the structure of new L-phenylalanine gene engineering bacteria; And to the genetic stability of this new L-phenylalanine gene engineering bacteria and produce acid and verify.

Further, the concrete operations of described step 3 are as follows: get PMDpckA carrier and PBV220 plasmid and make up the PBVpckA carrier by enzyme cutting method, get this PBVpckA carrier and pET28a plasmid afterwards and make up the pETpckA28 carrier by enzyme cutting method; Then with this pETpckA28 carrier as template, and design the primer of a pair of fusion PCR; Then this template and primer are mixed laggard performing PCR amplification, obtain complete pckA gene expression element, verify the integrity of the pckA gene expression element that is obtained at last by T-A cloning and enzyme cutting method.

Further, the concrete operations of described step 4 are as follows:

(1) pre-treatment of the original engineering plasmid MDphe-2 of complete pckA gene expression element and L-phenylalanine:

A. adopt round pcr that complete pckA gene expression element is increased, amplification procedure is a template with carrier pETpckA28, and utilizes fusion PCR primer to increase; Pcr amplification finishes the back and handles 1h, the template of digestion pcr amplification with Dpn I enzyme down at 37 ℃; At last the purpose fragment is carried out electrophoresis and reclaim, reclaim the flat end that fragment is complete pckA gene expression element;

B. the original engineering plasmid MDphe-2 with the L-phenylalanine carries out sequential analysis, and this project plasmid MDphe-2 carried out the single endonuclease digestion digestion reaction, reaction finishes the back it is carried out the electrophoresis recovery, reclaims fragment and is the MDphe-2 nucleotide fragments that contains flat terminal breach;

(2) structure of engineering plasmid MDphe-3 of new generation:

The flat end of the complete pckA gene expression element that A. will reclaim and the MDphe-2 nucleotide fragments that contains flat terminal breach adopt ligase enzyme to carry out ligation, and this reacts on and carries out under 16 ℃ and spend the night, after in ice, placing 15min earlier after reaction finishes, get 10 μ l and directly change into competence E.coli JM109 cell, and this competence E.coli JM109 cell is carried out the LB+Kana resistant panel screen, obtain the positive colony bacterial strain;

B. the positive colony bacterial strain of getting acquisition carries out plasmid extraction, and carry out Sty I enzyme and cut checking, enzyme carries out electrophoresis after cutting end, and electrophoresis result shows except that the flat terminal fragment that contains complete pckA gene expression element, also have another plasmid fragment simultaneously, be engineering plasmid MDphe-3 of new generation.

Further, the concrete operations of described step 5 are as follows:

(1) make up engineering plasmid MDphe-3 the whole metabolism network regulation of engineering host bacterium gene csrA knock out assembly: get the pKD13 plasmid as template, and the synthetic a pair of primer that contains the short homologous fragment sequence of gene csrA as PCR, then carry out and pcr amplification; After pcr amplification finishes, after Yu Bingzhong places 15min, amplified production is handled 1h with Dpn I enzyme down in 37 ℃, after finishing, the enzyme processing in ice, places 5min again, then carrying out electrophoresis reclaims, and will reclaim fragment and carry out sequence verification, sequence results shows that it conforms to theory, promptly the csrA gene knocks out component construction successfully;

(2) coding in the Red system is had the pKD46 plasmid of Red recombinase implant engineering host bacterium after, this project host bacterium is prepared into high efficiency electric transformed competence colibacillus cell;

(3) assembly that knocks out with the csrA gene imports the recovery operation of carrying out engineering host bacterium behind the competent cell mixing in (2), and it is induced to lose the pKD46 plasmid;

(4) csrA genetically deficient in the described engineering host bacterium is verified and the elimination of resistant gene.

Further, described engineering host bacterium is intestinal bacteria.

The beneficial effect of the method for a kind of L-of raising phenylalanine gene engineering bacteria of the present invention acid production rate is: introduce the expressed intact element of pckA gene on the basis that efficiently expresses the engineering plasmid MDphe-2 that produces L-phenylalanine key protein, strengthened the accumulation of the PEP of one of die aromatischen Aminosaeuren precursor; Simultaneously, utilize the engineering host bacterium of the homologous recombination gene knockout method structure csrA genetically deficient of Red mediation, make a large amount of the transforming of carbon metabolism flow to shikimic acid pathway, guarantee die aromatischen Aminosaeuren bigizationner of metabolism stream flow to the synthetic of L-phenylalanine, further improve the ability of engineering bacterium fermentation L-phenylalanine, thereby bring bigger benefit for suitability for industrialized production.

[description of drawings]

The invention will be further described in conjunction with the embodiments with reference to the accompanying drawings.

Fig. 1 is the structure schema of engineering plasmid MDphe-3 among the present invention.

Fig. 2 is the structure schema of the whole metabolism network regulation of engineering host bacterium gene csrA disappearance among the present invention.

[embodiment]

A kind of method that improves L-phenylalanine gene engineering bacteria acid production rate of the present invention, it specifically comprises following step:

Step 1: the structure of L-phenylalanine engineering plasmid MDphe-3 of new generation specifically comprises following construction step (seeing also Fig. 1):

1. the pcr amplification that has the pckA gene of restriction enzyme site

According to the complete pckA gene order (GenBank NO.EG10688) that NCBI announces, it is as follows to design the primer that has restriction enzyme site, and entrusts biotechnology (Shanghai) Co., Ltd. to utilize dna synthesizer synthetic:

pckA-F：5-ATGCGCGTTAACAATGGTTTG-3

pckA-R：5- GCTCAGCTTACAGTTTCGGACCAGCC-3

Wherein downstream primer has Esp 1 enzyme and cuts sequence, and the product length of pcr amplification is 1623bp, 57.5 ℃ of optimum annealing temperatures.The pcr amplification reaction system is as follows:

10×Ex?PCR?Buffer(2.5mM)

5μl

dNTP?Mix(2.5mM)

2.5μl

Forward primer (10uM)

2.5μl

Reverse primer (10uM)

2.5μl

Dna profiling

2μl

Ex?Taq?DNA?Polymerase(5

1μl

U/μl)

ddH20?to 50μl

Reaction conditions is as follows:

95℃ 5min

4 ℃ of insulations

2.PMDpckA the structure of carrier makes up and checking

The pckA gene fragment that has restriction enzyme site that pcr amplification is obtained is carried out agarose gel electrophoresis and is reclaimed, and reclaims fragment and directly carries out T-A with PMD18-T (TAKARA company) and clone, and method is with reference to the PMD18-T specification sheets.Positive colony carrier after the connection carries out the screening of closure, get the upstream primer of PMD18-T carrier primer and the downstream primer of pckA gene primer and carry out " forward " screening of positive colony as cross primer, PCR system and condition are the same, obtain the PMDpckA carrier that " forward " cloning vector is structure, simultaneously with PMDpckA carrier transformed competence colibacillus cell E.coli JM109, and carry out sequence verification, it is the pckA gene that sequential analysis confirms really, confirms successfully to make up the PMDpckA carrier.

3.pckA gene fusion has the acquisition of the expressed intact element of temperature control promotor Pl sequence and the strong terminator sequence of T7

(1) structure of pBVpckA carrier and checking

Get the PMDpckA carrier and carry out identical EcoR I and the double digestion of Pst I with the pBV220 plasmid, the row agarose gel electrophoresis of going forward side by side reclaims purpose fragment and plasmid fragment.It is as follows that enzyme is cut system (50 μ l enzymes cut back to close system):

10×H?buffer 5μl

EcoR?I 1μl

Pst?I 1μl

Plasmid 23 μ l (the about 2 μ g of plasmid)

dd?H2O?to 50μl

Behind 37 ℃ of water-bath endonuclease reaction 5h, reclaim test kit with the pillar dna gel and carry out the required dna fragmentation of electrophoresis rubber tapping recovery, the i.e. linear fragment of pckA gene and pBV220 respectively; Then the purpose fragment is carried out external connection with the T4DNA ligase enzyme and spend the night, product transformed competence colibacillus cell E.coli JM109, and picking clone extraction plasmid carry out same enzyme and cut checking, confirm successful carrier construction pBVpckA.

(2) structure of pETpckA28 carrier and checking

Get the pBVpckA carrier that successfully constructs and carry out Bgl II and Esp I double digestion with the pET28a plasmid, the row agarose gel electrophoresis of going forward side by side reclaims purpose fragment and plasmid fragment.It is as follows that enzyme is cut system:

10×K?buffer 5μl

0.1％BSA 5μl

Bgl?II 1μl

Esp?I 1μl

Plasmid 23 μ l (the about 2 μ g of plasmid)

dd?H2O?to 50μl

After 37 ℃ of water-bath enzymes are cut reaction overnight, reclaim test kit with the pillar dna gel and carry out the required dna fragmentation of electrophoresis rubber tapping recovery, the i.e. linear fragment of PlpckA gene and pET28a respectively; Then the purpose fragment is carried out external connection with the T4DNA ligase enzyme and spend the night, product transformed competence colibacillus cell E.coli JM109, and picking clone extraction plasmid carry out same enzyme and cut checking, confirm successful carrier construction pETpckA28.

(3) pcr amplification and the sequence verification thereof of the pckA gene of fusion Pl promoter sequence and T7 terminator sequence

Design the primer of a pair of fusion PCR, and its two ends have restriction enzyme site simultaneously so that checking, it is as follows that this merges the PCR design of primers, and entrust biotechnology (Shanghai) Co., Ltd. to utilize dna synthesizer synthetic:

PlpckAT7-F：5-CCTAGGAGATCTCTCACCTACCAAACAATG-3

PlpckAT7-R：5-CCTAGGATCCGGATATAGTTCCTCCTTTC-3

Wherein CCTAGG is the restriction endonuclease recognition sequence of Sty I, is mainly the checking use that goal gene inserts engineering plasmid.

With the pETpckA28 plasmid vector that makes up is template, and above-mentioned primer carries out the pcr amplification of pckA gene, Pl promotor and T7 terminator, reaction system and condition same (2) for the upstream and downstream primer.

The product of pcr amplification is through electrophoresis detection, utilize product to reclaim test kit then and carry out the segmental recovery of purpose, detect clip size and be about 2000bp, it is the pckA Expression element, pckA Expression element after reclaiming and pMD18-T carrier are carried out the T-A clone, to obtain transformed competence colibacillus E.coli JM109 cell, screening positive clone and to containing the positive colony subprocess order-checking of pckA Expression element, whether cut with checking pckA Expression element and to have sudden change or disappearance in the operating process, the integrity of the pckA Expression element after confirming simultaneously to merge at pcr amplification and enzyme.

4. make up engineering plasmid MDphe-3 of new generation

(1) pre-treatment of the original engineering plasmid MDphe-2 of complete pckA gene expression element and L-phenylalanine

The pre-treatment of complete pckA gene expression element: adopt round pcr that complete pckA gene expression element is increased, amplification procedure is a template with carrier pETpckA28, and utilizes fusion PCR primer that PlpckAT7-F/R is increased to this template; Pcr amplification finishes the back and handles 1h with Dpn I enzyme down at 37 ℃, and the template of digestion pcr amplification prevents the interference to follow-up screening of template plasmid; At last the purpose fragment is carried out agarose gel electrophoresis and reclaim, reclaim clip size and be about 2000bp, be the pckA gene expression element after the flat endization.

The pre-treatment of the original engineering plasmid MDphe-2 of L-phenylalanine: the original engineering plasmid MDphe-2 of L-phenylalanine is carried out sequential analysis, and plasmid MDphe-2 carried out the single endonuclease digestion digestion of Nhe I, Nhe I single endonuclease digestion system following (50 μ l enzymes cut back to close system):

dd?H2O?to 50μl

10×M?buffer 5μl

Nhe?I 2μl

Plasmid 24 μ l (containing plasmid 2ug approximately)

37 ℃ of water-bath 6h, after reaction finishes, 65 ℃ of water-bath termination reaction 10min, directly add the dNTP (2.5mM) of 10 * Klenow Buffer, 2 μ l of 6 μ l and the big fragment of Klenow of 2 μ l then, and under 37 ℃ of temperature, react 3h, reaction finishes the back and carry out the enzyme deactivation processing reaction in 65 ℃ of water-bath, and the 10 * Loading Buffer that adds 7 μ l behind the 10min again is with termination reaction and carry out the electrophoresis rubber tapping and reclaim; Reclaim enzyme by 0.7% low-melting agarose electrophoresis and cut the purpose fragment, reclaim fragment and be the MDphe-2 nucleotide fragments that contains flat terminal breach.

(2) structure of engineering plasmid MDphe-3 of new generation and checking thereof

With the pckA gene expression element after flat endization that reclaims and contain the MDphe-2 nucleotide fragments employing T4-DNA ligase enzyme of putting down terminal breach and carry out ligation, the ligation system is as follows:

dd?H2O?to 20μl

10×T4?lig?Buffer 2μl

PckA fragment 2 μ l (about 70ng)

Linearized vector fragment 6 μ l (about 180ng)

T4?DNALigase 1.5μl

And this reacts on and carries out under 16 ℃ and spend the night, after in ice, placing 15min earlier after reaction finishes, get 10 μ l and directly change into competence E.coli JM109 cell, and this competence E.coli JM109 cell is carried out the screening of LB+Kana resistant panel, obtain the positive colony bacterial strain.

The positive colony bacterial strain of getting acquisition carries out plasmid extraction, and carry out Sty I enzyme and cut checking, enzyme carries out electrophoresis after cutting end, electrophoresis result shows except that containing the flat terminal fragment that the purpose fragment that is about the 2000bp size is complete pckA gene expression element, also have another plasmid fragment simultaneously, be engineering plasmid MDphe-3 of new generation.

Because what take in plasmid construction process of new generation is flat terminal connection insertion method, so goal gene pckA Expression element does not have the selectivity of direction of insertion in being inserted into engineering plasmid MDphe-3, that utilizes carrier primer and fusion PCR primer carries out clone's evaluation of different directions as cross primer, qualification result is presented in the process of connection and has produced the different plasmid of goal gene pckA Expression element direction of insertion really, and different direction of insertion can be brought obvious influence to duplicating with transcriptional expression of whole plasmid; Confirm that by experiment (being called forward inserts) had comparatively good expression and produce sour effect when the direction of pckA Expression element in engineering plasmid MDphe-3 was consistent with the replication initiation sequence; PckA Expression element direction is inserted replication initiation sequence with engineering plasmid MDphe-3 when opposite, can influence the expression of functional gene in the host of engineering plasmid, and it is lower to produce acid, but concrete influence mechanism is also indeterminate.

Step 2: the homologous recombination function of employing Red system lacks (seeing also Fig. 2) to the whole metabolism network regulation of the engineering host bacterium gene csrA of engineering plasmid MDphe-3

With intestinal bacteria is the engineering host bacterium of engineering plasmid MDphe-3, and then gene csrA is the effector A of the colibacillary whole metabolism network regulation csr of system.

1.csrA the structure that knocks out assembly of gene

Announce colibacillary genome sequence according to NCBI, the a pair of primer that contains the short homologous fragment sequence of gene csrA of synthetic: the homologous sequence of upstream sequence 39bp, the homologous sequence of downstream 37bp, and entrust biotechnology (Shanghai) Co., Ltd. to utilize dna synthesizer synthetic:

P13csrA-F：5-TTTGAGGGTGCGTCTCACCGATAAAGATGAGACGCGG

AA GTGTAGGCTGGAGCTGCTTC-3

P13csrA-R：5-ATACAGAGAGACCCGACTCTTTTAATCTTTCAAGGAG

CTGTCAAACATGAGAATTAA-3

The partial sequence of wherein ruling has the segmental PCR primer of the resistant gene part that FRT sets the site for template plasmid pKD13.

With the pKD13 plasmid is template, above-mentioned primer (P13csrA-F/R) for primer to carrying out pcr amplification, amplification reaction system (50 μ l system) is as follows:

10×pfu?PCR?Buffer(2.5mM) 5μl

dNTP?Mix(2.5mM) 2.5μl

P1(10uM) 2.5μl

P2(10uM) 2.5μl

Dna profiling 2 μ l

pfu?DNA?Polymerase(5U/μl) 1μl

ddH20?to 50μl

Reaction conditions is as follows:

95℃ 5min

72℃ 10min

4 ℃ of insulations

After pcr amplification reaction finishes, after Yu Bingzhong places 15min, amplified production is handled 1h with Dpn I enzyme down in 37 ℃, in ice, place 5min again after the enzyme processing finishes, then carry out electrophoresis and reclaim.And utilize the pillar dna gel to reclaim test kit and carry out the dna fragmentation that the required 1300bp of being about is reclaimed in the electrophoresis rubber tapping respectively, will reclaim fragment simultaneously and carry out sequence verification, sequence results shows that it conforms to theory, what promptly confirm the csrA gene knocks out component construction successfully.

(2) Red system recombination function induces preparation with high efficiency electric transformed competence colibacillus cell

PKD46 plasmid in the Red system contains the replication orgin oriR101 of responsive to temperature type, can normal replication when the pKD46 plasmid is cultivated under 30 ℃ of conditions, and when temperature is higher than 37 ℃, just can lose automatically, on the pKD46 plasmid, also contain exo, the bet, the gam gene that are subjected to the ParaB promoter regulation, they can pass through the L-arabinose abduction delivering, and carry ampicillin resistance gene as selection markers.

The pKD46 plasmid that will contain coding Red recombinase plant to engineering host bacterium be in the middle of the intestinal bacteria after, place the LB substratum that contains 100 μ g/ml penbritins and 30 ℃ of following incubated overnight, the cultured culture of 0.5ml is joined in the 250ml Erlenmeyer flask that fills the 50mlLB substratum, and place this Erlenmeyer flask 30 ℃ to be cultured to OD ₆₀₀=015～0.20, and rotating speed 250rpm, the L-arabinose that then adds concentration and be 10mmol/L continues to be cultured to the dense OD of cell bacterium ₆₀₀=0.65～0.7, place the sterilization centrifuge tube of transferring to the 50ml of precooling behind the ice-water bath cooling 30min with obtaining the cell bacterium this moment, then this centrifuge tube being placed 4 ℃ and rotating speed is centrifugal 20min under the 5000rpm condition, remove supernatant after the centrifugal end, and the ice-cold aseptic high purity water washing of precipitation 2 times, again the precipitation of last gained is sub-packed in after resuspended in the sterilization EP pipe of precooling standby with the aseptic high purity water of 1ml precooling, and every pipe packing 50 μ l, this is the high efficiency electricity of engineering host bacterium changes competent cell.

(3) importing of csrA gene knockout assembly and inducing of recombinant plasmid pKD46 are lost

The about 100ng of linearizing csrA gene knockout assembly fragment is joined the engineering host bacterium electricity that fills prepared fresh changes mixing formation mixed liquor A in the competent cell, and Yu Bingzhong places 15min.

Earlier electroporation is transferred to 2.5KV, 2.5 μ F, pulse manipulator is transferred to 200-400 Ω, then mixed liquor A is transferred in the electric revolving cup, and the sample cell of putting into electroporation then directly carries out electricity and transforms; After transforming and finish, takes out electricity electric revolving cup rapidly, mixed liquor A after then electricity being changeed is drawn onto behind 900 μ lSOC substratum mixings in the middle of the EP pipe of 1.5ml, again the EP pipe is placed 37 ℃, 150rpm down concussion cultivate 60min to allow the recovery of engineering host bacterium, simultaneously, induce gradually and lose the pKD46 plasmid.

The engineering host bacterium bacterium liquid 200 μ l that get recovery are coated with screening on the LB resistant panel that contains 50 μ g/ml Kana, and with this LB resistant panel inversion cultivation 16h under 37 ℃, and when carrying out the resistant panel screening, further induce and lose the pKD46 plasmid, after treating that mono-clonal grows, choose mono-clonal and draw short-term respectively on Kana, Amp resistant panel, and Kana, Amp resistant panel respectively at 37 ℃ cultivate down observe behind the 12h mono-clonal growing state: the normal growth on Kana resistance plate; What can not grow on the Amp resistant panel is to confirm substantially that the pKD46 plasmid loses.

(4) checking of csrA gene knockout and the elimination of resistant gene in the engineering host bacterium

Be further complete the knocking out of checking csrA gene, adopt the method for bacterium colony PCR to verify, according to the genome sequence of announcing, the selection primer is right: select for use the cross primer of csrA genome both sides primer and the segmental primer of Kana resistant gene and two pairs of primer compositions right respectively, simultaneously not lack the negative control group of host's bacterium colony of csrA gene.Reaction system is as follows:

10×Taq?PCR?Buffer(2.5mM) 2μl

dNTP?Mix(2.5mM) 1μl

Primer upstream (10uM) 1 μ l

Primer downstream ((10uM) 1 μ l

Dna profiling list bacterium colony

Taq?DNA?Polymerase(5U/μl) 1μl

ddH20?to 20μl

Reaction conditions:

98℃ 1min

72℃ 10min

4 ℃ of insulations

The result shows, truly have the fragment of the Kana resistant gene clip size that is about 850bp, simultaneously cross primer also has and is about 1000bp, confirms that knocking out assembly has successfully carried out the replacement of Kana resistant gene fragment to the csrA gene, and csrA genetically deficient makes up initial success.

Because there is interference in the resistant gene fragment that has of engineering host cingula pair with the screening of plasmid, and the segmental existence of external source also has certain influence to engineering host bacterium, eliminates so be necessary the resistance fragment of carrying out knocking in the chromogene.

The pCP20 plasmid contains the replication orgin of responsive to temperature type, have penbritin and chlorampenicol resistant simultaneously, in addition, contain a Flippases recombinase (flipase recombination enzyme on the pCP20, FLP) gene, the FLP recombinase can combine with the FRT site, under the effect of FLP recombinase, homologous recombination takes place in FRT site self, thereby eliminates a FRT site and resistant gene.Recombinase FLP is abduction delivering in the time of 42 ℃, and the pCP20 plasmid also fades away simultaneously.

The electricity of preparation csrA gene knockout engineering host bacterium changes competent cell, making method is with aforementioned, the pCP20 plasmid electricity of getting 5 μ l changes cell over to, then electricity being changeed mixed solution after finishing and 900 μ l SOC substratum is mixed together to be placed on and allows engineering host bacterium recovery 2h under 30 ℃, the bacterial strain of getting respectively then after 200 μ l recover is applied on Amp and the Chl resistant panel, and it is sub to obtain the clone to cultivate 16h down in 30 ℃; Several clone sons of picking are inoculated in 3ml respectively and contain among the liquid LB of Amp and Chl resistance then, and at 30 ℃, after shaking table is cultivated 8h under the 250rpm condition, temperature is adjusted to 42 ℃ continues to cultivate 4～5h, the bacterium liquid that takes a morsel after cultivation finishes is coated on the sky LB flat board, and with this LB flat board in 37 ℃ of following incubated overnight, then mono-clonal that grows on this LB flat board is lined Kana respectively, Amp, on Chl resistant panel and the empty LB flat board, and each flat board is cultivated down respectively at 37 ℃, and cultivation results shows: the Kana plate, Amp, mono-clonal on the Chl resistance plate all can not be grown; And on empty LB plate can normal growth, this is the engineering host bacterium that csrA gene knockout and resistant gene are eliminated.

Step 3: the structure of L-phenylalanine gene engineering bacteria of new generation and genetic stability and fermentation and acid checking

1. the preparation of positive colony

Engineering plasmid MDphe-3 is changed into competence E.coli JM109 cell by electricity, then this competence E.coli JM109 cell is added among the electric shock recovery substratum SOB of 37 ℃ of preheatings and shake recovery 1h, and rotating speed is 150rpm, after cultivation finishes it is coated (Kan in the LB culture medium flat plate that contains the resistance selection ⁺) on, and place 37 ℃ be inverted to cultivate 14h this flat board to obtain positive colony.

2. the rapid screening of positive colony and enzyme are cut checking

10 positive colony of picking shaking table that spends the night under 37 ℃ is cultivated on the flat board from 1 at random, utilize the quick extraction process of plasmid to extract plasmid DNA afterwards, and this plasmid DNA carried out electrophoresis detection, then but the positive clone of the bacterium colony chosen of preliminary judgement is sub if the purpose band occurs, the plasmid that extracts positive colony again carries out Sty I enzyme and cuts checking, simultaneously with the negative contrast of original engineering plasmid MDphe-2, if the purpose band of the about 2000bp of size appears in the plasmid of positive colony, can confirm as positive colony that has plasmid MDphe-3.

3. recombinant plasmid MDphe-3 changes the through engineering approaches host of csrA genetically deficient over to

Wherein engineering host bacterium is the through engineering approaches host of the whole metabolism network regulation gene csrA defective type of process Red system modification, the positive colony bacterial strain that Transformed E .coli JM109 cell is obtained carries out plasmid extraction, adopts conventional molecule manipulation method that positive colony of plasmid MDphe-3 is imported in the engineering host bacterium of csrA genetically deficient then.

4. the stability checking of recombinant plasmid MDphe-3 in the engineering host bacterium of csrA genetically deficient

The plasmid stability of original plasmid MDphe-2 in former engineering host bacterium is through checking, has higher plasmid stability (97%), and in process of high-density fermentation (little formula, Chinese style and big formula), keeping high stability equally, done following experiment for guaranteeing recombinant plasmid MDphe-3 has plasmid equally in new engineering host bacterium (being the engineering host bacterium of csrA genetically deficient) this chamber of stability personnel:

Single bacterium colony (being positive colony) that one of picking contains plasmid MDphe-3 from 1 middle plateform is received 2mL and is contained Kan ⁺In the LB substratum of (50 μ g/ml), and this LB substratum placed 30 ℃ of following incubated overnight, get 20 μ l then and add to and do not contain Kan ⁺The LB substratum in, 35 ℃ of cultivations, make bacterial strain before thermal induction in the substratum of no Kan continuous growth 48h, breed more than 50 generations, when bacterial strain is growing into OD ₆₀₀Changed under 37 ℃ in=0.3 o'clock and induce, resampling is applied on the LB flat board that does not contain Kan, and next day, 100 points of picking colony contained Kan at random ⁺Flat board on, containing Kan at last ⁺The percentage ratio of growth bacterium colony is about 97% on the flat board.

Carry out the bacterium colony PCR checking of deletion mycopremna after respectively the new engineering host bacterium that contains plasmid MDphe-3 being gone down to posterity simultaneously again, the PCR qualification result shows that the engineering strain deletion fragment is the fragment for being designed to knock out really, and can genetic stability.

Experimental result proves: the plasmid MDphe-3 of reorganization has higher plasmid genetic stability equally in new engineering host bacterium.

5. the fermentation and acid checking of L-phenylalanine gene engineering bacteria of new generation

The L-phenylalanine gene engineering bacteria of new generation that plasmid MDphe-3 and new engineering host bacterium are made up is after shake flask fermentation is cultivated 48 hours, get fermented liquid supernatant and carry out the mensuration of L-phenylalanine content, simultaneously the negative control group of L-phenylalanine gene engineering bacteria that makes up with original engineering plasmid MDphe-2 and former engineering host bacterium; The shake flask fermentation experiment is triplicate under same processing condition, and the result shows: new L-phenylalanine gene engineering bacteria is compared with the bacterial strain of negative control group, and its glucose acid invert ratio has clear improvement, produces acid number and also is significantly improved, and wherein produces acid number and has improved 13%.

In addition, L-phenylalanine gene engineering bacteria of new generation is carried out the big jar production test of Chinese style and big formula, its produce acid number improve more remarkable, for suitability for industrialized production L-phenylalanine brings bigger benefit.

In sum, the present invention introduces the expressed intact element of pckA gene on the basis that efficiently expresses the engineering plasmid MDphe-2 that produces L-phenylalanine key protein, has strengthened the accumulation of the PEP of one of die aromatischen Aminosaeuren precursor; Simultaneously, utilize the engineering host bacterium of the homologous recombination gene knockout method structure csrA genetically deficient of Red mediation, make a large amount of the transforming of carbon metabolism flow to shikimic acid pathway, guarantee die aromatischen Aminosaeuren bigizationner of metabolism stream flow to the synthetic of L-phenylalanine, further improve the ability of engineering bacterium fermentation L-phenylalanine, thereby bring bigger benefit for suitability for industrialized production.

Claims (6)

1. a method that improves L-phenylalanine gene engineering bacteria acid production rate is characterized in that: adopt round pcr that the original engineering plasmid MDphe-2 of L-phenylalanine is built into engineering plasmid MDphe-3 of new generation; And the whole metabolism network regulation gene csrA of the engineering host bacterium of this project plasmid MDphe-3 is lacked structure by the Red system; Then engineering plasmid MDphe-3 is implanted in the engineering host bacterium of whole metabolism network regulation gene csrA disappearance to make up new L-phenylalanine gene engineering bacteria; At last the genetic stability and the product acid of this new L-phenylalanine gene engineering bacteria are verified.

2. a kind of according to claim 1 method that improves L-phenylalanine gene engineering bacteria acid production rate is characterized in that: comprise following step:

Step 1: the pckA gene that has restriction enzyme site that will design adopts round pcr to increase;

Step 2: the pckA gene that step 1 amplification back is obtained carries out electrophoresis and reclaims, and the fragment that reclaims is adopted T-A cloning clone, and the positive colony carrier after will connecting carries out the closure screening, with acquisition PMDpckA carrier;

Step 3:, finally obtain the expressed intact element of pckA gene by in the PMDpckA carrier, merging temperature control promotor Pl sequence and the strong terminator sequence of T7;

Step 4: the original engineering plasmid MDphe-2 of L-phenylalanine is carried out sequential analysis, pckA gene complete Expression element after the flat endization of insertion of then connecting again after the 1293bp site of this project plasmid MDphe-2 is equalled endization is to make up engineering plasmid MDphe-3 of new generation;

Step 5: the homologous recombination function of employing Red system lacks the whole metabolism network regulation of the engineering host bacterium gene csrA of engineering plasmid MDphe-3;

Step 6: express in the middle of the engineering host bacterium with engineering plasmid MDphe-3 implantation csrA genetically deficient, to finish the structure of new L-phenylalanine gene engineering bacteria; And to the genetic stability of this new L-phenylalanine gene engineering bacteria and produce acid and verify.

3. as a kind of method that improves L-phenylalanine gene engineering bacteria acid production rate as described in the claim 2, it is characterized in that: the concrete operations of described step 3 are as follows:

Get PMDpckA carrier and PBV220 plasmid and make up the PBVpckA carrier, get this PBVpckA carrier and pET28a plasmid afterwards and make up the pETpckA28 carrier by enzyme cutting method by enzyme cutting method; Then with this pETpckA28 carrier as template, and design the primer of a pair of fusion PCR; Then this template and primer are mixed laggard performing PCR amplification, obtain complete pckA gene expression element, verify the integrity of the pckA gene expression element that is obtained at last by T-A cloning and enzyme cutting method.

4. as a kind of method that improves L-phenylalanine gene engineering bacteria acid production rate as described in the claim 2, it is characterized in that: the concrete operations of described step 4 are as follows:

(1) pre-treatment of the original engineering plasmid MDphe-2 of complete pckA gene expression element and L-phenylalanine:

A. adopt round pcr that complete pckA gene expression element is increased, amplification procedure is a template with carrier pETpckA28, and utilizes fusion PCR primer to increase; Pcr amplification finishes the back and handles 1h, the template of digestion pcr amplification with Dpn I enzyme down at 37 ℃; At last the purpose fragment is carried out electrophoresis and reclaim, reclaim fragment and be flat terminal complete pckA gene expression element;

B. the original engineering plasmid MDphe-2 with the L-phenylalanine carries out sequential analysis, and this project plasmid MDphe-2 carried out the single endonuclease digestion digestion reaction, reaction finishes the back it is carried out the electrophoresis recovery, and flat endization processing, reclaims fragment and is flat terminal breach MDphe-2 nucleotide fragments;

(2) structure of engineering plasmid MDphe-3 of new generation:

The flat end of the complete pckA gene expression element that A. will reclaim and the MDphe-2 nucleotide fragments that contains flat terminal breach adopt ligase enzyme to carry out ligation, and this reacts on and carries out under 16 ℃ and spend the night, after in ice, placing 15min earlier after reaction finishes, get 10 μ l and directly change into competence E.coliJM109 cell, and this competence E.coli JM109 cell is carried out the LB+Kana resistant panel screen, obtain the positive colony bacterial strain;

B. the positive colony bacterial strain of getting acquisition carries out plasmid extraction, and carry out Sty I enzyme and cut checking, enzyme carries out electrophoresis after cutting end, and electrophoresis result shows except that the flat terminal fragment that contains complete pckA gene expression element, also have another plasmid fragment simultaneously, be engineering plasmid MDphe-3 of new generation.

5. as a kind of method that improves L-phenylalanine gene engineering bacteria acid production rate as described in the claim 2, it is characterized in that: the concrete operations of described step 5 are as follows:

(1) make up engineering plasmid MDphe-3 the whole metabolism network regulation of engineering host bacterium gene csrA knock out assembly: get the pKD13 plasmid as template, and the synthetic a pair of primer that contains the short homologous fragment sequence of gene csrA as PCR, then carry out pcr amplification; After pcr amplification finishes, after Yu Bingzhong places 15min, amplified production is handled 1h with Dpn I enzyme down in 37 ℃, after finishing, the enzyme processing in ice, places 5min again, then carrying out electrophoresis reclaims, and will reclaim fragment and carry out sequence verification, sequence results shows that it conforms to theory, promptly the csrA gene knocks out component construction successfully;

(2) coding in the Red system is had the pKD46 plasmid of Red recombinase implant engineering host bacterium after, this project host bacterium is prepared into high efficiency electric transformed competence colibacillus cell;

(3) assembly that knocks out with the csrA gene imports the recovery operation of carrying out engineering host bacterium behind the competent cell mixing in (2), and it is induced to lose the pKD46 plasmid;

(4) csrA genetically deficient in the described engineering host bacterium is verified and the elimination of resistant gene.

6. a kind of according to claim 1 method that improves L-phenylalanine gene engineering bacteria acid production rate is characterized in that: described engineering host bacterium is intestinal bacteria.

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