CN103276011A - Primary pathway transformation method under guidance of FK506 strain genome-scale metabolic network model - Google Patents
Primary pathway transformation method under guidance of FK506 strain genome-scale metabolic network model Download PDFInfo
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Abstract
The invention discloses a primary pathway transformation method under the guidance of an FK506 strain genome-scale metabolic network model, which comprises the following steps of: adding the characteristic reaction and thallus synthesis reaction of FK506 biosynthesis according to the streptomyces tsukubaensis genome sequence, and performing manual refining on the network reaction to obtain an FK506 strain streptomyces tsukubaensis genome-scale metabolic network model; and analyzing the metabolic network model, and determining the primary pathway precursor key gene of FK506 biosynthesis through a flux equilibrium analysis and minimum metabolic regulation analysis method. According to the method disclosed by the invention, the strain production level after the transformation is improved by 80-100%. The method provides efficient guidance to the establishment, research and analysis of the primary pathway of the FK506 strain, and has great application prospects in the research process of FK506 strain transformation.
Description
Technical field
The invention belongs to microorganism strains metabolic engineering molecular modification technical field, particularly based on the elementary pathways metabolism remodeling method of microorganism strains genome yardstick Metabolic Network Model.
Background technology
Have inhibiting 23 yuan of polyketone macrolides compounds of hyperimmunization as a kind of, FK506(has another name called tacrolimus) stronger 100 times than the dissimilar para-immunity inhibitor of structure S-Neoral, thereby be widely used in immunologic rejection treatment after allosome kidney, liver, the bone marrow transplantation, also for the treatment of inflammation dermatosis and eczema.Increase along with organ transplantation in recent years is exponential type, FK506 has become an a kind of very important line clinical medicine.
Once many scientific research personnel had done the High-efficient Production process that FK506 is devoted in a large amount of work, and these work are also based on random mutation and screening method.Because the main bottleneck of precursor level deficiency in the born of the same parents, the relatively low output of fermenting process has seriously limited the further application of FK506.By the genetic modification to original strain, can construct the precursor balance bacterial strain that guarantees that thalli growth and product generate.These genetic modification researchs can significantly improve the synthetic of precursor, yet this method for the identification of target gene always blindly and uncertain, and because pathways metabolism is highly interactive, topological framework is very complicated, operates a gene merely and may produce unknown result to other approach even whole cell.Importantly, because engineered normally local, there is limitation in the violent result of bacterial strain optimization.Because cell is the accurate complex system with highly cross-linked metabolism network, holding the global behavior of cell and accurately identify target from system level is a great challenge.
Systems biology has been brought into play vital role by providing system strategy to change in the extracellular microbial flux in the metabolic engineering field.Along with increasing species gene group sequence announces out that genome yardstick Metabolic Network Model has become the important platform that obtains the microorganism physiological property
[1]Genome yardstick Metabolic Network Model can be used for reflecting the response of hereditary disturbance, assists to disclose the potential cause of unfavorable phenotype.On this basis, the biosynthesizing behavior of systematic study bacterial strain, in conjunction with the model analysis means, can be quick, accurately identification improves the target spot gene of strain bio synthesis capability
[2]This method is time saving and energy saving, has reduced research cost.Genome yardstick Metabolic Network Model successfully has been used for the metabolic engineering target spot identification of important industrial production such as biofuel, VITAMIN, amino acid and secondary metabolites at present.
Elementary pathways metabolism precursor has material impact to microbial secondary metabolites synthetic.Elementary pathways metabolism is the main supply mode of precursor, and precursor can synthesize by utilizing different carbon substrates such as lipid acid, monose or protein.The ATP that elementary pathways metabolism generates can directly be used as body internal reaction energy needed; NADH can enter respiratory chain and produce ATP; The NADPH that pentose-phosphate pathway produces is usually as the reducing power of redox reaction; Some small-molecule substances such as the pyruvic acid that produce, acetyl-CoA etc. can be used as the synthetic precursor substance of microbiotic.By the carbon distributions that can regulate center carbon metabolism network to the identification of elementary pathway key gene and key enzyme, improve the interior level of born of the same parents of particular precursor thing.Phosphopentose pathway is connected by crucial metabolism node 6-glucose 1-phosphate1-with glycolytic pathway in the elementary pathways metabolism of glucose, has glucose phosphate isomerase, glucose-6-phosphate dehydrogenase and phosphoglucomutase to participate in.Hear people such as Jianping
[3]Strengthened phosphopentose pathway by the amplification glucose-6-phosphate dehydrogenase, the Streptomyces roseosporus genetic engineering bacterium of structure has improved 18% than the output of the bacterial classification that sets out, and the output of daptomycin is up to 700mg/L.Li and Townsend
[4]By knocking out glyceraldehyde 3-phosphate dehydro-genase gene gap1 and the gap2 of glycolytic pathway, with glyceraldehyde 3-phosphate to 1, the carbon stream of 3-diphosphoglyceric acid interrupts, and makes more precursor for the synthesis of clavulanic acid, finally under interpolation arginine condition clavulanic acid output has been improved 3.1 times.The coenzyme A class precursor of engineered elementary approach can promote the production of some polyketides such as erythromycin, oligomycin, monensin B and actinorhodin.By methylmalonyl CoA metabolism node is transformed, red saccharopolyspora has significantly improved the fermentation level of synthesis of erythromycin
[5]People such as Ryu
[6]After acetyl-CoA carboxylase encoding gene accA2, the accB of streptomyces coelicolor and accE carried out expressing, acetyl-CoA is strengthened to the malonyl coenzyme A flux, malonyl coenzyme A and acetyl-CoA all are the composite structure precursors as actinorhodin, therefore actinorhodin have been improved 6 times.The reducing power cofactor also plays a key effect to the synthetic of secondary metabolites in the born of the same parents of elementary approach, in order to improve NADPH reducing power level in the born of the same parents, people such as Asadollahi
[7]Behind the glutamate dehydrogenase that inactivation NADPH relies on, reduced NADPH consumption too much when this approach synthesizes L-glutamic acid, caused that the sesquiterpene output of bacterial strain has improved 85%.
And so far, do not see and utilize FK506 strain gene group yardstick Metabolic Network Model to predict elementary pathway key gene key enzyme, and then instruct relevant patent and the bibliographical information of the elementary approach molecular modification of engineering strain.The patented invention point of this paper is that elementary pathway key gene according to FK506 strain gene group yardstick Metabolic Network Model prediction is as transforming target spot, comprise that from the elementary pathways metabolism of streptomycete L-glutamic acid metabolic pathway of synthesizing, phosphoenolpyruvic acid anaplerotic sequence, shikimic acid pathway, reducing power balance approach, lipid acid metabolic pathway of synthesizing, phosphopentose pathway carry out molecular modification, improve the production level of FK506 with this.Therefore, by making up FK506 bacterial strain high precision genome yardstick metabolism network, determine that elementary approach knocks out key gene gdhA, ppc and increase key gene dahp, pntAB, accA2, zwf2, and then by the molecular modification of bacterial strain being reduced the consumption of reducing power NADPH, phosphoenolpyruvic acid precursor, stiffener rings hexane dicarboxylic acid, reducing power NADPH, propionyl coenzyme A, erythrose-4-phosphate precursor synthesize.
[1]Kim,T.Y.,S.B.Sohn,Y.B.Kim,et?al.,Recent?advances?in?reconstruction?and?applications?of?genome-scale?metabolic?models,Current?Opinion?in?Biotechnology,2012,23(4):617-623.
[2] hear Jianping, Huang Di, Li Shanshan, Jia Xiaoqiang, isopropylcarbinol synthesis bacterium genome yardstick Metabolic Network Model and molecular modification method, 201210194678.9
[3] hear the Jianping, space light sea, Jia Xiaoqiang crosses and expresses method and the application thereof that the zwf2 gene improves daptomycin output, 201110190206.1
[4]Li,R.and?C.A.Townsend,Rational?strain?improvement?for?enhanced?clavulanic?acid?production?by?genetic?engineering?of?the?glycolytic?pathway?in?Streptomyces?clavuligerus,Metabolic?Engineering,2006,8(3):240-252.
[5]Reeves,A.R.,I.A.Brikun,W.H.Cernota,et?al.,Engineering?of?the?methylmalonyl-CoA?metabolite?node?of?Saccharopolyspora?erythraea?for?increased?erythromycin?production,Metabolic?Engineering,2007,9(3):293-303.
[6]Ryu,Y.-G.,M.J.Butler,K.F.Chater,et?al.,Engineering?of?primary?carbohydrate?metabolism?for?increased?production?of?actinorhodin?in?Streptomyces?coelicolor,Applied?and?Environmental?Microbiology,2006,72(11):7132-7139.
[7]Asadollahi,M.A.,J.Maury,K.R.Patil,et?al.,Enhancing?sesquiterpene?production?in?Saccharomyces?cerevisiae?through?in?silico?driven?metabolic?engineering,Metabolic?Engineering,2009,11(6):328-334.
Summary of the invention
The objective of the invention is precursor synthetic gene according to the elementary approach of FK506 strain gene group yardstick Metabolic Network Model prediction as transforming target spot, replace the structure knock-out bacterial strain by the homology of resistance box, the conversion of integrated plasmid makes up the amplification bacterial strain, with this elementary approach (L-glutamic acid metabolic pathway of synthesizing, phosphoenolpyruvic acid anaplerotic sequence, shikimic acid pathway, reducing power balance approach, lipid acid metabolic pathway of synthesizing, phosphopentose pathway) precursor synthetic gene of transforming bacterial strain, can improve FK506 output and reduce production costs.
Technical scheme of the present invention is as follows:
A kind of FK506 strain gene group yardstick Metabolic Network Model instructs elementary approach remodeling method down, and step is as follows:
1) according to the streptomyces tsukubaensis genome sequence, add the biosynthetic characteristic reaction of FK506 and thalline building-up reactions and to network reaction carry out manual refining, obtained FK506 bacterial strain streptomyces tsukubaensis genome yardstick Metabolic Network Model;
2) by this Metabolic Network Model is analyzed, utilize flux equilibrium analysis and minimum metabolism to regulate analytical procedure, determine the biosynthetic elementary approach precursor key gene of FK506.
Adding the biosynthetic characteristic reaction of FK506 and thalline building-up reactions in FK506 bacterial strain Metabolic Network Model, is sole carbon source with starch.
The biosynthetic elementary approach precursor key gene of FK506 is gdhA, ppc, dahp, pntAB, accA2 and zwf2.
The biosynthetic elementary approach precursor key gene of FK506 carries out molecular modification, and step is as follows:
1) elementary approach precursor synthetic gene gdhA and the ppc that prediction is drawn carries out gene knockout, dahp, pntAB, accA2 and zwf2 carried out the molecular modification of gene amplification;
2) the precursor synthetic gene is knocked out and the bacterial strain that increases carries out shake flask fermentation and cultivates, investigate bacterial strain transform before and after the FK506 change of production.
To the key gene that dopes, adopt the molecular modification method of gene knockout, structure knocks out plasmid p Δ GDH and p Δ PPC, to contain the target gene plasmid and change intestinal bacteria ET12567 over to, utilize the conjugal transfer method to change among the streptomyces tsukubaensis D852, utilize kantlex (thiostrepton) resistance screening and PCR verification method to obtain genetically deficient bacterial strain HT-Δ GDH and HT-Δ PPC.
To the key gene that dopes, adopt the molecular modification method of gene amplification, make up amplification plasmid pACC, pDAHP, pPNT and pZWF, to contain the target gene plasmid and change intestinal bacteria ET12567 over to, utilize the conjugal transfer method to change among the streptomyces tsukubaensis D852, utilize apramycin resistance screening and PCR verification method to obtain gene amplification bacterial strain HT-ACC, HT-DAHP, HT-PNT and HT-ZWF.
The bacterial strain of transforming is carried out shake flask fermentation cultivate, 28 ° of C, 220rpm cultivated 168 hours in shaking bottle; The rate ratio wild-type has improved 80%-100%; The substratum that uses consists of: starch 60g/L, yeast extract 2g/L, peptone 2.5g/L, soybean cake powder 5g/L, K
2HPO
40.5g/L, CaCO
30.5g/L, MgSO
40.5g/L, pH6.8.
It is as follows to describe principle and method in detail:
According to the streptomyces tsukubaensis genome sequence, add the biosynthetic characteristic reaction of FK506 and thalline building-up reactions and to network reaction carry out manual refining (reject redundant reaction, add the reaction cofactor, the reversible direction of conditioned reaction, trim reaction, add reactant transport, analyze fill a vacancy) after, obtained FK506 bacterial strain streptomyces tsukubaensis genome yardstick Metabolic Network Model; Model mainly comprises center carbon metabolism (glycolytic pathway, phosphopentose pathway, tricarboxylic acid cycle), the pyruvic acid metabolism, the glyoxylate cycle metabolism, the synthetic and consumption metabolism of amino acid, nucleotide metabolism, lipid metabolism, peptidoglycan is synthetic, and coenzyme is synthetic, and the nitrogen sulfo-is thanked and correlated response such as porphyrin, comprise the route of synthesis reaction of FK506 and by product FK520, FK506D in addition, network contains 865 reactions and 621 metabolites altogether.
By this Metabolic Network Model is analyzed, predicted the target gene that can improve FK506 output.Actual physiological metabolism state according to bacterial strain, screen rational genetic modification strategy from elementary approach, comprise that the single-gene to glutamate dehydrogenase and phosphoric acid enol pyruvic acid carboxylase knocks out, to the single-gene amplification of the Arabic glycosyl of 3-deoxidation-D--heptulosonate-7-phosphate synthase, niacinamide nucleosides transhydrogenase, acetyl-CoA carboxylase, glucose-6-phosphate dehydrogenase.
Adding the biosynthetic characteristic reaction of FK506 and thalline building-up reactions in FK506 bacterial strain Metabolic Network Model, is sole carbon source with starch.
Utilize flux equilibrium analysis and minimum metabolism to regulate analytical procedure, determine the biosynthetic elementary approach precursor key gene of FK506.
FK506 strain gene group yardstick Metabolic Network Model of the present invention is carried out the precursor-gene transformation of elementary approach, and step is as follows:
1. elementary approach precursor synthetic gene gdhA and the ppc that prediction is drawn carries out gene knockout, dahp, pntAB, accA2 and zwf2 carried out the molecular modification of gene amplification;
2. the precursor synthetic gene is knocked out and the bacterial strain that increases carries out shake flask fermentation and cultivates, investigate bacterial strain transform before and after the FK506 change of production.
For the key gene that dopes---knock out gene, the primer that utilizes the homology left arm increases to the left arm of gdhA-LF and the goal gene gdhA of gdhA-LR, obtains the left arm fragment of gene gdhA.The primer that utilizes the homology right arm increases to the right arm of gdhA-RF and the goal gene gdhA of gdhA-RR, obtains the right arm fragment of gene gdhA.Two sections homologous fragments are sent to sequence verification, with two sections homologous fragments respectively with being connected in pUC119-kana successively behind XbaI – BamHI and the KpnI – EcoRI double digestion.Above-mentioned carrier construction is connected in E.coli-Streptomyces shuttle plasmid pKC1139 after with XbaI – EcoRI double digestion, obtains the knockout carrier p Δ GDH of gdhA.The knockout carrier building process of gene ppc is similar with p Δ GDH, and the primer of homology left and right arms is to being respectively ppc-LF/ppc-LR and ppc-RF/ppc-RR.Two sections homologous fragments are sent to sequence verification, with two sections homologous fragments respectively with being connected in pKC1139 successively behind HindIII – XbaI and the KpnI – EcoRI double digestion.Utilize primer tsr-F and tsr-R that thiostrepton tsr gene is increased from plasmid pGH112, through being connected in the plasmid that previous step makes up behind the XbaI – KpnI double digestion, obtain the knockout carrier p Δ PPC of ppc.To contain the target gene plasmid and change intestinal bacteria ET12567 over to, and utilize the conjugal transfer method to change among the streptomyces tsukubaensis D852, and utilize kantlex (thiostrepton) resistance screening and PCR verification method to obtain genetically deficient bacterial strain HT-Δ GDH and HT-Δ PPC.
For the key gene that dopes---amplification gene, be template with the S.roseosporus genome, respectively with accA2-F and accA2-R, dahp-F and dahp-R are primer (underscore is restriction enzyme site), amplification accA2 gene and dahp gene; Be template with the S.coelicolor genome, with pntAB-F and pntAB-R, accA2-F and accA2-R are primer, amplification pntAB gene and zwf2 gene, PCR product comprise each gene self with ribosome bind site.Respectively with the accA2 gene, the dahp gene, pntAB gene and zwf2 gene PCR product are connected into the pIB139 that same enzyme was cut after with the NdeI-XbaI double digestion, connect product and change competent escherichia coli cell JM109 or the DH5 α for preparing over to, be applied to incubated overnight on the screening flat board that contains 50 μ g/mL apramycins after the conversion.Double digestion or PCR checking are done in the activation of picking list bacterium colony, upgrading grain, finally obtain plasmid pACC, pDAHP, pPNT, pZWF.To contain the target gene plasmid and change intestinal bacteria ET12567 over to, and utilize the conjugal transfer method to change among the streptomyces tsukubaensis D852, and utilize apramycin resistance screening and PCR verification method to obtain gene amplification bacterial strain HT-ACC, HT-DAHP, HT-PNT and HT-ZWF.
The bacterial strain of transforming is carried out shake flask fermentation cultivate, 28 ° of C, 220rpm cultivated 168 hours in shaking bottle; The rate ratio wild-type has improved 80%-100%; The substratum that uses consists of: starch 60g/L, yeast extract 2g/L, peptone 2.5g/L, soybean cake powder 5g/L, K
2HPO
40.5g/L, CaCO
30.5g/L, MgSO
40.5g/L, pH6.8.
Transform strain fermentation rate ratio wild-type and improved 80%-100%, the illustrative experiment effect improves significantly; Maximum FK506 output reaches 200-300mg/L.
The present invention utilizes FK506 strain gene group yardstick Metabolic Network Model to predict that elementary approach precursor-gene is to improve the bacterial strain production level, the approach molecular modification method of realization bacterial strain.Actual physiological metabolism state according to bacterial strain, screen rational genetic modification strategy from elementary approach, comprise that the single-gene to glutamate dehydrogenase and phosphoric acid enol pyruvic acid carboxylase knocks out, to the single-gene amplification of the Arabic glycosyl of 3-deoxidation-D--heptulosonate-7-phosphate synthase, niacinamide nucleosides transhydrogenase, acetyl-CoA carboxylase, glucose-6-phosphate dehydrogenase.Key gene in six critical paths that dope is carried out genetic modification; The bacterial strain of transforming is carried out shake flask fermentation cultivate, the rate ratio wild-type has improved 80%-100%, and the illustrative experiment effect improves significantly; Maximum FK506 output reaches 200-300mg/L.The present invention adopts the remodeling method of the elementary approach precursor of genome yardstick Metabolic Network Model prediction FK506 bacterial strain synthetic gene, and the production level of transforming the back bacterial strain has improved 80%-100%.Should be based on the method for elementary approach precursor route of synthesis (L-glutamic acid metabolic pathway of synthesizing, phosphoenolpyruvic acid anaplerotic sequence, shikimic acid pathway, reducing power balance approach, lipid acid metabolic pathway of synthesizing, phosphopentose pathway) transformation, instruct for structure, the research and analysis of FK506 bacterial strain provide efficiently, in the research process of FK506 bacterial strain transformation, have great application prospect.
Description of drawings
The elementary pathway gene of Fig. 1 streptomyces tsukubaensis FK506 route of synthesis and model prediction (gdhA, ppc, accA2, dahp, pntAB and zwf2).
Fig. 2 target gene knocks out plasmid (p Δ GDH and p Δ PPC) and amplification plasmid (pACC, pDAHP, pPNT, structure pZWF).
Embodiment
The present invention will be further described below in conjunction with specific embodiment.
According to the streptomyces tsukubaensis genome sequence, add the biosynthetic characteristic reaction of FK506 and thalline building-up reactions and to network reaction carry out manual refining (reject redundant reaction, add the reaction cofactor, the reversible direction of conditioned reaction, trim reaction, add reactant transport, analyze fill a vacancy) after, obtained FK506 bacterial strain streptomyces tsukubaensis genome yardstick Metabolic Network Model (Fig. 1).Model mainly comprises center carbon metabolism (glycolytic pathway, phosphopentose pathway, tricarboxylic acid cycle), the pyruvic acid metabolism, the glyoxylate cycle metabolism, the synthetic and consumption metabolism of amino acid, nucleotide metabolism, lipid metabolism, peptidoglycan is synthetic, and coenzyme is synthetic, and the nitrogen sulfo-is thanked and correlated response such as porphyrin, comprise the route of synthesis reaction of FK506 and by product FK520, FK506D in addition, network contains 865 reactions and 621 metabolites altogether.Based on genome yardstick Metabolic Network Model, utilize flux equilibrium analysis and minimum metabolism to regulate analyses and prediction and can improve the target gene of FK506 output.Actual physiological metabolism state according to bacterial strain, screen rational genetic modification strategy from elementary approach, comprise that the single-gene to glutamate dehydrogenase and phosphoric acid enol pyruvic acid carboxylase knocks out, to the single-gene amplification (Fig. 1) of the Arabic glycosyl of 3-deoxidation-D--heptulosonate-7-phosphate synthase, niacinamide nucleosides transhydrogenase, acetyl-CoA carboxylase, glucose-6-phosphate dehydrogenase.
According to embodiment 1 prediction precursor L-glutamic acid metabolic pathway of synthesizing gene gdhA and phosphoenolpyruvic acid anaplerotic sequence gene ppc, the FK506 bacterial strain is carried out gene knockout.Utilize the primer of homology left arm to gdhA-LF and gdhA-LR(table 1) left arm of goal gene gdhA is increased, obtain the left arm fragment of gene gdhA.Utilize the primer of homology right arm to gdhA-RF and gdhA-RR(table 1) right arm of goal gene gdhA is increased, obtain the right arm fragment of gene gdhA.Two sections homologous fragments are sent to sequence verification, with two sections homologous fragments respectively with being connected in pUC119-kana successively behind XbaI – BamHI and the KpnI – EcoRI double digestion.Above-mentioned carrier construction is connected in E.coli-Streptomyces shuttle plasmid pKC1139 after with XbaI – EcoRI double digestion, obtains knockout carrier p Δ GDH(Fig. 2 of gdhA).The knockout carrier building process of gene ppc is similar with p Δ GDH, and the primer of homology left and right arms is to being respectively ppc-LF/ppc-LR and ppc-RF/ppc-RR(table 1).Two sections homologous fragments are sent to sequence verification, with two sections homologous fragments respectively with being connected in pKC1139 successively behind HindIII – XbaI and the KpnI – EcoRI double digestion.Utilize primer tsr-F and tsr-R(table 1) thiostrepton tsr gene is increased from plasmid pGH112, through being connected in the plasmid that previous step makes up behind the XbaI – KpnI double digestion, obtain knockout carrier p Δ PPC(Fig. 2 of ppc).To contain the target gene plasmid and change intestinal bacteria ET12567 over to, and utilize the conjugal transfer method to change among the streptomyces tsukubaensis D852, and utilize kantlex (thiostrepton) resistance screening and PCR verification method to obtain genetically deficient bacterial strain HT-Δ GDH and HT-Δ PPC.
The primer of using in this research of table 1
Underscore is restriction enzyme site
According to embodiment 1 prediction precursor shikimic acid pathway gene dahp, reducing power balance pathway gene pntAB, lipid acid metabolic pathway of synthesizing gene accA2, phosphopentose pathway gene zwf2, the FK506 bacterial strain is carried out molecular modification.Be template with the S.roseosporus genome, respectively with accA2-F and accA2-R, dahp-F and dahp-R are primer (table 1), amplification accA2 gene and dahp gene; Be template with the S.coelicolor genome, with pntAB-F and pntAB-R, accA2-F and accA2-R are primer (table 1), amplification pntAB gene and zwf2 gene, PCR product comprise each gene self with ribosome bind site.Respectively with the accA2 gene, the dahp gene, pntAB gene and zwf2 gene PCR product are connected into the pIB139 that same enzyme was cut after with the NdeI-XbaI double digestion, connect product and change competent escherichia coli cell JM109 or the DH5 α for preparing over to, be applied to incubated overnight on the screening flat board that contains 50 μ g/mL apramycins after the conversion.Double digestion or PCR checking are done in the activation of picking list bacterium colony, upgrading grain, finally obtain plasmid pACC, pDAHP, pPNT, pZWF(Fig. 2).To contain the target gene plasmid and change intestinal bacteria ET12567 over to, and utilize the conjugal transfer method to change among the streptomyces tsukubaensis D852, and utilize apramycin resistance screening and PCR verification method to obtain gene amplification bacterial strain HT-ACC, HT-DAHP, HT-PNT and HT-ZWF.
The precursor L-glutamic acid metabolic pathway of synthesizing gene gdhA knock-out bacterial strain HT-Δ GDH that embodiment 2 is transformed carries out the shake flask fermentation cultivation, and 28 ° of C, 220rpm cultivated 168 hours in shaking bottle; The substratum that uses consists of: starch 60g/L, yeast extract 2g/L, peptone 2.5g/L, soybean cake powder 5g/L, K
2HPO
40.5g/L, CaCO
30.5g/L, MgSO
40.5g/L, pH6.8.The maximum growth amount of L-glutamic acid metabolic pathway of synthesizing gene gdhA knock-out bacterial strain HT-Δ GDH is about the 70%-80% of original strain, and FK506 rate ratio wild-type has improved 85%-95%.
The precursor phosphoenolpyruvic acid anaplerotic sequence gene ppc knock-out bacterial strain HT-Δ PPC that embodiment 2 is transformed carries out the shake flask fermentation cultivation, and 28 ° of C, 220rpm cultivated 168 hours in shaking bottle; The substratum that uses consists of: starch 60g/L, yeast extract 2g/L, peptone 2.5g/L, soybean cake powder 5g/L, K
2HPO
40.5g/L, CaCO
30.5g/L, MgSO
40.5g/L, pH6.8.The maximum growth amount of phosphoenolpyruvic acid anaplerotic sequence gene ppc knock-out bacterial strain HT-Δ PPC is about the 60%-70% of original strain, and FK506 rate ratio wild-type has improved 80%-90%.
The precursor lipid acid metabolic pathway of synthesizing gene accA2 amplification bacterial strain HT-ACC that embodiment 3 is transformed carries out the shake flask fermentation cultivation, and 28 ° of C, 220rpm cultivated 168 hours in shaking bottle; The substratum that uses consists of: starch 60g/L, yeast extract 2g/L, peptone 2.5g/L, soybean cake powder 5g/L, K
2HPO
40.5g/L, CaCO
30.5g/L, MgSO
40.5g/L, pH6.8.The maximum growth amount of lipid acid metabolic pathway of synthesizing gene accA2 amplification bacterial strain HT-ACC is about the 90%-100% of original strain, and FK506 rate ratio wild-type has improved 85%-95%.
The precursor shikimic acid pathway gene dahp amplification bacterial strain HT-DAHP that embodiment 3 is transformed carries out the shake flask fermentation cultivation, and 28 ° of C, 220rpm cultivated 168 hours in shaking bottle; The substratum that uses consists of: starch 60g/L, yeast extract 2g/L, peptone 2.5g/L, soybean cake powder 5g/L, K
2HPO
40.5g/L, CaCO
30.5g/L, MgSO
40.5g/L, pH6.8.The maximum growth amount of shikimic acid pathway gene dahp amplification bacterial strain HT-DAHP is about the 95%-105% of original strain, and FK506 rate ratio wild-type has improved 90%-100%.
Embodiment 8
The precursor reducing power balance pathway gene pntAB amplification bacterial strain HT-PNT that embodiment 3 is transformed carries out the shake flask fermentation cultivation, and 28 ° of C, 220rpm cultivated 168 hours in shaking bottle; The substratum that uses consists of: starch 60g/L, yeast extract 2g/L, peptone 2.5g/L, soybean cake powder 5g/L, K
2HPO
40.5g/L, CaCO
30.5g/L, MgSO
40.5g/L, pH6.8.The maximum growth amount of reducing power balance pathway gene pntAB amplification bacterial strain HT-PNT is about the 90%-100% of original strain, and FK506 rate ratio wild-type has improved 80%-90%.
Embodiment 9
The precursor phosphopentose pathway gene zwf2 amplification bacterial strain HT-ZWF that embodiment 3 is transformed carries out the shake flask fermentation cultivation, and 28 ° of C, 220rpm cultivated 168 hours in shaking bottle; The substratum that uses consists of: starch 60g/L, yeast extract 2g/L, peptone 2.5g/L, soybean cake powder 5g/L, K
2HPO
40.5g/L, CaCO
30.5g/L, MgSO
40.5g/L, pH6.8.The maximum growth amount of phosphopentose pathway gene zwf2 amplification bacterial strain HT-ZWF is about the 90%-100% of original strain, and FK506 rate ratio wild-type has improved 80%-90%.
Through overtesting, the present invention can reach the effect that needs in following condition and range.
Make up FK506 strain gene group Metabolic Network Model, carry out the metabolic flux analysis, obtain elementary approach and screen rational genetic modification strategy, comprise that the single-gene to glutamate dehydrogenase and phosphoric acid enol pyruvic acid carboxylase knocks out, to the single-gene amplification of the Arabic glycosyl of 3-deoxidation-D--heptulosonate-7-phosphate synthase, niacinamide nucleosides transhydrogenase, acetyl-CoA carboxylase, glucose-6-phosphate dehydrogenase.Precursor-gene to prediction carries out molecular modification, transforming bacterial strain shakes in the bottle at the 500mL that the 200mL liquid fermentation medium is housed, 160-220rpm, cultivate 120-196h under the 25-30 ° of C condition, can improve the output 80%-100% of FK506, verify that model is to elementary approach precursor synthetic gene prediction accuracy.
Claims (7)
1. a FK506 strain gene group yardstick Metabolic Network Model instructs elementary approach remodeling method down, and its characterization step is as follows:
1) according to the streptomyces tsukubaensis genome sequence, add the biosynthetic characteristic reaction of FK506 and thalline building-up reactions and to network reaction carry out manual refining, obtained FK506 bacterial strain streptomyces tsukubaensis genome yardstick Metabolic Network Model;
2) by this Metabolic Network Model is analyzed, utilize flux equilibrium analysis and minimum metabolism to regulate analytical procedure, determine the biosynthetic elementary approach precursor key gene of FK506.
2. the method for claim 1 is characterized in that: adding the biosynthetic characteristic reaction of FK506 and thalline building-up reactions in FK506 bacterial strain Metabolic Network Model, is sole carbon source with starch.
3. method as claimed in claim 1 is characterized in that the biosynthetic elementary approach precursor key gene of FK506 is gdhA, ppc, dahp, pntAB, accA2 and zwf2.
4. method as claimed in claim 3 is characterized in that the biosynthetic elementary approach precursor key gene of FK506 carries out molecular modification, and step is as follows:
1) elementary approach precursor synthetic gene gdhA and the ppc that prediction is drawn carries out gene knockout, dahp, pntAB, accA2 and zwf2 carried out the molecular modification of gene amplification;
2) the precursor synthetic gene is knocked out and the bacterial strain that increases carries out shake flask fermentation and cultivates, investigate bacterial strain transform before and after the FK506 change of production.
5. method as claimed in claim 4, it is characterized in that: to the key gene that dopes, adopt the molecular modification method of gene knockout, structure knocks out plasmid p Δ GDH and p Δ PPC, to contain the target gene plasmid and change intestinal bacteria ET12567 over to, utilize the conjugal transfer method to change among the streptomyces tsukubaensis D852, utilize kantlex (thiostrepton) resistance screening and PCR verification method to obtain genetically deficient bacterial strain HT-Δ GDH and HT-Δ PPC.
6. method as claimed in claim 4, it is characterized in that: to the key gene that dopes, adopt the molecular modification method of gene amplification, make up amplification plasmid pACC, pDAHP, pPNT and pZWF, to contain the target gene plasmid and change intestinal bacteria ET12567 over to, utilize the conjugal transfer method to change among the streptomyces tsukubaensis D852, utilize apramycin resistance screening and PCR verification method to obtain gene amplification bacterial strain HT-ACC, HT-DAHP, HT-PNT and HT-ZWF.
7. method as claimed in claim 4 is characterized in that: the bacterial strain of transforming is carried out shake flask fermentation cultivate, 28 ° of C, 220rpm cultivated 168 hours in shaking bottle; The rate ratio wild-type has improved 80%-100%; The substratum that uses consists of: starch 60g/L, yeast extract 2g/L, peptone 2.5g/L, soybean cake powder 5g/L, K
2HPO
40.5g/L, CaCO
30.5g/L, MgSO
40.5g/L, pH6.8.
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