CN117264862A - Genetically engineered bacterium for high yield of D-pantothenic acid, construction method and application thereof - Google Patents
Genetically engineered bacterium for high yield of D-pantothenic acid, construction method and application thereof Download PDFInfo
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- GHOKWGTUZJEAQD-UHFFFAOYSA-N pantothenic acid Chemical compound OCC(C)(C)C(O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-UHFFFAOYSA-N 0.000 title claims abstract description 103
- 241000894006 Bacteria Species 0.000 title claims abstract description 80
- 238000010276 construction Methods 0.000 title claims abstract description 27
- 235000014469 Bacillus subtilis Nutrition 0.000 claims abstract description 44
- 244000063299 Bacillus subtilis Species 0.000 claims abstract description 42
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- 101150076071 panD gene Proteins 0.000 claims abstract description 26
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 24
- 101150005925 aspC gene Proteins 0.000 claims abstract description 21
- 101150007902 ASPA gene Proteins 0.000 claims abstract description 18
- 101100242684 Mesorhizobium japonicum (strain LMG 29417 / CECT 9101 / MAFF 303099) panD1 gene Proteins 0.000 claims abstract description 17
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- 101150116541 nadB gene Proteins 0.000 claims abstract description 13
- 241000186226 Corynebacterium glutamicum Species 0.000 claims abstract description 12
- 101100492609 Talaromyces wortmannii astC gene Proteins 0.000 claims abstract description 12
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- UCMIRNVEIXFBKS-UHFFFAOYSA-N beta-alanine Chemical compound NCCC(O)=O UCMIRNVEIXFBKS-UHFFFAOYSA-N 0.000 abstract description 79
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- 229940055726 pantothenic acid Drugs 0.000 abstract description 17
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 abstract description 14
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- CKLJMWTZIZZHCS-UHFFFAOYSA-N D-OH-Asp Natural products OC(=O)C(N)CC(O)=O CKLJMWTZIZZHCS-UHFFFAOYSA-N 0.000 abstract description 13
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of genetic engineering, and discloses a genetic engineering bacterium for high-yield D-pantothenic acid, and a construction method and application thereof. The invention uses bacillus subtilis as chassis fungus, reforms its metabolic pathway, firstly heterologously expresses ppC, aspC genes from colibacillus, then overexpresses panD, panC, aspB genes in beta-alanine synthetic pathway, strengthens the synthesis of beta-alanine which is a precursor of pantothenic acid synthesis; knocking out a nadB gene to reduce branch metabolic flow in a beta-alanine precursor L-aspartic acid synthesis path; heterologous expression of the aspA gene of the escherichia coli, and continuous enhancement of beta-alanine accumulation; fourthly, the gabT gene is knocked out, and the metabolic flow of the branched-chain organic acid synthesis pathway is further weakened on the basis of reducing the competitive branch metabolic flow; finally, the bacillus subtilis genetic engineering strain with high D-pantothenic acid yield is constructed by using plasmids to heterologously express panD genes from escherichia coli and panC genes from corynebacterium glutamicum.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a genetic engineering bacterium for high-yield D-pantothenic acid, and a construction method and application thereof.
Background
Pantothenate is an important precursor for acyl carrier protein and acetyl-CoA biosynthesis in organisms. In addition, pantothenic acid can be converted to acetyl-CoA under the catalysis of pantothenate kinase and pyruvate dehydrogenase, providing an important substrate for the tricarboxylic acid cycle of the cell. At the same time, pantothenic acid also plays a central role in the anabolic and catabolic pathways of many substances, such as histone acetylation, carbohydrates and fatty acids, to assist in the functioning of cells. In addition, pantothenic acid plays an important role in antagonizing stress, maintaining hair, skin and blood health, and also contributes to the secretion of anti-stress hormones in the body, and also can maintain the health of skin and hair, etc.
Products which are pantothenic acid and derivatives thereof have a wide variety of applications. In the medical field, pantothenic acid has the function of producing antibodies, and the pantothenic acid product can be externally used for treating various skin diseases, such as ichthyosis, psoriasis, contact dermatitis and the like; pantothenic acid is often used as a vitamin additive in animal feed, added into feed for poultry, pigs, young ruminants, fish, etc., and is helpful for growth and development of animals, synthesis and decomposition of fat, and can improve color and luster of animal hair, feathers, scales, etc.; pantothenic acid has the main functions of moisturizing agent and skin conditioner in cosmetics and skin care products, has the functions of relieving and resisting allergy, and is safer.
D-pantothenic acid (also known as D-calcium pantothenate, vitamin B5), is a water-soluble vitamin. The production methods of D-pantothenic acid are mainly physical induction crystallization, chemical resolution, chemical synthesis, biological methods, etc. The physical induced crystallization process is mature, and the principle is that the solubility of D-type calcium pantothenate and L-type calcium pantothenate which are mixed together is larger than that of single calcium pantothenate, so that the induced crystallization is carried out, but the method can only produce calcium pantothenate simply, can not be used for derivative products of pantothenic acid, and has a narrow application range. The chemical resolution method is the most widely used production method at present, and the chemical resolution method is to use chiral resolving agent to resolve to obtain D-pantothenic acid, but the chemical resolution method also has a series of problems of difficult separation after resolution, difficult treatment of residual reaction liquid, expensive chiral resolving agent and the like. The chemical synthesis method utilizes two precursors of pantothenic acid beta-alanine to react with D-pantolactone to obtain D-pantothenic acid, but the method has higher requirements on equipment when producing D-pantothenic acid, and toxic substances can be produced in the precursor synthesis process, which does not meet the green development requirement.
The D-pantothenic acid product is produced by a biological fermentation method, and can be obtained by using inexpensive industrial raw materials such as glucose and the like and through the self-metabolic reaction of organisms. The biological fermentation method utilizes cheap raw materials to obtain a product with high added value, which not only accords with development benefits to reduce production cost, but also has the advantages of low environmental cost, high production efficiency and the like. However, the biological fermentation method has the problems of unstable fermentation process, low yield and the like.
Disclosure of Invention
In order to solve the technical problems of unstable fermentation process and low yield of D-pantothenic acid produced by the biological fermentation method, the invention provides a genetically engineered bacterium for high-yield D-pantothenic acid, and a construction method and application thereof. The invention strengthens the synthesis of the precursor beta-alanine for pantothenic acid synthesis and reduces the branch metabolic flow of the precursor beta-alanine by modifying the metabolic pathway of bacillus subtilis, so as to strengthen the accumulation of beta-alanine, and further, increase the yield of D-pantothenic acid.
The specific technical scheme of the invention is as follows:
first, the invention provides a genetically engineered bacterium for high yield of D-pantothenic acid. The genetic engineering bacteria for high yield of D-pantothenic acid are constructed by taking bacillus subtilis as chassis bacteria, and the construction method comprises the following steps:
(1) Overexpression of panD, panC, aspB gene in chassis fungus genome;
(2) Knocking out the nadB and gabT genes in the chassis fungus genome;
(3) Heterologous expression of ppC, aspC, aspA, panD gene from E.coli in Chaetomium;
(4) Heterologous expression of the panC gene from Corynebacterium glutamicum in Chaetomium.
The synthetic precursors of D-pantothenic acid are mainly pantolactone and beta-alanine. In the beta-alanine synthesis pathway, phosphoenolpyruvate is obtained after glycolysis, and then, phosphoenolpyruvate is synthesized into oxaloacetate under the action of phosphoenolpyruvate carboxylase, and then, the oxaloacetate generates aspartic acid, which is converted into beta-alanine. In the synthetic pathway of D-pantothenate, the precursor β -alanine is an important factor affecting D-pantothenate production. According to the invention, bacillus subtilis is taken as chassis bacteria, the nadB and gabT genes of the chassis bacteria are knocked out by over-expressing panD, panC, aspB genes of the chassis bacteria, ppC, aspC, aspA, panD genes derived from escherichia coli are expressed in the chassis bacteria in a heterologous manner, and panC genes derived from corynebacterium glutamicum are expressed in the chassis bacteria in a heterologous manner, so that D-pantothenic acid can be efficiently produced under the condition that beta-alanine is not required to be added after the bacillus subtilis is reformed by modifying a beta-alanine synthesis path. Specifically, the transformation principle is as follows:
the invention is characterized in that: (1) Introducing exogenous ppC and aspC genes of escherichia coli to strengthen the direct conversion of phosphoenolpyruvic acid into oxaloacetic acid and then into L-aspartic acid, and strengthening the precursor accumulation of beta-alanine metabolic pathway; (2) Enhancing the expression of the gene panD, panC, aspB, a key gene in the D-pantothenate synthesis pathway, and enhancing the conversion of L-aspartic acid to beta-alanine; (3) Knocking out the nadB gene in the metabolic bypass of L-aspartic acid, so that the β -alanine precursor L-aspartic acid is further accumulated; (4) Introducing a key gene aspA gene in the production of L-aspartic acid by exogenous escherichia coli fumarate, and increasing accumulation in the process of L-aspartic acid recharging; (5) Knocking out the gabT gene in the metabolic bypass of beta-alanine, so that the beta-alanine is further accumulated; (6) The panC gene of exogenous corynebacterium glutamicum and panD gene from colibacillus are introduced to continuously increase the synthesis of D-pantothenic acid, and finally the bacillus subtilis genetic engineering strain with high yield of D-pantothenic acid is constructed.
Meanwhile, the invention provides a construction method of the genetically engineered bacterium for high yield of D-pantothenic acid, which comprises the following steps: step S1: introducing ppC genes derived from escherichia coli into a chassis fungus genome by taking bacillus subtilis as the chassis fungus to obtain engineering bacteria DPA1;
step S2: introducing aspC genes derived from escherichia coli into an engineering bacterium DPA1 genome to obtain engineering bacterium DPA2;
step S3: over-expressing panD genes in the engineering bacteria DPA2 genome to obtain engineering bacteria DPA3;
step S4: over-expressing the panC gene in the engineering bacterium DPA3 genome to obtain engineering bacterium DPA4;
step S5: overexpressing aspB gene in engineering bacteria DPA4 genome to obtain engineering bacteria DPA5;
step S6: knocking out a nadB gene in the engineering bacterium DPA5 genome to obtain engineering bacterium DPA6;
step S7: inserting aspA genes from escherichia coli into an engineering bacterium DPA6 genome to obtain engineering bacterium DPA7;
step S8: knocking out the gabT gene in the engineering bacterium DPA7 genome to obtain engineering bacterium DPA8;
step S9: and (3) using engineering bacteria DPA8 as chassis bacteria, and overexpressing panC genes derived from corynebacterium glutamicum and panD genes derived from escherichia coli to obtain the genetically engineered bacteria with high D-pantothenic acid yield.
The above steps can be achieved by genetic engineering means, such as insertion or knockout of genes using Cre/loxP gene editing system.
According to the invention, bacillus subtilis is used as chassis bacteria, the chassis bacteria are modified through steps S1-S9, the metabolic synthesis path of D-pantothenic acid is regulated, and the yield of D-pantothenic acid is improved. Firstly, introducing an exogenous escherichia coli ppC gene to strengthen the supply of oxaloacetic acid, introducing an exogenous escherichia coli aspC gene to strengthen the supply of converting oxaloacetic acid into L-aspartic acid, and strengthening the precursor accumulation of a beta-alanine metabolic pathway; then, the expression of a key gene panD, panC, aspB in the D-pantothenic acid synthesis pathway is enhanced, and the conversion from L-aspartic acid to beta-alanine is enhanced; knocking out the nadB gene in the metabolic bypass of L-aspartic acid to reduce the metabolic loss; the aspA gene of the exogenous escherichia coli is continuously introduced to continuously enhance the metabolic accumulation of the escherichia coli; knocking out the gabT gene, and reducing the metabolic loss of L-aspartic acid; the panC gene of exogenous corynebacterium glutamicum and panD gene from escherichia coli are introduced, the beta-alanine metabolic pathway is continuously increased to synthesize D-pantothenic acid, and the loss of metabolic flow of the beta-alanine pathway is reduced; and (3) enhancing the expression of the key genes of the beta-alanine metabolic pathway, and finally constructing the bacillus subtilis genetic engineering strain with high D-pantothenic acid yield.
In the preferred embodiment of the present invention, in step S1, the bacillus subtilis is Bacillus subtilis and 168.
As a preferable mode of the above technical scheme of the present invention, in the step S1, the manner of introducing ppC gene derived from Escherichia coli into Chaetomium is as follows: the glmS gene in the genome of Chaetomium was replaced with a ppC gene derived from E.coli.
Further preferably, the promoter of the ppC gene derived from Escherichia coli is P 43 A promoter.
As a preferable mode of the technical scheme, in the step S2, the aspC gene derived from the escherichia coli is introduced into the engineering bacteria DPA1 genome in the following way: the ybdG gene in the engineering bacterium DPA1 genome is replaced by aspC gene from escherichia coli.
Further preferably, the promoter of the aspC gene derived from Escherichia coli is P 43 A promoter.
Compared with the prior art, the invention has the following technical effects:
the invention uses bacillus subtilis as chassis fungus, through modifying its metabolic pathway, firstly heterologously express ppC, aspC genes from colibacillus, then overexpress panD, panC, aspB genes in beta-alanine synthetic pathway, strengthen the synthesis of pantothenic acid synthetic precursor beta-alanine; knocking out a nadB gene to reduce branch metabolic flow in a beta-alanine precursor L-aspartic acid synthesis path; heterologous expression of the aspA gene of the escherichia coli, and continuous enhancement of beta-alanine accumulation; fourthly, the gabT gene is knocked out, and the metabolic flow of the branched-chain organic acid synthesis pathway is further weakened on the basis of reducing the competitive branch metabolic flow; finally, the bacillus subtilis genetic engineering strain with high D-pantothenic acid yield is constructed by using plasmids to heterologously express panD genes from escherichia coli and panC genes from corynebacterium glutamicum. The construction of the genetic engineering strain solves the problem of insufficient pantothenic acid precursor beta-alanine in the metabolic process, reduces the level of exogenously added beta-alanine, and reduces the production cost.
D-pantothenate production in 5L fermentation by the starting strain was barely detectable from the fermentation without exogenously adding beta-alanine. The genetically engineered bacterium for high yield of D-pantothenic acid provided by the invention can improve the yield of 72. 72h D-pantothenic acid to 70.6g/L in 5L fermentation, and the yield is greatly increased.
Drawings
FIG. 1 is a schematic diagram of a construction strategy of a genetically engineered bacterium for high D-pantothenic acid production provided by the invention;
FIG. 2 is a graph showing the fermentation result of recombinant Bacillus subtilis DPA9 in example 10 of the present invention.
Detailed Description
The invention is further described below with reference to examples. Those of ordinary skill in the art will be able to implement the invention based on these descriptions. In addition, the embodiments of the present invention referred to in the following description are typically only some, but not all, embodiments of the present invention. Therefore, all other embodiments, which can be made by one of ordinary skill in the art without undue burden, are intended to be within the scope of the present invention, based on the embodiments of the present invention.
The chassis strain used in the examples of the present invention was Bacillus subtilis (b.subtilis 168).
The genes involved in Gene editing and their corresponding metabolic pathways in the present invention are shown in Table 1, wherein the Gene ID is an identifier for uniquely identifying the Gene from Entrez Gene database of National Center for Biotechnology Information (NCBI).
TABLE 1 Gene involved in Gene editing and corresponding pathways
The beta-alanine content of the embodiment of the invention is measured by adopting an HPLC method, and the detection method is as follows:
(1) chromatographic conditions: c18 column (250 x 4.6mm x 5 μm, agilent Technologies co., santa Clara, CA, USA), detection wavelength: 338nm, column temperature: detecting the flow rate at 40 ℃ at 1.0mL/min;
(2) sample treatment: diluting the sample with ultrapure water, keeping the beta-alanine content between 0.1g/L and 0.80g/L, and then filtering through a 0.22 mu m microporous filter membrane;
(3) mobile phase: phase A: weighing a certain amount of sodium acetate, preparing 1000mL of buffer solution with the concentration of 0.02mol/L, adding 180 mu L of triethylamine, adjusting the pH to 7.20+/-0.05 by using acetic acid, and adding 3mL of tetrahydrofuran for uniform mixing; and B phase: 0.02mol/L sodium acetate in acetonitrile in methanol=1:2:2 (V/V), pH was adjusted to 7.20.+ -. 0.05.
(4) Data acquisition time: 30min.
(5) OPA derivatizing agent configuration: 250mg of phthalic dicarboxaldehyde, 5mL of absolute ethanol and 100. Mu.L of 2-mercaptoethanol were mixed and dissolved in 0.4mol/L boric acid buffer (pH=9.5) to 25mL in the absence of light.
The D-pantothenic acid content of the embodiment of the invention is measured by adopting an HPLC method, and the detection method is as follows:
(1) chromatographic conditions: c18 column (250 x 4.6mm x 5 μm, agilent Technologies co., santa Clara, CA, USA), detection wavelength: 200nm, column temperature: detecting the flow rate at 30 ℃ at 1mL/min;
(2) sample treatment: diluting the sample with purified water, keeping D-pantothenic acid content between 0.1g/L and 0.80g/L, and filtering with 0.22 μm microporous filter membrane;
(3) mobile phase: acetonitrile/water/phosphoric acid volume ratio of 50:949:1;
(4) data acquisition time: 20min.
General examples
The embodiment provides a genetically engineered bacterium for high yield of D-pantothenic acid, B.subtilis 168 is taken as chassis bacterium, the construction strategy is shown in figure 1, and the genetically engineered bacterium is constructed according to the following method:
(1) The bacillus subtilis168 is taken as chassis fungus, a Cre/loxP gene editing method is applied, and the glmS (ineffective gene) gene in the genome is replaced by ppC gene in the escherichia coli genome, so that engineering bacteria B.subilis (delta glmS:: P) is obtained 43 ppC), noted DPA1;
(2) The Cre/loxP gene editing method is used to replace ybdG (ineffective gene) gene in engineering bacteria DPA1 genome with aspC gene in colibacillus genome to obtain engineering bacteria B.subilis (delta glmS:: P) 43 -ppCΔybdG::P 43 aspC), noted as DPA2;
(3) The Cre/loxP gene editing method is applied to over-express panD gene in engineering bacteria DPA2 genome to construct strain B.subilis (delta glmS:: P) 43 -ppCΔybdG::P 43 aspC-panD), noted DPA3;
(4) The Cre/loxP gene editing method is applied to over-express panC gene in engineering bacteria DPA3 genome to construct strain B.subilis (delta glmS:: P) 43 -ppCΔybdG::P 43 aspC-panDpanC), noted DPA4;
(5) The Cre/loxP gene editing method is used to over-express aspB gene in engineering bacteria DPA4 genome to construct strain B 43 -ppCΔybdG::P 43 aspC-panDpanCaspB), noted DPA5;
(6) The Cre/loxP gene editing method is applied to knock out the nadB gene in the engineering bacterium DPA5 genome to construct a strain B.subilis (delta glmS:: P) 43 -ppCΔybdG::P 43 aspC-panDpanCaspBΔnandB), noted as DPA6;
(7) Introducing aspA gene from escherichia coli into engineering bacterium DPA6 genome by using Cre/loxP gene editing method, and constructing strain B
(ΔglmS::P 43 -ppCΔybdG::P 43 -aspC-panDpanCaspBΔnadB::P 43 aspA), noted DPA7;
(8) The Cre/loxP gene editing method is applied to knock out the gabT gene in the engineering bacterium DPA7 genome to construct a strain B.subilis (delta glmS:: P) 43 -ppCΔybdG::P 43 -aspC-panDpanCaspBΔnadB::P 43 aspA Δgabt), noted DPA8;
(9) The panC gene from corynebacterium glutamicum and panD gene from colibacillus are constructed on PHT01 plasmid to obtain PHT01-panC-panD, panC and panD genes are overexpressed in engineering bacterium DPA9 genome, and strain B.subilis (delta glmS:: P) is constructed 43 -ppCΔybdG::P 43 -aspC-panDpanCaspBΔnadB::P 43 And (3) aspA delta gabT-panCD), and marking as DPA9, namely the genetically engineered bacterium for high-yield D-pantothenic acid.
EXAMPLE 1 construction of D-pantothenate-producing Chassis Strain DPA1 (. DELTA.glmS:: P 43 -ppC)
According to the upstream sequence and the downstream sequence of the glmS gene of bacillus subtilis168 published on NCBI, a ppC gene sequence and the sequence lox71-zeo-lox66 of a bleomycin resistance gene are inserted, wherein the knockout frame sequence constructed according to the glmS gene and the ppC gene sequence is shown in SEQ ID NO. 1.
The constructed insertion frame is transformed into bacillus subtilis168 chassis strain, correct bacterial colonies are screened out by a bleomycin resistance plate, and then the bacteria colonies are electrotransferred into pDG148 plasmid with a cyclase Cre gene, so that the resistance gene is eliminated, and the specific operation method is as follows:
inoculating transformant containing pDG148 plasmid into LB liquid culture medium containing 0.2mmol/L IPTG, culturing at 37deg.C for 24 hr, inoculating 1 μl culture solution into 2mL sterilized LB liquid culture medium, shake culturing at 50deg.C for 10 hr, streaking on non-anti-LB solid plate, and then respectively plating bacterial colony grown on non-anti-LB solid plate on bleomycin, kanamycin and non-anti-LB solid plate, and engineering B.subti for removing pDG148 plasmid only on non-anti-solid plate growth for knocking out target genelis engineering Strain (. DELTA.glmS:: P 43 ppC), designated DPA1, DPA1 is a plasmid-free recombinant Bacillus subtilis.
EXAMPLE 2 construction of D-pantothenate-producing Chassis Strain DPA2 (. DELTA.glmS:: P 43 -ppCΔybdG::P 43 -aspC)
The sequence lox71-zeo-lox66 and aspC genes of bleomycin resistance genes are inserted in the middle of the upstream and downstream sequences of ybdG genes of bacillus subtilis B.subilis 168 published on NCBI, wherein the knockout frame sequences constructed according to the sequences of the ybdG genes and aspC genes are shown in SEQ ID NO. 2.
The constructed insert was transformed into Bacillus subtilis DPA1 (. DELTA.glmS:: P 43 ppC) and verifying correct colonies by bleomycin resistance plate screening and colony PCR, and recovering bleomycin resistance gene, the specific steps are the same as in example 1, to obtain plasmid-free recombinant bacillus subtilis (ΔglmS: P) 43 -ppCΔybdG::P 43 aspC), noted as DPA2.
EXAMPLE 3 construction of D-pantothenate-producing Chassis Strain DPA3 (. DELTA.glmS:: P 43 -ppCΔybdG::P 43 aspC-panD) the bleomycin resistance gene lox71-zeo-lox66 gene was ligated based on the panD gene of B.subtilis 168 published on NCBI, with the panD gene as the inserted frame sequence constructed.
The constructed insert was transformed into Bacillus subtilis DPA2 (. DELTA.glmS:: P 43 -ppCΔybdG::P 43 -aspC), the correct colony was verified for bleomycin resistance gene recovery by bleomycin resistance plate screening and colony PCR verification, and plasmid-free recombinant Bacillus subtilis (. DELTA.glmS:: P) was obtained in the same manner as in example 1 43 -ppCΔybdG::P 43 aspC-panD), noted DPA3.
EXAMPLE 4 construction of D-pantothenate-producing Chassis Strain DPA4 (. DELTA.glmS:: P 43 -ppCΔybdG::P 43 aspC-panDpanC) the bleomycin resistance gene lox71-zeo-lox66 gene was ligated according to the panC gene of Bacillus subtilis B.subtilis 168 published on NCBI, with the panC gene as the constructed insertion frame sequence.
Transforming the constructed insertion frame into bacillus subtilis DPA3
(ΔglmS::P 43 -ppCΔybdG::P 43 aspC-panD), correct colonies were verified for bleomycin resistance gene recovery by bleomycin resistance plate screening and colony PCR verification, and plasmid-free recombinant Bacillus subtilis (. DELTA.glmS: P) was obtained in the same manner as in example 1 43 -ppCΔybdG::P 43 aspC-panDpanC), noted as DPA4.
EXAMPLE 5 construction of D-pantothenate-producing Chassis Strain DPA5
(ΔglmS::P 43 -ppCΔybdG::P 43 -aspC-panDpanCaspB)
The bleomycin resistance gene lox71-zeo-lox66 gene was ligated according to the aspB gene of Bacillus subtilis168 published on NCBI, and the aspB gene was used as the insertion frame sequence.
Transforming the constructed insertion frame into bacillus subtilis DPA4
(ΔglmS::P 43 -ppCΔybdG::P 43 aspC-panDpanC), bleomycin resistance gene recovery was performed on correct colonies by bleomycin resistance plate screening and colony PCR verification, and plasmid-free recombinant Bacillus subtilis (. DELTA.glmS:: P) was obtained in the same manner as in example 1 43 -ppCΔybdG::P 43 aspC-panDpanCaspB), noted DPA5.
EXAMPLE 6 construction of D-pantothenate-producing Chassis Strain DPA6
(ΔglmS::P 43 -ppCΔybdG::P 43 -aspC-panDpanCaspBΔnadB)
The insertion frame sequence is constructed by connecting the bleomycin resistance gene lox71-zeo-lox66 gene with the nadB gene on the upper and lower streams of the nadB gene of bacillus subtilis168 published on NCBI.
Transforming the constructed knockout frame into bacillus subtilis DPA5
(ΔglmS::P 43 -ppCΔybdG::P 43 -aspC-panDpanCaspB), bleomycin resistance gene recovery was performed on correct colonies by bleomycin resistance plate screening and colony PCR verification, and plasmid-free recombinant Bacillus subtilis (. DELTA.glmS:: P) was obtained by the same specific procedures as in example 1 43 -ppCΔybdG::P 43 -aspC-panDpanCaspB Δnadb), noted as DPA6.
EXAMPLE 7 construction of D-pantothenate-producing Chassis Strain DPA7
(ΔglmS::P 43 -ppCΔybdG::P 43 -aspC-panDpanCaspBΔnadB::P 43 -aspA)
The bleomycin resistance gene lox71-zeo-lox66 gene is connected on the upstream and downstream of the aspA gene of bacillus subtilis168 published on NCBI, and the aspA gene is used as an insertion frame sequence.
Transforming the constructed knockout frame into bacillus subtilis DPA6
(ΔglmS::P 43 -ppCΔybdG::P 43 aspC-panDpanCaspBΔnandB), correct bacterial colony is verified to be subjected to bleomycin resistance gene recovery by bleomycin resistance plate screening and bacterial colony PCR verification, and plasmid-free recombinant bacillus subtilis (ΔglmS:: P) is obtained by the specific steps as in example 1 43 -ppCΔybdG::P 43 -aspC-panDpanCaspBΔnadB::P 43 aspA), noted as DPA7.
EXAMPLE 8 construction of D-pantothenate-producing Chassis Strain DPA8
(ΔglmS::P 43 -ppCΔybdG::P 43 -aspC-panDpanCaspBΔnadB::P 43 -aspAΔgabT)
The bleomycin resistance gene lox71-zeo-lox66 gene is connected on the upstream and downstream of the gabT gene of bacillus subtilis B.subtilis 168 published on NCBI, and the gabT gene is used as a constructed insertion frame sequence.
Transforming the constructed knockout frame into bacillus subtilis DPA7
(ΔglmS::P 43 -ppCΔybdG::P 43 -aspC-panDpanCaspBΔnadB::P 43 -aspA), the correct colony was verified to be subjected to bleomycin resistance gene recovery by bleomycin resistance plate screening and colony PCR verification, and plasmid-free recombinant Bacillus subtilis (. DELTA.glmS:: P) was obtained in the same manner as in example 1 43 -ppCΔybdG::P 43 -aspC-panDpanCaspBΔnadB::P 43 aspA Δgabt), noted as DPA8.
EXAMPLE 9 construction of high-yield D-pantothenate Gene engineering bacterium DPA9
(ΔglmS::P 43 -ppCΔybdG::P 43 -aspC-panDpanCaspBΔnadB::P 43 -aspAΔgabT-panCD)
The panC gene coded by corynebacterium glutamicum and the panD gene coded by escherichia coli published on NCBI are subjected to PCR to obtain a panCD expression frame shown as SEQ ID NO.3, and the panCD expression frame is connected to PHT01 plasmid by a one-step cloning method (ClonExpress IIOne Step Cloning Kit kit, purchased from Nanjinouzan biotechnology Co., ltd.) to construct PHT01-panCD plasmid. Transforming the constructed plasmid into bacillus subtilis DPA8
(ΔglmS::P 43 -ppCΔybdG::P 43 -aspC-panDpanCaspBΔnadB::P 43 AspaΔgabt), by kanamycin resistance plate screening and colony PCR validation. Recombinant Bacillus subtilis (. DELTA.glmS:: P) containing the plasmid was obtained 43 -ppCΔybdG::P 43 -aspC-panDpanCaspBΔnadB::P 43 aspaΔgabT-panCD), noted as DPA9.
EXAMPLE 10 engineering bacteria DPA 9D-pantothenic acid fermentation verification
For recombinant bacillus subtilis DPA9
(ΔglmS::P 43 -ppCΔybdG::P 43 -aspC-panDpanCaspBΔnadB::P 43 aspaΔgabT-panCD) to perform fermentation verification of D-pantothenic acid. The formula of the fermentation medium is as follows:
LB liquid medium: 10g/L peptone, 5g/L yeast extract, 10g/L LNaCl, deionized water as solvent, and natural pH.
MS fermentation medium: glucose 20g/L, yeast extract 3g/L, (NH) 4 ) 2 SO 4 16 g/L、KH 2 PO 4 1 g/L、MgSO 4 0.5 g/L、CaCO 3 15g/L, 2.5g/L beta-alanine and 1ml/L microelement solution are dissolved by deionized water, and the pH value is not required to be regulated. The microelement solution comprises the following components: 0.15g/LNa 2 MoO 4 ·2H 2 O、2.5g/L H 3 BO 3 、0.7g/LCoCl 2 ·6H 2 O、0.25g/L CuSO 4 ·5H 2 O、1.6g/L MnCl 2 ·4H 2 O、0.3g/LZnSO 4 ·7H 2 O, the solvent is deionized water.
5L fermenter Medium: glucose 10g/L, yeast powder 8g/L and tryptone 12g/L; tripotassium phosphate 4g/L, sodium chloride 3g/L, citric acid monohydrate 2.1g/L, ferric ammonium citrate 0.3g/L, glycerin 10g/L, ammonium sulfate 2.5g/L, magnesium sulfate heptahydrate 0.5g/L, isoleucine solution (40 g/L) 4mL, microelement solution 1mL and L, and the pH is natural by dissolving with deionized water.
Feed medium: glucose 500g/L, yeast extract 12g/L, peptone 5g/L, (NH) 4 ) 2 SO 4 10 g/L、KH 2 PO 4 14 g/L、K 2 HPO 4 ·4H 2 O 5g/L、MgSO 4 8g/L, 10g/L of ammonium sulfate and 4g/L of anhydrous betaine are dissolved by deionized water.
The recombinant bacillus subtilis DPA9 of the genetically engineered bacteria is streaked on an LB solid medium plate with kanamycin resistance to separate single colonies, and the single colonies are cultured for 8 hours at 37 ℃. The single colony was transferred to a 50mL Erlenmeyer flask containing 10mL of LB liquid medium with kanamycin resistance, and cultured at 37℃for 8 hours to obtain a seed solution. 1% of the amount was inoculated into a 500mL Erlenmeyer flask containing 50mL of a MS fermentation medium having kanamycin resistance, and the supernatant was collected by centrifugation after 48 hours of fermentation culture at 37℃to measure the D-pantothenic acid yield of 6.98g/L.
Further fermentation was performed in a 5L fermenter: single colonies were inoculated into 500mL Erlenmeyer flasks containing 100mL of LB liquid medium with kanamycin resistance and cultured at 37℃for 8 hours to obtain seed solutions. 2L of fermentation medium, 1000mL of feed medium, is prepared in a 5L fermentation tank, the pH value is regulated to 6.8 by using 50% ammonia water, the seed liquid in the upper tank is inoculated according to the volume ratio of 10%, the feed medium is subjected to combined control of dissolved oxygen, the dissolved oxygen is controlled to be 20%, and the culture is carried out for 72 hours. The fermentation curve in the fermentation process is shown in FIG. 2, and the yield of D-pantothenic acid after 72 hours is 70.6g/L.
The wild-type strain B.subtilis 168 was used as a reference, and B.subtilis 168 was inoculated to ferment in a 5L fermenter in the same manner as recombinant Bacillus subtilis DPA9. Strain B.sub.168 gave a D-pantothenate yield of 0.7g/L after 72h fermentation.
The invention uses bacillus subtilis168 as chassis fungus, through modifying its metabolic pathway, firstly heterologously express ppC, aspC gene from colibacillus, then overexpress panD, panC, aspB gene in beta-alanine synthetic pathway, strengthen the synthesis of beta-alanine of precursor of pantothenic acid synthesis; secondly, a Cre/loxP gene editing technology is used for knocking out a nadB gene, so that the branch metabolic flow in the synthesis path of the beta-alanine precursor L-aspartic acid is reduced; heterologous expression of the aspA gene of the escherichia coli, and continuous enhancement of beta-alanine accumulation; fourthly, the Cre/loxP gene editing technology is used for knocking out the gabT gene, and the metabolic flow of the branched-chain organic acid synthesis pathway is further weakened on the basis of reducing the competitive branch metabolic flow; finally, the bacillus subtilis genetic engineering strain with high D-pantothenic acid yield is constructed by using plasmids to heterologously express panD genes from escherichia coli and panC genes from corynebacterium glutamicum. The construction of the genetic engineering strain solves the problem of insufficient pantothenic acid precursor beta-alanine in the metabolic process, reduces the level of exogenously added beta-alanine, and reduces the production cost. The wild-type strain B.subtilis 168 produced only 2.77g/L D-pantothenate in a 5L fermentation. The genetically engineered bacterium for high yield of D-pantothenic acid provided by the invention can improve the yield of 72. 72h D-pantothenic acid to 70.6g/L in 5L fermentation, and the yield is greatly increased.
In the bleomycin resistance plate of the present invention, the bleomycin concentration is 30 μg/mL.
The raw materials and equipment used in the invention are common raw materials and equipment in the field unless specified otherwise; the methods used in the present invention are conventional in the art unless otherwise specified.
The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and any simple modification, variation and equivalent transformation of the above embodiment according to the technical substance of the present invention still fall within the scope of the technical solution of the present invention.
Claims (8)
1. A genetically engineered bacterium for high yield of D-pantothenic acid is characterized in that: the construction method of the bacillus subtilis chassis fungus comprises the following steps:
(1) Overexpression of panD, panC, aspB gene in chassis fungus genome;
(2) Knocking out the nadB and gabT genes in the chassis fungus genome;
(3) Heterologous expression of ppC, aspC, aspA, panD gene from E.coli in Chaetomium;
(4) Heterologous expression of the panC gene from Corynebacterium glutamicum in Chaetomium.
2. The method for constructing genetically engineered bacteria of claim 1, wherein: the method comprises the following steps:
step S1: introducing ppC genes derived from escherichia coli into a chassis fungus genome by taking bacillus subtilis as the chassis fungus to obtain engineering bacteria DPA1;
step S2: introducing aspC genes derived from escherichia coli into an engineering bacterium DPA1 genome to obtain engineering bacterium DPA2;
step S3: over-expressing panD genes in the engineering bacteria DPA2 genome to obtain engineering bacteria DPA3;
step S4: over-expressing the panC gene in the engineering bacterium DPA3 genome to obtain engineering bacterium DPA4;
step S5: overexpressing aspB gene in engineering bacteria DPA4 genome to obtain engineering bacteria DPA5;
step S6: knocking out a nadB gene in the engineering bacterium DPA5 genome to obtain engineering bacterium DPA6;
step S7: inserting aspA genes from escherichia coli into an engineering bacterium DPA6 genome to obtain engineering bacterium DPA7;
step S8: knocking out the gabT gene in the engineering bacterium DPA7 genome to obtain engineering bacterium DPA8;
step S9: engineering bacteria DPA8 are used as chassis bacteria to overexpress panC genes from corynebacterium glutamicum and panD genes from escherichia coli.
3. The method of construction of claim 2, wherein: in step S1, the bacillus subtilis is Bacillus subtilis and 168.
4. The method of construction of claim 2, wherein: in step S1, the ppC gene derived from Escherichia coli is introduced into Chaetomium in the following manner: the glmS gene in the genome of Chaetomium was replaced with a ppC gene derived from E.coli.
5. The construction method according to claim 2 or 4, wherein: the promoter of the ppC gene from Escherichia coli is P 43 A promoter.
6. The method of construction of claim 2, wherein: in the step S2, the aspC gene derived from the escherichia coli is introduced into the engineering bacteria DPA1 genome in the following manner: the ybdG gene in the engineering bacterium DPA1 genome is replaced by aspC gene from escherichia coli.
7. The construction method according to claim 2 or 6, wherein: the promoter of aspC gene derived from Escherichia coli is P 43 A promoter.
8. The genetically engineered bacterium of claim 1 or the genetically engineered bacterium constructed by the construction method of any one of claims 2 to 7
Use of a program for producing D-pantothenic acid.
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