CN114134094A - Construction method of escherichia coli engineering bacteria for synthesizing riboflavin from head - Google Patents

Construction method of escherichia coli engineering bacteria for synthesizing riboflavin from head Download PDF

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CN114134094A
CN114134094A CN202111482186.5A CN202111482186A CN114134094A CN 114134094 A CN114134094 A CN 114134094A CN 202111482186 A CN202111482186 A CN 202111482186A CN 114134094 A CN114134094 A CN 114134094A
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seq
riboflavin
escherichia coli
artificial sequence
genes
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姚泉洪
彭日荷
邓永东
田永生
张文慧
许晶
王波
高建杰
韩红娟
王丽娟
王宇
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Shanghai Academy of Agricultural Sciences
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Abstract

The invention provides a construction method of an escherichia coli engineering bacterium for synthesizing riboflavin de novo, which relates to the field of genetic engineering and comprises the following steps: carrying out structure optimization on 26 genes for synthesizing riboflavin from bacillus subtilis, and carrying out chemical synthesis to obtain 26 optimized genes; selecting a T7 promoter and a terminator to control the expression of the optimized 26 genes, and completing the splicing of the optimized 26 gene prokaryotic expression units; two plasmids pBR326 and pYB8895 with different replication origins and resistance markers are constructed, and a large functional module of the riboflavin de novo biosynthetic pathway 4 is assembled on the basis of two compatible plasmids; the double plasmid with 4 large functional modules is transferred into the Escherichia coli with T7RNAP gene to obtain the Escherichia coli engineering strain which synthesizes riboflavin de novo and contains 26 gene prokaryotic expression units of bacillus subtilis. The capacity of the strain to generate riboflavin is checked by a shake flask fermentation experiment, and the yield of the riboflavin reaches 0.55g/L as finally measured by HPLC mass spectrometry.

Description

Construction method of escherichia coli engineering bacteria for synthesizing riboflavin from head
Technical Field
The invention relates to the field of genetic engineering, in particular to a construction method of an escherichia coli engineering bacterium for synthesizing riboflavin de novo.
Background
Riboflavin is one of 8B vitamins and is an essential nutrient for mammals. In the last 15 years, the chemical synthesis of riboflavin has been completely replaced by microbial synthesis, and the main production strains in the fermentation industry at present are Bacillus subtilis and Ashbya gossypii.
In recent years, the prices of domestic agricultural products and various chemical raw materials are obviously increased, and various expenses such as manpower, environmental protection and the like are increased, so that the production cost of the riboflavin is continuously increased. Therefore, it is urgent to further improve the riboflavin-producing strain and reduce the fermentation production cost.
Disclosure of Invention
The invention aims to provide a construction method of an engineering bacterium escherichia coli for synthesizing riboflavin de novo, and solves at least one technical problem proposed by the background.
A method for constructing an engineering bacterium of Escherichia coli for de novo synthesis of riboflavin, the method comprising:
carrying out structure optimization on 26 genes for synthesizing riboflavin from bacillus subtilis, and carrying out chemical synthesis to obtain 26 optimized genes;
selecting a T7 promoter and a terminator to control the expression of the optimized 26 genes, and completing the splicing of the optimized 26 gene prokaryotic expression units;
two plasmids pBR326 and pYB8895 with different replication origins and resistance markers are constructed, and a large functional module of the riboflavin de novo biosynthetic pathway 4 is assembled on the basis of two compatible plasmids;
the double plasmid with 4 large functional modules is transferred into the Escherichia coli with T7RNAP gene to obtain the Escherichia coli engineering strain which synthesizes riboflavin de novo and contains 26 gene prokaryotic expression units of bacillus subtilis.
Preferably, the 26 genes for synthesizing riboflavin of bacillus subtilis are: BsGLK1, BsZWF1, BsPgl, BsGND, BsYWLF, BsPRS, BspurF, BspurD, BspurN, BspurL, BspurQ, BspurS, BspurM, BspurK, BspurE, BspurC, BspurB, BspurH, BsguaA, BsguaB, BspMK, BssRibA, BsribB, BsribG and BsribH;
the sequence numbers thereof respectively correspond to: SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21, SEQ ID No. 23, SEQ ID No. 25, SEQ ID No. 27, SEQ ID No. 29, SEQ ID No. 31, SEQ ID No 33, SEQ ID No 35, SEQ ID No 37, SEQ ID No 39, SEQ ID No 41, SEQ ID No 43, SEQ ID No 45, SEQ ID No 47, SEQ ID No 49, SEQ ID No 51;
preferably, the 26 riboflavin-synthesizing genes of Bacillus subtilis are structurally optimized according to codon preference.
Preferably, the optimized 26 genes are:
BsGLK1S, BsZWF1S, BsPglS, BsGNDS, BsYWLFS, BsPRSS, BspurFS, BspurDS, BspurNS, BspurLS, BspurQS, BspurSS, BspurMS, BspurKS, BspurES, BspurCS, BspurBS, BspurHS, BsguaAS, BsguaBS, BspGMKS, BstNDKS, BsribAS, BsribBS, BsribGS, and BsribHS;
the sequence numbers thereof respectively correspond to: SEQ ID No 2, SEQ ID No 4, SEQ ID No 6, SEQ ID No 8, SEQ ID No 10, SEQ ID No 12, SEQ ID No 14, SEQ ID No 16, SEQ ID No 18, SEQ ID No 20, SEQ ID No 22, SEQ ID No 24, SEQ ID No 26, SEQ ID No 28, SEQ ID No 30, SEQ ID No 32, SEQ ID No 34, SEQ ID No 36, SEQ ID No 38, SEQ ID No 40, SEQ ID No 42, SEQ ID No 44, SEQ ID No 46, SEQ ID No 48, SEQ ID No 50, SEQ ID No 52.
Preferably, the 4-large function module comprises: ribulose-5-phosphate biosynthesis, hypoxanthine nucleotide biosynthesis, GTP biosynthesis and riboflavin RIB biosynthesis.
Preferably, plasmids pBR326 and pYB8895, which construct two different origins of replication and resistance markers, are two compatible plasmids: two vectors were selected, pBR322 Ori and the broad host pBBR1MCS plasmid Ori.
Preferably, the construction method further comprises verifying the integrity and expression of the introduced foreign gene and verifying the riboflavin-producing ability thereof.
The technical effects are as follows:
the invention carries out structural optimization and chemical synthesis on 26 genes of a bacillus subtilis riboflavin de novo biosynthesis pathway, creates escherichia coli engineering bacteria containing 4 functional modules and 26 gene prokaryotic expression units, finally verifies that the 26 exogenous genes carry out correct expression on transcription level through RT-PCR, further tests the riboflavin generating capacity through a shake flask fermentation experiment, and finally tests the riboflavin yield to reach 0.55g/L through HPLC mass spectrometry.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1, pBR326 vector map;
FIG. 2, pYB889 map of vectors;
FIG. 3, plasmid transformation map;
FIG. 4, PCR identification chart;
FIG. 5, graph of shake flask fermentation experiment;
FIG. 6, standard quality spectra;
FIG. 7, transformant fermentation liquid mass spectrum.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The features and properties of the present invention are described in further detail below with reference to examples.
The embodiment of the invention provides a construction method of genetic engineering escherichia coli capable of expressing and producing riboflavin, which comprises the following steps:
carrying out structure optimization on 26 genes for synthesizing riboflavin from bacillus subtilis, and carrying out chemical synthesis to obtain 26 optimized genes;
specifically, the bacillus subtilis riboflavin is selected to synthesize 26 genes BsGLK1, BsZWF1, BsPgl, BsGND, BsYWLF, BsPRS, BspurF, BspurD, BspurN, BspurL, BspurQ, BspurS, BspurM, BspurK, Bspure, BspurcC, Bspure B, BspurH, BspuaA, BsguaB, BsGMPK, BsNDK, BsRibA, BsRibB, BsRibG and BsRibH from head;
the corresponding sequence numbers are respectively: SEQ ID No 1, SEQ ID No 3, SEQ ID No 5, SEQ ID No7, SEQ ID No 9, SEQ ID No 11, SEQ ID No 13, SEQ ID No 15, SEQ ID No 17, SEQ ID No 19, SEQ ID No 21, SEQ ID No 23, SEQ ID No 25, SEQ ID No 27, SEQ ID No 29, SEQ ID No 31, SEQ ID No 33, SEQ ID No 35, SEQ ID No 37, SEQ ID No 39, SEQ ID No 41, SEQ ID No 43, SEQ ID No 45, SEQ ID No 47, SEQ ID No 49, SEQ ID No 51.
26 genes for synthesizing riboflavin of the bacillus subtilis are structurally optimized according to codon preference; gene
The chemical synthesis follows the following principles: optimizing gene codons and improving the gene translation efficiency; eliminating recognition sites of common restriction enzymes in the gene, and facilitating the construction of an expression cassette; reverse repeat sequences, stem-loop structures and transcription termination signals are eliminated, GC/AT in the gene is balanced, and the stability of RNA is improved; the intron recognition sequence is eliminated, so that the intron splicing in a coding region is avoided, and the loss of gene function is avoided; RNA makes gene-encoded protein conform to N-terminal principle (Tobias 1991) to improve the stability of translated protein; avoiding 6 or more consecutive a + T sequences, 5 or more G + C sequences; the CG and TA double oligonucleotides are used at the 2 and 3 positions, and the sequences are easy to cause methylation in plants, so that gene silencing is caused; the design improves the free energy of the 5 'end of the gene and reduces the free energy of the 3' end so as to improve the gene translation efficiency.
The optimized 26 genes are: BsGLK1S, BsZWF1S, BsPglS, BsGNDS, BsYWLFS, BsPRSS, BspurFS, BspurDS, BspurNS, BspurLS, BspurQS, BspurSS, BspurMS, BspurKS, BspurES, BspurCS, BspurBS, BspurHS, BsguaAS, BsguaBS, BspGMPKS, BstKS, BsRibAS, BsRibBS, BsRibGS, and BsRibHS;
the sequence numbers thereof respectively correspond to: SEQ ID No 2, SEQ ID No 4, SEQ ID No 6, SEQ ID No 8, SEQ ID No 10, SEQ ID No 12, SEQ ID No 14, SEQ ID No 16, SEQ ID No 18, SEQ ID No 20, SEQ ID No 22, SEQ ID No 24, SEQ ID No 26, SEQ ID No 28, SEQ ID No 30, SEQ ID No 32, SEQ ID No 34, SEQ ID No 36, SEQ ID No 38, SEQ ID No 40, SEQ ID No 42, SEQ ID No 44, SEQ ID No 46, SEQ ID No 48, SEQ ID No 50, SEQ ID No 52.
Selecting a T7 promoter and a terminator to control the expression of the optimized 26 genes, and completing the splicing of the optimized 26 gene prokaryotic expression units;
in particular, the T7 bacteriophage late transcription system is a special expression system and is the first choice system for expressing exogenous genes. The promoter is a III type promoter, and the Escherichia coli RNA polymerase cannot identify and can only be specifically identified and regulated by T7RNA polymerase (T7RNAP) coded by the phage; the T7 promoter is one of the strongest prokaryotic promoters, and the highly active T7RNAP synthesizes mRNA 5 times faster than E.coli RNA polymerase. The invention selects T7 promoter and terminator to control the expression of the above 26 chemically synthesized genes, splices the escherichia coli expression units respectively, and inserts the units into T-vector.
Two different origins of replication and resistance marker plasmids pBR326 were constructed (pBR326 vector map is shown in FIG. 1)
And pYB8895(pYB8895 vector map is shown in FIG. 2), assembling the large functional module of the de novo riboflavin biosynthesis pathway 4 on the basis of two compatible plasmids;
specifically, the embodiment of the invention obtains a full-length 4523bp plasmid pYB8895 by a whole-gene chemical synthesis technology, the plasmid has a wide-host replication origin Rep, a mob sequence of a conjugative plasmid and a Km resistance gene for bacterial screening, and the copy number of the plasmid is about 20; meanwhile, a plasmid pBR326 with the full length of 1860bp is obtained by PCR technology amplification and in vitro recombination, and the plasmid carries pBR322 ori and Ap resistance marker genes with the copy number of about 30.
The embodiment of the invention is based on two compatible plasmids pBR326 and pYB8895 to carry out functional module assembly, and utilizes an in vitro recombination technology to assemble a ribulose-5-phosphate biosynthesis module (T7PRPP) and an inosinic acid (IMP) biosynthesis functional module (T7BsIMP) between EcoRI and HindIII of a pYB326 carrier to form a plasmid pA 6428; meanwhile, a GTP biosynthesis function module (T7BSGTP) and a riboflavin RIB biosynthesis function module (T7Bsrib) are assembled between EcoRI and HindIII of pYB8895 vector, and the formed plasmid is pA 8101. The pA6428 and pA8101 are transformed into an Escherichia coli strain with a T7RNAP gene on a chromosome, and a biosynthesis system and an engineering strain for synthesizing riboflavin from the head of the Escherichia coli strain containing 4 functional modules and 26 gene prokaryotic expression units are obtained.
On the basis of completing the construction of the expression unit, 4 large functional modules [ including: ribulose-5-phosphate biosynthesis, hypoxanthine nucleotide (IMP) biosynthesis, GTP biosynthesis and riboflavin RIB operon ] were assembled from several prokaryotic expression units, the product was inserted into a T-vector, and the above 4 functional modules were cloned and nucleotide full sequence analyzed. Finally, the 4 assembled riboflavin biosynthesis functional modules are respectively named as: t7PRPP, T7 BsAP, T7BSGTP and T7 Bsrib.
The double plasmid with 4 large functional modules is transferred into the Escherichia coli with T7RNAP gene to obtain the Escherichia coli engineering strain which synthesizes riboflavin de novo and contains 26 gene prokaryotic expression units of bacillus subtilis. And analyzing and determining the riboflavin biosynthesis yield of the constructed escherichia coli engineering bacteria.
The construction method also comprises the steps of verifying the integrity and the expression of the transferred exogenous gene, verifying the riboflavin generating capacity of the exogenous gene, verifying the integrity and the expression of the exogenous gene by RT-PCR, and further verifying the riboflavin generating capacity of the exogenous gene by adopting a shake flask fermentation experiment.
The following detailed description is given with reference to specific examples:
example 1:
26 genes for synthesizing riboflavin from bacillus subtilis are subjected to structural optimization and chemical synthesis, and the optimized 26 genes are obtained:
used in the synthesis was a Phanta Max Super-Fidelity DNA Polymerase suitable for long-gene high Fidelity amplification from tokyo kexuan biotechnology limited (Vazyme Biotech co., Ltd). The PCR amplification procedure was: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 15s, annealing at 56-72 ℃ for 15s, extension at 72 ℃ for 30s-10min, and amplification for 25-35 cycles; final extension at 72 ℃ for 5 min.
Wherein, the 26 genes for synthesizing riboflavin of the bacillus subtilis are wild type genes.
The genetic chemical synthesis follows the following principles: optimizing gene codons and improving the gene translation efficiency; eliminating recognition sites of common restriction enzymes in the gene, and facilitating the construction of an expression cassette; reverse repeat sequences, stem-loop structures and transcription termination signals are eliminated, GC/AT in the gene is balanced, and the stability of RNA is improved; the intron recognition sequence is eliminated, so that the intron splicing in a coding region is avoided, and the loss of gene function is avoided; RNA makes gene-encoded protein conform to N-terminal principle (Tobias 1991) to improve the stability of translated protein; avoiding 6 or more consecutive a + T sequences, 5 or more G + C sequences; the CG and TA double oligonucleotides are used at the 2 and 3 positions, and the sequences are easy to cause methylation in plants, so that gene silencing is caused; the design improves the free energy of the 5 'end of the gene and reduces the free energy of the 3' end so as to improve the gene translation efficiency.
Example 2:
splicing gene expression unit and assembling functional module
Splicing the 26 gene prokaryotic expression units synthesized after the structure is optimized adopts an improved overlap extension PCR technology, and the improved overlap extension PCR technology is specifically disclosed in the following references: (Rihe Pen, Aisheng Xiong, Quanhong Yao; A direct and effective PAGE-mediated overlap extension PCR method for gene multiple-site mutagenesis, Applied Microbiology Biotechnology.2006,73:234-40), using the Phanta Max Super-Fidelity DNA Polymerase from Nanjing Nodezak Biotechnology Ltd (Vazyme Biotechnology Co., Ltd.) suitable for long-gene high Fidelity amplification. The PCR amplification procedure was: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 45s, annealing at 56-72 ℃ for 45s, extension at 72 ℃ for 5-20Min (according to fragment length), and amplification for 25-35 cycles; final extension at 72 ℃ for 10 min.
Then, the spliced 26 gene prokaryotic expression units are respectively connected with a pMD18-ST vector of the company TAKARA in Japan, the connector is transformed into an escherichia coli DH5 alpha competent cell, an escherichia coli transformant is selected for liquid culture, plasmid DNA is extracted for enzyme digestion identification, and the complete nucleotide sequence of an inserted fragment in the identified correct positive plasmid is determined. Finally, 26 genes of prokaryotic expression units with completely correct sequences are obtained.
The names of 26 prokaryotic expression units of synthetic genes are respectively named as: : bsglk1S, BsZWF1S, BspglS, BsgnDS, ByWLFS, BspPRSS, BspurFS, BspurDS, BspurNS, BspurLS, BspurQS, BspurSS, BspurMS, BspurKS, BspurES, BspurCS, BspurBS, BspuhS, BspuaAS, BsguaBS, BspGMPKS, BstRibAS, BsribBS, BsribGS, and BsribHS;
the sequence numbers thereof respectively correspond to: SEQ ID No 2, SEQ ID No 4, SEQ ID No 6, SEQ ID No 8, SEQ ID No 10, SEQ ID No 12, SEQ ID No 14, SEQ ID No 16, SEQ ID No 18, SEQ ID No 20, SEQ ID No 22, SEQ ID No 24, SEQ ID No 26, SEQ ID No 28, SEQ ID No 30, SEQ ID No 32, SEQ ID No 34, SEQ ID No 36, SEQ ID No 38, SEQ ID No 40, SEQ ID No 42, SEQ ID No 44, SEQ ID No 46, SEQ ID No 48, SEQ ID No 50, SEQ ID No 52.
The assembly of the individual riboflavin biosynthesis function modules likewise uses the modified overlap-extension PCR technique. Phanta Max Super-Fidelity DNA Polymerase suitable for long-gene high Fidelity amplification from Nanjing Novowed Biotech Co., Ltd is used. The PCR amplification procedure was: pre-denaturation at 95 ℃ for 30 s; denaturation at 95 ℃ for 45s, annealing at 56-72 ℃ for 45s, extension at 72 ℃ for 5-20Min (according to fragment length), and amplification for 25-35 cycles; final extension at 72 ℃ for 5 min.
Respectively connecting the assembled 4 functional module DNA long fragments with a pMD18-ST vector of TAKARA company in Japan, transforming a connector into escherichia coli DH5 alpha competent cells, selecting an escherichia coli transformant for liquid culture, extracting plasmid DNA for enzyme digestion identification, sequencing positive plasmid committee bio-engineering (Shanghai) Limited company, ensuring that a sequencing result is completely consistent with a designed sequence, and finally obtaining 4 functional modules with completely correct sequences, wherein EcoRI and HindIII cutting points are arranged at two ends of each functional module.
Embodiment 3
Coli compatible vector construction
In the present example, the entire Bacillus subtilis riboflavin biosynthesis system was constructed on two compatible plasmids, and we selected two vectors of pBR322 Ori and the broad-host pBBR1MCS plasmid Ori. Firstly, a pair of PCR amplification primers is designed and synthesized by taking a Pichia pastoris PPIC9K vector (Genbank No: Z46234) as a template:
r47312:5 '-AAG, CTT, ACC, GAA, TTC, ATG, GTT, TCT, TAG, ACG, TCA, GGT-3') and R47313 (5 '-GAA, TT C, GGT, AAG, CT T, GGA, TAA, CGC, AGG, AAA, GAA, CAT, G-3') were PCR amplified with the shaded areas of the primers as homologous regions and the amplification procedure was pre-denatured at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 45s, extension at 72 ℃ for 2min, and 35 cycles; finally, extension is carried out for 10min at 72 ℃. Detecting by 1% agarose gel electrophoresis, recovering PCR product, performing recombination cyclization by GenRec Assembly Master Mix Kit of general biological System (Anhui), Inc., preserving heat at 50 deg.C for 15-60min, placing in ice bath for 5min, and storing at-20 deg.C. When needed, the escherichia coli DH5 alpha is unfrozen and transformed, EcoRI is subjected to single enzyme digestion to identify positive clones, the committee organism engineering (Shanghai) limited company carries out sequencing on positive plasmids, the sequencing result is completely the same as the original sequence, the plasmids are named as pBR326, the length of the plasmids is only 1860bp, the plasmids are provided with pBR322 ori and Ap resistance marker genes, the copy number is higher (about 30), after chloramphenicol amplification, 3000 copies of 1000-fold can be accumulated in each cell, and the characteristic provides convenience for the preparation of recombinant plasmid DNA.
Example 4
Construction of riboflavin biosynthesis System on E.coli-compatible vectors
Firstly, respectively carrying out long primer PCR amplification on a ribulose-5-phosphate biosynthesis module (T7PRPP) and an hypoxanthine nucleotide (IMP) biosynthesis function module (T7 BsAP), using GenRec Assembly Master Mix Kit of a general biological System (Anhui) Limited company, inserting two PCR amplification products with a 24bp overlapping region between EcoRI and HindIII of a pYB326 vector through in vitro recombination, transforming escherichia coli DH5 alpha through the recombination products, extracting plasmids, carrying out double enzyme digestion identification through EcoRI and HindIII to obtain positive clones, carrying out sequencing on a committee bioengineering (Shanghai) Limited company, wherein the sequencing result is completely consistent with the designed sequence, and the finally formed plasmid is named as pA 6428.
Respectively carrying out long primer PCR amplification on a GTP biosynthesis function module (T7BSGTP) and a riboflavin RIB biosynthesis function module (T7Bsrib), using a general biological system (Anhui) Limited GenRec Assembly Master Mix Kit, inserting two PCR amplification products with a 24bp overlapping region between EcoRI and HindIII of pYB8895 vector through in vitro recombination, transforming Escherichia coli DH5 alpha through the recombination products, extracting plasmids, carrying out double enzyme digestion identification through EcoRI and HindIII to obtain positive clones, sequencing by a committee biological engineering (Shanghai) Limited company, completely conforming sequencing results to designed sequences, and finally obtaining the plasmid named pA 8101.
These two plasmids were then simultaneously transformed into BL21-AI (Cat No.6070-03) [ F' ompT hsdSB (rB-mB-) gal dcm araB:: T7RNAP-tetA ] (named EG61) E.coli competence, double plasmid transformants were selected on LB + Km + Ap solid plates and growth of the strain was observed after 12h culture at 37 ℃ as shown in FIG. 3. It is evident from the figure that the culture medium has a yellowish color, which primarily explains the biosynthesis and secretion of riboflavin.
Example 5
Further verifying the integrity and expression of the transferred exogenous gene and verifying the riboflavin generating capacity of the exogenous gene.
In order to further verify the integrity of the transferred foreign gene, 26 pairs of primers are respectively designed to perform RT-PCR verification on the transferred 26 genes. The designed primer sequences are as follows:
r50580 BsgLKSZ1 Tm 60, GGT, CTG, GTG, CTG, GGT, AAT, CTG and
r50581 BsGLKSF1 Tm 60, GCA, GTT, CTG, ATG, CTT, CAG, CCA. The product is as follows: 237 bp.
R50582 BsZWF1SZ1 Tm 60, CTG, AAC, ACT, CCT, GAA, GCA, TAC and
r50583 BsZWF1SF1 Tm 60, GTT, CCA, ATG, CAG, ACC, ATC. The product is as follows: 225bp
5.R50584＝BsPglSZ1:Tm＝60,GTG,TTC,GAA,GTG,AAC,CAG,TAC
R50585 BsPglSF1 Tm 60, CAC, TTG, ATG, CAG, GAA, CTT, CAC. The product is as follows: 237bp
R50586 BsgnDSZ1 Tm 60 ATC, GCA, CCA, TAC, TTC, ACT, GAC and
r50587 BsGNDSF1 Tm 60 AGA, CCA, GTT, AGT, ATG, GAA, CAC. The product is as follows: 237bp
R50588 BsYWLFSZ1 Tm 60, TGT, GGT, ACT, GGT, ATC, GGT, ATG and
r50589 BsYWLFSF1 Tm 60 ACC GAT ACG AGT CTG ATG ACG. The product is as follows: 222bp
R50590-BsPRSSZ 1 Tm-60 GAG, AAC, GGT, GCT, AAG, GAA, GTG and
r50591 BsPRSSF1 Tm 60 AGA, CTG, TTC, ATG, AAC, ACG. The product is as follows: 210bp
R50592 BspurFSZ1 Tm 60, ACT, CAT, GAA, GAA, CTG, ATC, GCA and
r50593 BspurFSF1 Tm 60 AGT, CAG, CAC, AGC, CTC, CAC. The product is as follows: 225bp
R50594 BspurSZ 1 Tm 60, ACT, CCT, ATC, GGT, TCA, CTT, GCT and
r50595 BspurDSF1 Tm 60, CTG, TGC, AGC, CTT, CAG, TGC, ACG. The product is as follows: 4740bp
R50596 BspurNSZ1 Tm 60, AAC, ATC, CAT, CCA, TCA, CTT, CTG and
r50597 BspurNSF1 Tm 60, ACG, GTT, CAA, ACC, CAA, CAG. The product is as follows: 234bp
R50598 BspurLSZ1 Tm 60 GAG, AAC, CTT, GGT, GCT, AAC, GTG and
r50599 BspurLSF1 Tm 60, TGC, CTT, TGA, CTT, CAG, GCA. The product is as follows: 267bp
R50600 ═ BspurQSZ1: Tm ═ 60, ACT, ATC, CCT, GTT, GCA, CAT, GGT and
r50601 ═ BspurQSF1: Tm ═ 60, TGC, AGT, CAC, ATG, AGT, CTC. The product is as follows: 273bp
R50602 ═ BspurSSZ1: Tm ═ 60, AAG, GTG, TAT, GTG, TCA and
r50603 ═ BspurSSF1: Tm ═ 60, CTG, TGC, CAC, AAC, CTC, CAC. The product is as follows: 246bp
R50604 BspurMSZ1 Tm 60 CCA, CGT, ATG, CTG, CCT, GAA, GGT and
r50605 ═ BspurMSF1: Tm ═ 60, TGC, AGC, ACC, GAA, AGT, CAC. The product is as follows: 264bp
R50606 ═ BspurKSZ1: Tm ═ 60, GAA, CAG, CAT, ATC, CGT, GCT, GTG and
r50607 ═ BspurKSF1: Tm ═ 60, CTC, AGC, TTG, ACC, ATC, ACG. The product is as follows: 270b
R50608 BspurESZ1 Tm 60, GGT, GGT, GCT, GCA, CAT, CTT, CCT and
r50609 ═ BspurESF1: Tm ═ 60, GAA, TCA, TCT, GAC, CAG, CTT, GTG. The product is as follows: 291bp
R50610 ═ BspurCSZ1: Tm ═ 60, GCA, ACT, CCT, GAA, CAG, GTG, GAG and
r50611 ═ BspurCSF1 Tm ═ 60, ATG, GAT, ACC, CAG, ACG. The product is as follows: 288bp
R50612 BspurBSZ1 Tm 60, GAG, AAC, ATG, AAG, CGT, AAC, ATG and
r50613 BspurBSF1 Tm 60 ACC CAA ACG TTC GAA GAT CAG. The product is as follows: 273bp
R50614 ═ BspurHSZ1 Tm ═ 60, GTG, GTG, AAG, CAT, GTG, AAG, TCA and
r50615 ═ BspurHSF1: Tm ═ 60, ATG, CTT, GAA, ATG, ACG, GAT, ACC. The product is as follows: 312bp
R50616 BsguaASZ1 Tm 60 GAG, ATC, GCA, AAT, CAT, GGT, CTG and
r50617 ═ BsguaASF1 ═ Tm 60, CTC, CCA, CTC, GAT, AGT, TGC, AGG. The product is as follows: 273bp
R50618 BsguaBSZ1 Tm 60, CGT, GGT, ATG, GGT, TCT, GTT, GCT and
r50619 ═ BsguaBSF1 Tm ═ 60, GAT, AGT, GTA, GTT, AGG, TGA, CTC. The product is as follows: 297bp
R50620 ═ BsGMPKSZ1 ═ Tm 60, GCA, TTC, CCT, GAG, GGT, CTG, TTC, and
r50621 ═ BsGMPKSF1 Tm ═ 60, CTC, CAG, CAT, CTT, GTA, ACG. The product is as follows: 267bp
43, R50622 ═ BstDKSZ 1: Tm ═ 60, GGT, GAA, CTG, GTT, GAG, TTC, ATC and
r50623 BsNDKSF1 Tm 60, CAT, CAG, CTG, GTA, TGA, CAC. The product is as follows: 255bp
45.R50624 BsribASZ1 Tm 60 GAG, GGT, CGT, GGT, ATC, GGT, CTG and
r50625 BsribASF1 Tm 60, GAA, ATG, CAG, ATG, ACC, CAG. The product is as follows: 315bp
R50626 bsrbsz 1 Tm 60, AAG, TCT, AAC, GCT, GTG, TAC and
r50627 ═ bsibsf 1: Tm ═ 60, ACC, GTT, CTC, AGA, CAA, GAA, TGC. The product is as follows: 303bp
R50628 ═ bsibgsz 1 Tm ═ 60, CGT, CTG, TCT, GCA, TTC, GGT, GTG and
r50629 ═ bsibbsf 1: Tm ═ 60, CTT, AGT, TGG, CTT, AGC, AGT, CAG. The product is as follows: 321bp
51, R50630 BsribHSZ1 Tm 60, ATG, GCT, GAG, ACT, AAG, AAG, TAC and
r50631 ═ bsibhsf 1: Tm ═ 60, GAA, TGA, ACG, ATT, CAG, GTT, AGC. The product is as follows: 264bp
For RNA extraction of E.coli transformants, reference is made to the molecular cloning protocols. Thereafter, cDNA was obtained using a takara RNA reverse transcription kit. Then, RT-PCR amplification was performed using cDNA as a template. Reaction system: 1. mu.L of plasmid, 4. mu.L of 2.5mmol/L dNTPs, 5. mu.L Buffer, 0.5U of Ex-Taq (Toyobo Japan), 1. mu.L of each primer, and ddH2O to 50. mu.L; the reaction procedure is as follows: 30s at 94 ℃, 30s at 54 ℃ and 30s at 72 ℃ for 45 cycles, re-extension at 72 ℃ for 10min, and photographing by gel electrophoresis (FIG. 4). Confirming that the transferred exogenous gene is correctly expressed at the transcription level.
To further verify riboflavin biosynthesis, which was possible to infer riboflavin biosynthesis, from the light yellow medium on the above-described transformed plates (FIG. 3), we picked a single colony of transformants and inoculated it into a fermentation medium: 40g/L glucose, 10g/L yeast powder, 10g/L tryptone, 13.5g/L potassium dihydrogen phosphate, 2g/L magnesium sulfate heptahydrate, 2g/L sodium chloride, 2g/L citric acid, 5mL of 200 multiplied trace elements, and adjusting the pH value to be 7; rotating speed of a shaking table: 220rpm, temperature: fermenting at 37 deg.C for 12h, sampling, performing HPLC mass spectrometry, and mass spectrometry for reference to Cabernet Sauvignon (Cabernet saururi. metabolism engineering research of riboflavin-producing engineering bacteria B. subtilis PY [ D ]. Tianjin university, 2007). As a result, as shown in FIGS. 5, 6 and 7, it was finally determined that the engineered strain could produce riboflavin of about 0.55 g/L.
The foregoing has described the general principles, principal features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are given by way of illustration of the principles of the present invention, and that various changes and modifications may be made without departing from the spirit and scope of the invention as defined by the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Sequence listing
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Claims (7)

1. A method for constructing an engineering bacterium of Escherichia coli for synthesizing riboflavin de novo, which is characterized by comprising the following steps:
carrying out structure optimization on 26 genes for synthesizing riboflavin from bacillus subtilis, and carrying out chemical synthesis to obtain 26 optimized genes;
selecting a T7 promoter and a terminator to control the expression of the optimized 26 genes, and completing the splicing of the optimized 26 gene prokaryotic expression units;
two plasmids pBR326 and pYB8895 with different replication origins and resistance markers are constructed, and a large functional module of the riboflavin de novo biosynthetic pathway 4 is assembled on the basis of two compatible plasmids;
the double plasmid with 4 large functional modules is transferred into the Escherichia coli with T7RNAP gene to obtain the Escherichia coli engineering strain which synthesizes riboflavin de novo and contains 26 gene prokaryotic expression units of bacillus subtilis.
2. The method for constructing engineering bacteria of Escherichia coli for the de novo synthesis of riboflavin according to claim 1, wherein said 26 genes of Bacillus subtilis for the synthesis of riboflavin are: BsGLK1, BsZWF1, BsPgl, BsGND, BsYWLF, BsPRS, BspurF, BspurD, BspurN, BspurL, BspurQ, BspurS, BspurM, BspurK, BspurE, BspurC, BspurB, BspurH, BsguaA, BsguaB, BspMK, BssRibA, BsribB, BsribG and BsribH;
the sequence numbers thereof respectively correspond to: SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, SEQ ID No. 11, SEQ ID No. 13, SEQ ID No. 15, SEQ ID No. 17, SEQ ID No. 19, SEQ ID No. 21, SEQ ID No. 23, SEQ ID No. 25, SEQ ID No. 27, SEQ ID No. 29, SEQ ID No. 31, SEQ ID No 33, SEQ ID No 35, SEQ ID No 37, SEQ ID No 39, SEQ ID No 41, SEQ ID No 43, SEQ ID No 45, SEQ ID No 47, SEQ ID No 49, SEQ ID No 51.
3. The method for constructing an engineered Escherichia coli for the de novo synthesis of riboflavin according to claim 1, wherein 26 genes of said Bacillus subtilis for the synthesis of riboflavin are structurally optimized according to codon preference.
4. The method for constructing engineering bacteria of Escherichia coli for the de novo synthesis of riboflavin according to claim 1, wherein said optimized 26 genes are:
BsGLK1S, BsZWF1S, BsPglS, BsGNDS, BsYWLFS, BsPRSS, BspurFS, BspurDS, BspurNS, BspurLS, BspurQS, BspurSS, BspurMS, BspurKS, BspurES, BspurCS, BspurBS, BspurHS, BsguaAS, BspUGWKS, BstNDKS, BsRibAS, BsRibBS, BsRibGS, and BsRibHS;
the sequence numbers thereof respectively correspond to: SEQ ID No 2, SEQ ID No 4, SEQ ID No 6, SEQ ID No 8, SEQ ID No 10, SEQ ID No 12, SEQ ID No 14, SEQ ID No 16, SEQ ID No 18, SEQ ID No 20, SEQ ID No 22, SEQ ID No 24, SEQ ID No 26, SEQ ID No 28, SEQ ID No 30, SEQ ID No 32, SEQ ID No 34, SEQ ID No 36, SEQ ID No 38, SEQ ID No 40, SEQ ID No 42, SEQ ID No 44, SEQ ID No 46, SEQ ID No 48, SEQ ID No 50, SEQ ID No 52.
5. The method for constructing engineering bacteria of Escherichia coli for the de novo synthesis of riboflavin according to claim 1, wherein said 4 large functional modules comprise: ribulose-5-phosphate biosynthesis, hypoxanthine nucleotide biosynthesis, GTP biosynthesis and riboflavin RIB biosynthesis.
6. The method for constructing engineered Escherichia coli for the de novo synthesis of riboflavin according to claim 1, wherein two plasmids pBR326 and pYB8895 compatible with each other, having two different origins of replication and resistance markers, are constructed: two vectors were selected, pBR322 Ori and the broad host pBBR1MCS plasmid Ori.
7. The method for constructing engineering bacteria of Escherichia coli for the de novo synthesis of riboflavin according to claim 1, further comprising verifying the integrity and expression of the introduced foreign gene and verifying its ability to produce riboflavin.
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