CN114181963A

CN114181963A - Method for improving riboflavin production capacity of escherichia coli engineering bacteria by DNA shuffling

Info

Publication number: CN114181963A
Application number: CN202111482246.3A
Authority: CN
Inventors: 邓永东; 姚泉洪; 田永生; 张文慧; 彭日荷; 许晶; 王波; 高建杰; 韩红娟; 王丽娟; 王宇
Original assignee: Shanghai Academy of Agricultural Sciences
Current assignee: Shanghai Academy of Agricultural Sciences
Priority date: 2021-12-07
Filing date: 2021-12-07
Publication date: 2022-03-15

Abstract

The invention provides a method for improving the riboflavin production capacity of escherichia coli engineering bacteria by DNA reorganization, which relates to the field of genetic engineering and comprises the following steps: constructing 26 genes of escherichia coli engineering bacteria producing riboflavin; rearranging and directionally screening the DNA of the expression unit of the T7RNA polymerase gene, and extracting to obtain a first batch of positive plasmids; DNA rearrangement and directional screening of a riboflavin biosynthesis and transport system, and extracting to obtain a second batch of positive plasmids; DNA rearrangement and directional screening of the sigma factor gene of the escherichia coli, and extracting to obtain a third batch of positive plasmids; introducing the third batch of plasmids into different chassis cells to obtain recombinant strains; selecting the positive strain with the highest riboflavin yield. Effectively eliminating the toxicity problem of T7RNAP to an escherichia coli host, the feedback inhibition problem of a product and the moderate and coordinated expression problem of a constructed riboflavin biosynthesis gene in the construction process of the riboflavin-producing escherichia coli engineering bacteria. Further improving the efficiency and the yield of riboflavin biosynthesis by the engineering strain of the escherichia coli.

Description

Method for improving riboflavin production capacity of escherichia coli engineering bacteria by DNA shuffling

Technical Field

The invention relates to the field of genetic engineering, in particular to a method for improving the riboflavin production capacity of escherichia coli engineering bacteria by DNA shuffling.

Background

On the basis of the construction of the engineering bacteria of escherichia coli for synthesizing riboflavin, two existing problems are encountered:

in a first aspect: t7 toxicity of RNAP to E.coli host. The T7 promoter is one of the strongest prokaryotic promoters, and the high-activity T7RNA polymerase can synthesize mRNA 5 times faster than the Escherichia coli RNA polymerase; meanwhile, the T7 promoter cannot be recognized by the RNA polymerase of escherichia coli, and can only be specifically recognized and regulated by the T7RNAP coded by the phage; plus the short length of both the T7 promoter and terminator. In view of the characteristics and advantages, the T7 expression system becomes the first choice for constructing a high-expression exogenous gene and prokaryotic synthetic biology system. However, T7RNAP is highly toxic to E.coli, which makes the application of this system difficult.

In a second aspect: feedback inhibition of the product, moderation and coordinated expression of the riboflavin biosynthesis genes. In the process of creating a complex synthetic biology system, it is very important to relieve the feedback inhibition of product accumulation, in addition, the high expression of exogenous genes is often unfavorable for the biosynthesis of final products, and the expression of each gene cannot be at the same level, but exists in a certain proper proportion.

In order to further improve the efficiency and the yield of the riboflavin biosynthesis of the escherichia coli engineering strain, the invention further optimizes a DNA rearrangement and directional screening system to improve the reservoir building capacity and the screening efficiency, and simultaneously carries out genome DNA rearrangement and directional screening of the engineering strain to solve the problems of toxicity of T7RNAP to an escherichia coli host, the feedback inhibition of products and the moderate and coordinated expression of the riboflavin biosynthesis gene.

Disclosure of Invention

The invention aims to provide a method for improving the riboflavin production capacity of escherichia coli engineering bacteria by DNA shuffling, and solves at least one technical problem provided by the background.

A method for improving the riboflavin production capacity of engineering bacteria of Escherichia coli by using DNA shuffling, which comprises the following steps:

constructing 26 genes of escherichia coli engineering bacteria producing riboflavin;

rearranging and directionally screening DNA of a T7RNA polymerase gene expression unit to obtain a first batch of positive strains, and extracting to obtain a first batch of positive plasmids;

DNA rearrangement and directional screening of a riboflavin biosynthesis and transport system to obtain a second batch of positive strains, and extracting to obtain a second batch of positive plasmids;

DNA rearrangement and directional screening of the sigma factor gene of the escherichia coli to obtain a third batch of positive strains, and extracting to obtain a third batch of positive plasmids;

introducing the third batch of plasmids into different chassis cells to obtain recombinant strains; and (3) performing test tube liquid culture on the recombinant strains, analyzing and determining the riboflavin biosynthesis yield of each engineering strain, and selecting the positive strain with the highest yield.

Preferably, the method for DNA rearrangement and directional screening of the expression unit of the T7RNA polymerase gene comprises the following steps:

chemically synthesizing a T7RNAP gene expression unit under the control of a lac promoter with a lacI repressor protein and a T1 terminator;

performing primary screening according to the yellow shade of the transformant colony;

selecting dark yellow escherichia coli positive colonies for liquid culture, and accurately determining the riboflavin content in the culture solution by using an HPLC (high performance liquid chromatography) technology;

obtaining a first batch of positive strains through rearrangement, library establishment and directional screening, and extracting to obtain a first batch of positive plasmids.

Preferably, the method for DNA rearrangement and targeted screening of the riboflavin biosynthesis and transport system comprises:

mixing the first batch of positive plasmids for enzyme digestion, and constructing a mutation element library on the mixed vector; transforming escherichia coli, and performing primary screening according to the yellow shade of a transformant colony;

and obtaining a second batch of positive strains with obviously improved riboflavin yield by rearrangement, library establishment and directional screening, and extracting to obtain a second batch of positive plasmids.

amplifying and cloning the sigma factor EcRpoD gene of the escherichia coli by a PCR technology to complete nucleotide complete sequence analysis;

eliminating common restriction enzyme cutting points inside the gene;

carrying out high-efficiency mutation on the gene by using a DNA shuffling technology;

constructing a mutant gene library on a second set of positive plasmids;

e, transforming the escherichia coli by electric shock, and performing primary screening according to the light color of a colony of a transformant; then selecting dark yellow escherichia coli positive colonies for liquid culture, and accurately determining the riboflavin content in the culture solution by using an HPLC (high performance liquid chromatography) technology;

and obtaining a third batch of positive strains with remarkably improved riboflavin yield by rearrangement and directional screening.

Preferably, the escherichia coli underplate cells comprise:

DH5 alpha, HB101, JM105, JM109-DE3, C600, RR1, XL1-Blue, BL21, BL21-DE3, EG61-BL21-AI, BL21-DE3-PlysS, ER2566, IJ1127, MV1190, DH10B, MC8, JM83, SURE, electrochen-Blue, XL10-Gold, Omnimax 2T1 and TOP 10;

the technical effects are as follows:

the invention effectively eliminates the toxicity problem of T7RNAP to the escherichia coli host, the feedback inhibition problem of the product and the moderate and coordinated expression problem of the constructed riboflavin biosynthesis gene in the construction process of the riboflavin-producing escherichia coli engineering bacteria. Further improving the efficiency and the yield of the riboflavin biosynthesis by the escherichia coli engineering strain, and finally improving the riboflavin yield by 161.8 percent compared with the original strain.

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 shows DNA rearrangement of lacpol1 gene

FIG. 2 screening of mutant lacpol1 transformation plates

FIG. 3 shows riboflavin content of lacpol1 mutant gene

FIG. 4 shows DNA rearrangement of T7Guarib gene

FIG. 5 mutant operon transformation plate screening

FIG. 6 shows the riboflavin content of the mutant gene

FIG. 7 shows DNA rearrangement of E.coli EcRpoDS gene

FIG. 8 mutant transformation plate Screen

FIG. 9 shows the riboflavin content of the mutant gene

FIG. 10 transformation of shake flask fermentation broth by obstructed base plate cells

FIG. 11.5 is a graph showing the content of riboflavin in the cells of the dark background

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 DNA rearrangement and directional screening method for improving the yield of riboflavin-producing escherichia coli engineering bacteria, which comprises the following steps:

DNA rearrangement and directional screening of a riboflavin biosynthesis and transport system to obtain a second batch of positive strains with significantly improved riboflavin yield, and extracting to obtain a second batch of positive plasmids;

DNA rearrangement and directional screening of the sigma factor gene of the escherichia coli to obtain a third batch of positive strains with obviously improved riboflavin yield, and extracting to obtain a third batch of positive plasmids;

The T7 promoter was used to control the expression of genes in the riboflavin biosynthetic pathway of Bacillus subtilis. Since Escherichia coli does not contain T7RNA polymerase, and needs to introduce exogenous T7RNA polymerase into host bacteria, the whole de novo biosynthesis pathway of riboflavin is totally controlled by T7RNA polymerase, and the expression level of enzyme and riboflavin transporter can be controlled by controlling the expression level and activity of T7RNA polymerase. Since the highly active T7RNA polymerase synthesizes mRNA 5 times faster than the E.coli RNA polymerase, the target protein can usually account for more than 50% of the total cell protein after several hours of induced expression. Therefore, in order for the T7 system to be commercially useful in synthetic biology, it is necessary to precisely regulate the activity of T7RNA polymerase. The embodiment of the invention firstly chemically synthesizes a lac promoter with lacI repressor protein and a T7RNAP gene expression unit controlled by a T1 terminator, carries out DNA rearrangement of the T7RNAP expression unit, constructs a mutation unit library on the basis of pA8101 plasmid, converts the competence of Escherichia coli DH10B [ pA6428 ], and carries out primary screening according to the yellow shade of a transformant colony; selecting a relatively dark yellow escherichia coli positive bacterial colony for liquid culture, and accurately determining the riboflavin content in the culture solution by using an HPLC (high performance liquid chromatography) technology; finally, a batch of positive strains are obtained through mutation, library establishment and directional screening for several rounds, and a batch of positive plasmids are obtained through extraction.

In order to reduce the feedback inhibition of the product on the enzyme in the riboflavin biosynthesis pathway, simultaneously realize the precise coordinated expression of the enzyme in the riboflavin biosynthesis pathway and further improve the riboflavin yield of the escherichia coli positive strain. The invention designs the long DNA rearrangement of the bacillus subtilis riboflavin biosynthesis system; mixing the first batch of positive plasmids for enzyme digestion, and constructing a mutation element library on the mixed vector; transforming escherichia coli, and performing primary screening according to the yellow shade of a transformant colony; then selecting a dark yellow escherichia coli positive bacterial colony for liquid culture, and accurately determining the riboflavin content in the culture solution by using an HPLC (high performance liquid chromatography) technology; and obtaining a second batch of positive strains with obviously improved riboflavin yield through several rounds of recombination, library establishment and directional screening, and extracting to obtain a second batch of positive plasmids.

Coli sigma factor is a cofactor for all RNA polymerases, which is an intrinsic component of DNA-dependent RNA polymerases, and sigma factor usually binds to DNA and searches along the DNA until it hits the core enzyme on the promoter, thereby effecting regulation of the overall transcription level of e. In order to realize the optimization of the overall transcription level of the escherichia coli genome, the invention designs that the rapid optimization of a biosynthesis pathway is realized through a global transcription regulation mechanism, the overall transcription level of cells is changed by changing the transcription efficiency of RNA polymerase and the affinity to a promoter, and the positive genes of which the overall transcription level of the genome is favorable for riboflavin biosynthesis are finally obtained by screening in combination with the directional screening of the riboflavin content. Firstly, amplifying and cloning a sigma factor EcRpoD gene of escherichia coli by a PCR technology to complete nucleotide complete sequence analysis; eliminating common restriction enzyme cutting points inside the gene; carrying out high-efficiency mutation on the gene by using a DNA shuffling technology; constructing a mutant gene library on a second set of positive plasmids; e, transforming the escherichia coli by electric shock, and performing primary screening according to the yellow shade of a transformant colony; then selecting a dark yellow escherichia coli positive bacterial colony for liquid culture, and accurately determining the riboflavin content in the culture solution by using an HPLC (high performance liquid chromatography) technology; a third batch of positive strains with significantly improved riboflavin production was obtained by several rounds of mutation, pooling and directed screening.

Selecting 22 Escherichia coli underpan cells [ including DH5 alpha, HB101, JM105, JM109(DE3), C600, RR1, XL1-Blue, BL21, BL21(DE3), EG61[ BL21(AI) ], BL21(DE3) PlySS, ER2566, IJ1127, MV1190, DH10B, MC8, JM83, SURE, ElectroTen-Blue, XL10-Gold, Omnimax 2T1 and TOP10] which comprise EG61([ BL21(AI) ], transforming the constructed plasmid of the riboflavin biosynthesis system into different underpan cells to obtain recombinant strains; and (3) performing test tube liquid culture on each recombinant strain, and analyzing and determining the riboflavin biosynthesis yield of each engineering strain to finally obtain a positive strain with remarkably improved yield.

The following is further illustrated in conjunction with the detailed examples:

the specific method for constructing the riboflavin-producing escherichia coli engineering bacteria containing 26 genes comprises the following steps:

the 26 genes required for the first synthesis of riboflavin were synthesized by Nanjing Nodezam Biotech Co., Ltd (VazymeBiotech Co., Ltd.) and 26 genes were Bsglk1, BszWF1, Bspgl, BsgD, ByWLF, BspS, BspurF, BspurD, BspurN, BspurL, BspuQ, Bspus, BspumM, BspurK, Bspure, BspurcC, BspurB, BspurH, BsguaA, BsguaB, BspGMPK, BstNDK, BsriA, BsribB, BsribG and BsribH, and then the spliced 26 gene prokaryotic expression units were ligated to pMD 18-KAST vectors of Japanese TAKARA, and two vectors of pBR322 Ori and pBBR1 Ori were selected, respectively. These two plasmids were then simultaneously transformed into BL21-AI (Cat No.6070-03) [ F' ompT hsdSB (rB-mB-) gal dcm araB:: T7RNAP-tetA ] (nomenclature EG61) E.coli competence and double plasmid transformants were selected on LB + Km + Ap solid plates.

Splicing 26 gene prokaryotic expression units and assembling 4 riboflavin biosynthesis function modules, selecting a T7 promoter and a terminator to control the expression of the 26 chemical synthesis genes, and completing the splicing of the 26 gene prokaryotic expression units; two plasmids pBR326(1860bp, Apr) and pYB8895(4523bp, Kmr) with different origins of replication 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, and comprises the following components: ribulose-5-phosphate biosynthesis, hypoxanthine nucleotide (IMP) biosynthesis, GTP biosynthesis and the riboflavin RIB operon. Assembling a ribulose-5-phosphate biosynthesis module (T7PRPP) and an inosinic acid (IMP) biosynthesis function module (T7BsIMP) between EcoRI and HindIII of pYB326 carrier by using an in vitro recombination technology 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 Escherichia coli strain with a T7RNAP gene on the chromosome is transformed by pA6428 and pA8101, so that the Escherichia coli engineering strain of a biosynthesis system for synthesizing riboflavin from the beginning, which contains 4 functional modules and 26 prokaryotic gene expression units, is obtained, and the engineering strain is used as an original strain.

Further, the method for rearranging and directionally screening the DNA of the expression unit of the T7RNA polymerase gene comprises the following steps:

the embodiment of the invention firstly designs an expression unit LacRNAP controlled by a prokaryotic lac promoter, and firstly carries out the codon and structure optimization of escherichia coli on a lacI repressor gene and a T7RNAP gene, and the optimization follows the following principle: 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; the protein coded by the RNA initiation gene conforms to the N-terminal principle (Tobias1991) so as to improve the stability of the 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, 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 codon and structure optimized lacI and T7RNAP genes are respectively named as lacIS and T7 RNAPS, on the basis of completing the design of the two genes, a T7 RNAPS gene expression unit lacpol1 is chemically synthesized, the lacIS gene expression is controlled by the lacI original promoter PlacI and terminator lacI-ter, the T7 RNAPS gene expression is controlled by the lac promoter and T1 terminator, the whole expression unit has the full length of 4536bp (SEQ ID No.1), and the full-length lacpol1 sequence only contains one NdeI cut point inside, so that the subsequent genetic operation is convenient.

To achieve commercial application of the T7 system in synthetic biology, the activity of T7RNAP was precisely regulated. Based on the above known sequence (SEQ ID No.1), two primers were designed for the construction of the mutation library, and the PCR amplification primers were R45521(5 '-GTC, TTG, AGG, GGT, TTT, TTG, GTC, GAC, CCA, GAA, GCA, TTG, GTG, CAC, CG T, GC-3'), and R45522(5 '-CGC, TGG, CGA, TTC, AGG, TTC, AAG, CTT, GGT, GTC, GAC, CTA, CTC, AGG, AGA, GCG, TTC, ACC, GAC-3'), and the italic part was the homologous fragment, and the present example used Ex-taq for PCR amplification using the following PCR reaction program: pre-denaturation at 94 ℃ for 10min, denaturation at 94 ℃ for 45s, annealing at 60 ℃ for 50s and extension at 72 ℃ for 5min for 30 cycles, and final extension at 72 ℃ for 10 min. PCR product 0.7% Agrose electrophoresis, and a product of about 4.5kb was recovered by the transpipette bag method. The recovered lacpol1 expression unit was dissolved in 100. mu.L of DNase I buffer (50mmol/L Tris-ClpH7.4+1mmol/L MgCl2), 0.1U of DNase I was added, treated at 25 ℃ for 15min, heated at 70 ℃ for 10mon to inactivate the DNase I, the mixture was subjected to 10% acrylamide gel electrophoresis, and 500-and 1000-bp fragments were recovered by the transpipette-bag method, as shown in FIG. 1 left and FIG. 1. The pellet was dissolved with 10. mu.L of 10 × Primerless PCR Buffer (Primerless PCR Buffer) (50mmol/LKCl +10mmol/LTris-Cl pH9.0+ 1% Triton). Then Primerless PCR amplification is carried out, and the reaction system: mu.L of small fragment DNA +4. mu.L of 2.5mmol/L dNTPs + 4.5. mu.L of 25mmol/L MgCl₂+Taq2U+ddH₂O to 50μL；

The reaction procedure is as follows: 94 ℃ 40S, 40 ℃ 50S, 72 ℃ 1min, 20 cycles in total), and whether the PCR amplification product is accurate or not is detected by 0.7% agarose electrophoresis. And finally performing Primer PCR amplification. Reaction system: mu.L Primerless PCR product + R455210.2ng + R455220.2ng +10 XPCR Buffer 5. mu.L +2.5mmol/L dNTPs 4. mu.L + Ex-taq2U + ddH2O to 50. mu.L. The reaction procedure is as follows: at 94 ℃ for 1min, at 60 ℃ for 1min and at 72 ℃ for 10min, for 35 cycles, carrying out 0.7% agarose electrophoresis detection, and recovering a 4.5Kb DNA fragment, as shown in the right part of the figure 1.

The recovered PCR product was recombined in vitro with the carrier pA8101 ═ pYB8895[ T7BSGTP + T7BsRib ] for riboflavin biosynthesis by the in vitro recombination method and SalI + HindIII double-digested E.coli using the Clonexpress one-step seamless cloning kit from Vazyme Biotech Co according to the instructions, and the reaction system: 15-20bp perfect consensus sequence, linearized vector: 50-200ng insert 20-200ng, 5 XCEII Buffer 4. mu.L, Exnase II 2. mu.L, supplemented with ddH2O to 20. mu.L. After 30min at 37 ℃ the cells were placed in an ice bath for 5min and subsequently stored at-20 ℃ and, if necessary, thawed for transformation.

The recombinant product obtained above was transformed into chemical competence of E.coli DH10B [ pA6428 ] carrying no T7RNAP gene on its chromosome, and the transformant was plated on LB solid medium and grown overnight at 37 ℃ as shown in FIG. 2. Further, a batch of mutants are obtained by primary screening according to the yellow shade of the colony of the mutant, and are randomly named as mutant 1, mutant 3, mutant 6, mutant 9 and mutant 12.

Carrying out shake flask liquid culture on the escherichia coli positive mutant 1, the mutant 3, the mutant 6, the mutant 9 and the mutant 12, taking the original strains as a control group, and carrying out liquid culture on a culture 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; and the riboflavin content was measured by HPLC, as a method reference (research on metabolic engineering of Scirpus canescens B. subtilis PY [ D ]. Tianjin university, 2007), as shown in FIG. 3, it was calculated that the highest of the mutant strains 9 among these mutant strains could produce riboflavin of about 0.68 g/L. Then, a plasmid of mutant strain 9 was extracted, and the lacPol1 mutant gene of this mutant strain (mutant strain 9) was subjected to nucleotide full sequence analysis (SEQ ID NO.2) and designated as lacPol1 a. Through sequence alignment, two amino acid site mutations are observed inside the lacI, namely S93L and S288L, and the mutation of the two amino acid sites is supposed to be related to the super repression of the lacI.

The bacterial liquid of a plurality of selected mutant strains is subjected to riboflavin content measurement, and the calculation shows that the mutant strain 9 can produce the highest riboflavin of about 0.68 g/L. The lacPol1 mutant gene of this mutant strain (mutant strain 3) was then subjected to nucleotide full sequence analysis (SEQ ID No.2) and designated lacPol1 a. Through sequence alignment, two amino acid site mutations are observed inside the lacI, namely S93L and S288L, and the mutation of the two amino acid sites is supposed to be related to the super repression of the lacI.

Further, the DNA rearrangement and directional screening method of the riboflavin biosynthesis system comprises the following steps:

the embodiment of the invention chemically synthesizes a long operon T7Guarib (SEQ ID No.3), which comprises 8 genes in 2 functional modules (T7BSGTP and T7Bsrib) of a subtillis riboflavin biosynthesis system, and comprises the following steps: BsguaS, BsguaBS, BsgmPGS, BstNDKS, BsribAS, BsribBS, BsribGS and BsribHS, and also BsribU gene which functions as efflux of riboflavin efflux, a total of 9 genes, the long operon controlled by T7 promoter and terminator, and T7 SD sequence connection between the genes.

First of all by

R47390(5 '-CGC, TCT, CCT, GAG, TAG, GTC, GAC, CGA, TCC, CGC, GAA, ATT, AAT, ACG, ACT, CA-3') and R47399(5 '-CGC, TGG, CGA, TTC, AGG, TTC, AAG, CTT, GGT, GTC, GAC, GGA, TCC, AAA, AAA, CCC, CTC, AAG, ACC, CGT-3') pair of primers, PCR amplified using Ex-taq, the reaction conditions being: pre-denaturation at 94 deg.C for 10min, denaturation at 94 deg.C for 1min, annealing at 60 deg.C for 1min and extension at 72 deg.C for 12min for 30 cycles, and final extension at 72 deg.C for 10 min. The PCR product was subjected to 0.7% agarose electrophoresis and the 8319bp PCR product was recovered by the transpipette bag method. The recovered T7Guarib DNA fragment was digested with 100. mu.L of DNase I buffer (50mmol/L Tris-ClpH7.4+1mmol/L MgCl)₂) Dissolve, add 0.1U DNase I, and treat for 15 minutes at 25 ℃. The treatment was carried out at 70 ℃ for 10 minutes. 10% acrylamide electrophoresis, and recovering the 500-1500bp fragment by the transsuction bag method (FIG. 4, left and FIG. 4). The pellet was dissolved with 10. mu.L of 10 × Primerless PCR Buffer (Primerless PCR Buffer) (50mmol/LKCl +10mmol/L Tris-Cl pH9.0+ 1% Triton). Then Primerless PCR amplification is carried out, and the reaction system: mu.l of small fragment DNA +4. mu.l of 2.5mmol/L dNTPs + 4.5. mu.l of 25mmol/LMgCl₂+Taq2U+ddH₂O to 50. mu.l; the reaction procedure is as follows: 94 ℃ 40S, 40 ℃ 50S, 72 ℃ 1min, 20 cycles in total), and detecting the PCR amplification result by 0.7% agarose electrophoresis. Finally, PrimerPCR amplification reaction is carried out. Reaction system: mu.l Primerless PCR product + R473900.2ng + R473990.2ng +10 XPCR Buffer 5. mu.L +2.5mmol/L dNTPs 4. mu.L + Ex-taq2U + ddH₂O to 50. mu.L. The reaction procedure is as follows: at 94 ℃ for 1min, at 60 ℃ for 1min and at 72 ℃ for 10.0min for 35 cycles, carrying out 0.7% agarose electrophoresis detection, and recovering a DNA fragment of 8.3Kb (right in FIG. 4).

To facilitate DNA rearrangement, library construction and directed screening of the T7Guarib operon, the examples of the present invention re-PCR amplify the already screened lacpol1a mutant gene with primers R45581(5 '-CGA, ATT, TTA, ACA, AAA, TAT, TAA, CGC, GAA, TTC, CCA, GAA, GCA, TTG, GTG, CAC, CGT, GCA-3') and R45582(5 '-CGC, TGG, CGA, TTC, AGG, TTC, AAG, CTT, GGT, GTC, GAC, CTA, CTC, AGG, AGA, GCG, TTC, ACC, GAC-3') and insert pYB8895 plasmid by recombination and complete the full sequence determination to constitute lac plasmid pYB8895[ 1a ]. The recovered T7Guarib DNA rearrangement product was ligated with the pYB8895[ lacpol1a ] vector digested with SalI + HindIII by in vitro recombination, and E.coli DH10B [ pA6428 ] was transformed after 1h of recombination to obtain a batch of mutants as shown in FIG. 5.

Then, a batch of escherichia coli positive mutants with dark yellow colors are picked, randomly named as a mutant 21, a mutant 25, a mutant 29, a mutant 32 and a mutant 36, liquid culture is carried out, the mutant 9 and the original strain are used as controls, and the content of riboflavin in the culture solution of the mutant strains is accurately determined by using an HPLC (high performance liquid chromatography) technology. It was calculated that the highest of mutant 25 strains among these mutant strains could produce riboflavin of about 0.88g/L, as shown in FIG. 6.

Subsequently, plasmid extraction was performed on this mutant 25 strain with the highest riboflavin content, and nucleotide complete sequence analysis (SEQ ID No.4) was performed. Through sequence alignment, 9 amino acid site mutations are observed inside the gene, namely E127D of BsguaS gene, K125R of BsguaBS gene, K151Q of BsgGMPKS gene, N103K of BstNDKS gene, E175D of BsribAS gene, L132V and K79Q of BsribBS gene and Q129E of BsribGS gene, and the mutant amino acid sites are presumed to improve the yield of riboflavin in the mutant 1 strain through synergism, and the mutant operon is named as T7 Guaribm.

Further, the DNA rearrangement and directional screening method of the sigma factor gene of the escherichia coli comprises the following steps:

the E.coli sigma factor EcRpoD is a cofactor for all RNA polymerases, which is an intrinsic component of DNA-dependent RNA polymerases, and the sigma factor usually binds to DNA and searches along the DNA until it hits the core enzyme on the promoter, thus achieving regulation of the overall transcription level of E.coli. In order to realize the optimization of the overall transcription level of the escherichia coli genome, the research design realizes the rapid optimization of a biosynthesis pathway through a global transcription regulation mechanism, the overall transcription level of cells is changed by changing the transcription efficiency of RNA polymerase and the affinity to a promoter, and the positive genes of which the overall transcription level of the genome is favorable for riboflavin biosynthesis are finally obtained by screening in combination with the directional screening of the riboflavin content.

Firstly, designing a pair of primers:

(R27304:5 '-GAA, TTC, GGA, TCC, ATG, GAG, CAA, AAC, CCG, CAG, TCA, CAG-3' and R27305: 5 '-AAA, GAG, CTC, TTA, ATC, G TC, CAG, GAA, GCT, ACG, CAG-3'), using the extracted Escherichia coli genome DNA as a template, using Ex-Taq to amplify a product with a length of about 1800bp, the reaction program is: 50s at 94 ℃, 50s at 60 ℃ and 2min at 72 ℃ for 30 cycles, and final extension is 5min at 72 ℃. Subsequently, 0.7% Agrose electrophoresis detection is carried out, a DNA fragment of about 1.8Kb is recovered, a PCR product is subjected to 1% agarose gel electrophoresis, the product is recovered and is connected with a pMD18-ST vector of TAKARA company in Japan, a connector is transformed into escherichia coli DH5 alpha, bacterial liquid is coated on an LB solid plate, a white bacterial colony is selected to be cultured in an LB liquid culture medium overnight, thalli are collected by centrifugation, plasmids are extracted by an alkaline method, EcoRI + SacI are used for double enzyme digestion identification, and the correctly identified positive plasmids are sent to Hisheng Biotech limited company for nucleotide complete sequence analysis of an insert fragment. The result showed that the insert sequence of positive clone YN2098 completely agreed with the E.coli EcRpoD gene sequence.

Because a BamHI cleavage point exists inside the EcRPOD gene in the YN2098 plasmid, the subsequent genetic operation is influenced. Designing a pair of mutation primers near a BamHI cleavage point:

R38046(5’-GAT,CGC,CTG,ACG,AAT,CCA,CCA,GGT,TGC-3’)

and R38047(5-TGG, TGG, ATT, CGT, CAG, GCG, ATC, ACC, CGC, TCT-3'), eliminating the BamHI site inside the gene by the "overlap extension PCR technique" to obtain the correct positive plasmid YN5383, and the EcRpoD gene with the eliminated internal site is named as EcRpoDS (SEQ ID No. 5).

The EcRpoDS gene was subsequently amplified using Ex-taq using R27304 and R27305 primers under the following reaction conditions: pre-denaturation at 94 ℃ for 10min, denaturation at 94 ℃ for 50S, annealing at 60 ℃ for 50S and extension at 72 ℃ for 2min for 30 cycles, and final extension at 72 ℃ for 10 min. The PCR product is subjected to 0.7% Agrose electrophoresis, and the PCR product of about 1850bp is recovered by a transudatory bag method. The recovered DNA fragment was dissolved in 100. mu.L of DNase I buffer (50mmol/L of LTris-ClpH7.4+1mmol/L of MgCl2), 0.1U of DNase I was added, and the mixture was treated at 25 ℃ for 15 min. Treating at 70 deg.C for 10 min. 10% acrylamide electrophoresis and a suction bag method are adopted to recover the 50-100bp fragment. Using 10. mu.L of 10 Xprimerless PCR Buffer (Primerless PCR Buffer) (50mmol/L KCl +10mmol/LTris-Cl pH9.0+ 1% Triton) dissolving the precipitate. Then Primerless PCR amplification is carried out, and the reaction system: mu.L of small fragment DNA +4. mu.L of 2.5mmol/LdNTPs + 4.5. mu.L of 25mmol/LMgCl₂+ Taq2U + ddH2O to 50. mu.L; the reaction procedure is as follows: 94 ℃ 40S, 40 ℃ 50S, 72 ℃ 1min, 20 cycles in total), and 0.7% agarose electrophoresis to detect the PCR amplification results (FIG. 7, bottom left). Finally, PrimerPCR amplification reaction is carried out. Reaction system: mu.l Primerless PCR product + R273040.2ng + R273050.2ng +10 XPCR Buffer 5. mu.L +2.5mmol/L dNTPs 4. mu.L + Ex-taq2U + ddH2O to 50. mu.L. The reaction procedure is as follows: 50S at 94 ℃, 50S at 60 ℃ and 2.0min at 72 ℃ for 35 cycles, and recovering a DNA fragment of 1.84Kb by 0.7% agarose electrophoresis detection, as shown in the lower right part of FIG. 7.

BamHI + SacI double enzyme digestion is carried out on the gene rearrangement product overnight, a library is built on a prokaryotic expression vector G251 (the vector is a prokaryotic expression vector, a pBR322 replication origin, and has PGm promoter and T1 terminator, and a screening marker is Ap resistance), a DH10B escherichia coli strain with pYB8895[ lacT7po l1a + T7Guaribm ] is transformed, and a bacterial colony with dark yellow is screened on an LB + Km + Ap plate with 0.5mM IPTG, as shown in figure 8.

Selecting a batch of escherichia coli positive mutants with dark yellow, randomly naming the mutants as 42, 45, 49, 53 and 56 to be cultured in liquid, and taking the mutant 25 and the original strain as controls; and the riboflavin content in the culture solution is accurately determined by using an HPLC technology. It was calculated that riboflavin was produced at the highest in about 1.14g/L in the mutant 53 strain among these mutant strains, as shown in FIG. 9.

This mutant 53 strain was then subjected to plasmid extraction and nucleotide full sequence analysis (SEQ ID No. 6). Through sequence alignment, 2 amino acid site mutations are observed in the gene, namely D96H and S366A, and the two mutation sites are supposed to act synergistically so that the capacity of riboflavin biosynthesis is enhanced by changing the transcription efficiency of RNA polymerase and the affinity capacity to a promoter.

Further, the adaptation of a riboflavin biosynthesis system and the underpan cells, namely, introducing a third batch of plasmids into different underpan cells to obtain recombinant strains; and (3) performing test tube liquid culture on the recombinant strains, analyzing and determining the riboflavin biosynthesis yield of each engineering strain, and selecting the positive strain with the highest yield.

Taking the EcRpoDS mutant gene expression plasmid G251[ EcRpoDS45] obtained by the previous step of screening as a template, respectively designing a pair of primers at the 5 'end of a prokaryotic PGm promoter and the 3' end of a T1 terminator for PCR amplification, wherein the amplification product is a PGm + EcRpoDS45+ T1 expression unit, which is named as PGmEcRp oDS45T1, and the primers are as follows:

r45589(5 '-GTC, TTG, AGG, GGT, TTT, TTG, GTC, GAC, GCA, CAC, CGT, GGA, AAC, GGA, TG-3'), and

r45590(5 '-TGC, CGC, TGG, CGA, TTC, AGG, TTC, AAG, CTT, GGT, GTC, GAC, CTA, CTC, AGG, AGA, GCG, TTC, ACC, GAC-3'), using Phanta Max super R-Fidelity DNA Polymerase suitable for long gene high Fidelity amplification, PCR amplification program: pre-denaturation at 95 ℃ for 40 s; denaturation at 95 ℃ for 45s, annealing at 60 ℃ for 45s, extension at 72 ℃ for 3min, and amplification for 25 cycles; final extension at 72 ℃ for 10 min. The PCR product is subjected to 1% agarose gel electrophoresis, a product of about 2400bp is recovered, a plasmid pYB8895[ lacpol1a + T7Guaribm ] is inserted into the PCR product by an in vitro recombination method to form a new recombinant plasmid pYB8895[ lacpol1a + T7Guaribm + PGmEcRpoDS45T1 ], and the complete sequence determination is completed.

Finally pYB8895[ lacT7pol1a + T7Guaribm + PGmEcRpoDS45T1 ] and pA6428 were mixed for double plasmid transformation of E.coli. 22 E.coli underpan cells [ including DH 5. alpha., HB101, JM105, JM109-DE3, C600, RR1, XL1-Blue, BL21, BL21-DE3, EG61-BL21-AI, BL21-DE3-PlysS, JM 2566, IJ1127, MV1190, DH10B, MC8, JM83, SURE, electroTen-Blue, XL10-Gold, Omnimax 2T1, TOP10] which include EG61-BL21-AI different strains were selected and transformed to conduct suitability studies of the riboflavin biosynthesis system and underpan cells, wherein DH 5. alpha, HB101, JM105, JM109-DE3, TOP10 strains were kept in this laboratory. C600, RR1, XL1-Blue, BL21, BL21-DE3, EG61-BL21-AI, BL21-DE3PlysS were purchased from Shanghai Weidi Biotechnology Ltd. MV1190, DH10B, MC8, JM83, SURE, XL10-Gold, ElectroTen-Blue, ER2566, IJ1127, Omnimax 2T1 were purchased from Biotech, Inc., NEB and INVITROGEN. Streaking was then performed as per the instructions for the purchased strain, and the next day a single colony was picked for routine chemical competent preparation, followed by routine competent transformation. Individual transformants were picked and inoculated into 10mL LB medium, 37 ℃ overnight on a shaker. Inoculating 1mL of bacterial liquid into 50mL of LB liquid medium, culturing for 6-8h at 37 ℃ in a shaking table to enable the bacterial liquid OD600 to reach about 0.75, and preliminarily judging which strain has high riboflavin yield according to the shade of the bacterial liquid, as shown in figure 10.

As can be seen from the color of the bacterial liquid in FIG. 10, the riboflavin production of the five Escherichia coli Chassis cells BL21-DE3, BL21-DE3-plysS, ER2566 and JM109-DE3 is significantly higher than that of other cells. In order to further illustrate which strain is the most suitable underpan cell, the fermentation broth was subjected to HPLC to determine the riboflavin content in the culture broth. It was found that the BL21(DE3) Chassis cell strain produced riboflavin at a maximum of about 1.44g/L as shown in FIG. 11, and thus BL21-DE3 were found to be the most suitable Chassis cells, while the yield of the original strain was 0.55g/L, which was 161.8% higher than that of the original strain.

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

<110> Shanghai city academy of agricultural sciences

<120> a method for improving riboflavin production capacity of escherichia coli engineering bacteria by DNA shuffling

<141> 2021-12-06

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Claims

1. a method utilizing DNA shuffling to improve the ability of Escherichia coli engineering bacteria to produce riboflavin, is characterized in that, described method comprises:

Construction of riboflavin-producing Escherichia coli engineering bacteria containing 26 genes;

T7 RNA polymerase gene expression unit DNA rearrangement and directional screening to obtain the first batch of positive strains, and extract the first batch of positive plasmids;

DNA rearrangement and directional screening of the riboflavin biosynthesis and transport system to obtain the second batch of positive strains and extraction to obtain the second batch of positive plasmids;

DNA rearrangement and directional screening of Escherichia coli sigma factor gene to obtain the third batch of positive strains and extraction to obtain the third batch of positive plasmids;

The third batch of plasmids were introduced into different chassis cells to obtain recombinant strains; the recombinant strains were cultured in vitro, and the riboflavin biosynthesis yield of each engineered strain was analyzed and determined, and the positive strain with the highest yield was selected.

2. utilize DNA shuffling as claimed in claim 1 to obtain the method for the Escherichia coli engineering bacteria of high yield riboflavin, it is characterized in that, the method for described T7 RNA polymerase gene expression unit DNA rearrangement and directional screening comprises:

Chemical synthesis of T7RNAP gene expression unit controlled by lac promoter with lacI repressor and T1 terminator;

Preliminary screening is carried out according to the shade of yellow of the transformant colony;

Pick dark yellow Escherichia coli positive colonies for liquid culture, and use HPLC technology to accurately measure the riboflavin content in the culture solution;

The first batch of positive strains were obtained through rearrangement, library construction and directional screening, and the first batch of positive plasmids were obtained by extraction.

3. the method that utilizes DNA shuffling to obtain the Escherichia coli engineering bacteria of high yield riboflavin as claimed in claim 2, is characterized in that, the method for DNA rearrangement and directional screening of described riboflavin biosynthesis and transport system comprises:

Mix the first batch of positive plasmids for enzyme digestion, and construct a library of mutant elements on the mixed vector; transform E. coli, and conduct preliminary screening according to the shade of yellow of the transformant colony;

The second batch of positive strains with significantly improved riboflavin production were obtained by rearrangement, library construction and directional screening, and the second batch of positive plasmids were obtained by extraction.

4. the method for the Escherichia coli engineering bacteria that utilizes DNA shuffling to obtain high-yielding riboflavin as claimed in claim 3, is characterized in that, the method for DNA rearrangement and directional screening of described riboflavin biosynthesis and transport system comprises:

The sigma factor EcRpoD gene of Escherichia coli was amplified and cloned by PCR technology, and the complete nucleotide sequence analysis was completed;

Eliminate common restriction endonuclease sites within genes;

High-efficiency mutation of genes using DNA shuffling technology;

Construct mutant gene library on the second batch of positive plasmids;

Escherichia coli was transformed by electric shock, and preliminary screening was carried out according to the light color of the transformant colony; then the dark yellow Escherichia coli positive colonies were picked for liquid culture, and the riboflavin content in the culture solution was accurately determined by HPLC technology;

The third batch of positive strains with significantly increased riboflavin production was obtained by rearrangement and directional screening.

5. the method that utilizes DNA shuffling to obtain the Escherichia coli engineering bacteria of high riboflavin production as claimed in claim 4, it is characterized in that, described Escherichia coli chassis cell comprises:

DH5α, HB101, JM105, JM109-DE3, C600, RR1, XL1-Blue, BL21, BL21-DE3, EG61-BL21-AI, BL21-DE3-PlysS, ER2566, IJ1127, MV1190, DH10B, MC8, JM83, SURE, ElectroTen-Blue, XL10-Gold, OmniMAX 2T1 and TOP10.