WO2024045620A1 - 一种生产2'-岩藻糖基乳糖的重组菌株及构建方法和应用 - Google Patents

一种生产2'-岩藻糖基乳糖的重组菌株及构建方法和应用 Download PDF

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WO2024045620A1
WO2024045620A1 PCT/CN2023/087184 CN2023087184W WO2024045620A1 WO 2024045620 A1 WO2024045620 A1 WO 2024045620A1 CN 2023087184 W CN2023087184 W CN 2023087184W WO 2024045620 A1 WO2024045620 A1 WO 2024045620A1
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gene
encoding
mannose
fucosyllactose
encoding gene
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宗剑飞
李庆刚
李玉
肖卫华
路福平
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山东合成远景生物科技有限公司
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Definitions

  • the invention belongs to the technical field of genetic engineering, and specifically relates to a recombinant strain for producing 2'-fucosyllactose and its construction method and application.
  • 2'-fucosyllactose (2'-FL) is one of the important components of human milk oligosaccharides (HMOs). As one of the components of breast milk, 2'-fucosyllactose accounts for up to 30% of breast milk oligosaccharides and has high nutritional and medicinal value.
  • Current methods for producing 2'-fucosyllactose include chemical synthesis, enzymatic methods, fermentation methods, etc. Among them, the fermentation method to produce 2'-fucosyllactose has the advantages of low cost and environmental friendliness.
  • E. coli cells have the advantages of high metabolic activity and high reproduction rate, so E. coli of various genetic backgrounds are one of the most commonly used organisms in the fields of molecular biology and biotechnology.
  • the key enzymes involved in the de novo synthesis pathway and the salvage pathway include: phosphomannose isomerase ManA, phosphomannose mutase ManB, mannose-1-phosphate guanine transferase ManC, GDP-mannose-4,6- Dehydratase Gmd, GDP-fucose synthase WcaG, 2'-fucosyllactose synthase FutC, etc.
  • the cost of carbon sources required for fermentation to produce 2'-fucosyllactose through the salvage pathway is high, while the yield of 2'-fucosyllactose through fermentation through de novo synthesis is low.
  • the protein encoded by the ⁇ -galactosidase permease encoding gene lacY can transport galactoside into cells, and the encoding protein of the ⁇ -galactosidase encoding gene lacZ can cut off half of lactose. Lactosidic bond, the product of regulatory gene lacI is called lac repressor.
  • the proteins encoded by the UDP-glucose lipid carrier transferase encoding gene wcaj and the GDP-mannose hydrolase encoding gene nudd are enzymes involved in the degradation of the precursors GDP-L-fucose and GDP-mannose (Ni ZJ, et al.
  • Set is a transporter family discovered in Escherichia coli in 1999, including SetA, SetB and SetC. This family of transporters can transport glucose, lactose, certain monosaccharides and disaccharides, and inducers Efflux guide molecules such as isopropy- ⁇ -D-thiogalactoside (IPTG). Among them, SetA has broad substrate specificity and is preferred for glycosides or galactosides with alkyl or aryl substituents. However, under normal conditions, the expression level of setA gene is low and its transport activity for larger oligosaccharides such as heptose or trisaccharide is low (Liu JY, et al.
  • the present invention uses gene editing technology to edit the gene setA encoding the sugar efflux transporter A of Escherichia coli. It was found that in situ overexpression of setA gene unexpectedly increased the yield of 2'-fucosyllactose, and its effect was better than plasmid overexpression of setA gene, which was beneficial to improving the yield of 2'-fucosyllactose. production level.
  • gene overexpression uses expression control elements with different transcription or translation strengths to construct a plasmid library ex situ, and transform the plasmid into microbial cells. With the self-replication of the plasmid in the cytoplasm, the expression of the target gene can be quickly achieved.
  • In situ overexpression, or chromosomal in situ overexpression is a commonly used term in this field.
  • promoters for gene overexpression may be constitutive promoters and/or inducible promoters.
  • constitutive promoters and inducible promoters Promoters refer to promoters suitable for prokaryotic expression systems, especially promoters suitable for E. coli expression systems, including natural promoters and artificially constructed promoters.
  • the present invention first provides a recombinant strain that produces 2'-fucosyllactose, which has a higher yield of 2'-fucosyllactose than setA gene plasmid overexpression.
  • the recombinant strain is obtained through gene editing using Escherichia coli (scientific name: Escherichiacoli, usually abbreviated as E.coli) as the starting strain.
  • the starting strain can be E. coli with various genetic backgrounds.
  • E.coli E. coli with various genetic backgrounds.
  • a constitutive promoter was inserted in front of setA in the genome of the recombinant strain, thereby overexpressing setA in situ.
  • the constitutive promoter is selected from any one of P J23102 , P J23104 , P J23105 , P J23108 , P J23100 , P J23110 , P J23111 , P J23113 , P J23119 , P 637 and P 699 .
  • the constitutive promoter is selected from any one of P J23108 , P J23110 and P J23119 .
  • the promoter of the chloramphenicol resistance gene was inserted before setA in the genome of the recombinant strain.
  • the promoter of the chloramphenicol resistance gene was used to overexpress setA in situ. Or insert the promoter of the chloramphenicol resistance gene and a constitutive promoter before the setA gene for in situ overexpression.
  • a constitutive promoter When a constitutive promoter is inserted in front of setA, such as one of P J23102 , P J23104 , P J23105 , P J23108 , P J23100 , P J23110 , P J23111 , P J23113 , P J23119 , P 637 , and P 699 , and chlorine is inserted
  • dual promoters can be used to overexpress setA in situ.
  • the nucleotide sequence of the promoter of the chloramphenicol resistance gene is shown in SEQ ID NO: 19.
  • the starting strain is a recombinant strain that has been genetically modified with genes encoding enzymes related to the de novo synthesis pathway of 2'-fucosyllactose; preferably, it includes knocking out the ⁇ -half on the E.
  • lactosidase encoding gene lacZ and overexpression of the phosphomannose isomerase encoding gene manA, phosphomannose mutase encoding gene manB, GDP-fucose synthase encoding gene wcaG, GDP-mannose-4,6- Dehydratase encoding gene gmd, ⁇ -galactosyl permease encoding gene lacY, mannose-1-phosphate guanine transferase encoding gene manC; more preferably, it also includes 2'-fucosyllactose salvage pathway related enzymes
  • lactose lac operon sequence specifically, the P lac promoter sequence and regulatory genes lacI and lacZ in the lactose lac operon sequence of the starting strain are deleted
  • the 2'-fucosyllactose synthesis precursor is deleted Genetic modification of genes encoding degradation-related enzymes; further preferably,
  • M4 Insert the gene manA encoding mannose phosphate isomerase into the E. coli genome
  • M5 Insert the phosphomannose mutase encoding gene manB into the E. coli genome
  • M6 Insert the GDP-fucose synthase encoding gene wcaG into the E. coli genome
  • M7 Insert the GDP-mannose-4,6-dehydratase encoding gene gmd into the E. coli genome
  • M8 In situ overexpression of the ⁇ -galactoside permease encoding gene lacY;
  • M9 Overexpression of the mannose-1-phosphate guanine transferase encoding gene manC on the plasmid;
  • M10 Overexpression of the 2'-fucosyllactose synthase encoding gene futC on the plasmid.
  • genetic modification of genes encoding related enzymes of the salvage pathway includes knocking out genes involved in fucose decomposition, such as Fuc I, Fuck k, araA, rhaA; and/or genes related to fucose phosphorylation, such as fkp , fuc T2 was overexpressed.
  • the core is L-fucokinase/GDP-fucose pyrophosphorylase (FKP), a bifunctional enzyme that can independently complete the synthesis of L-fucose through the GDP-fucose rescue synthesis pathway. The process of synthesizing GDP-fucose. Therefore, when using the remedial approach, only expensive L-fucose needs to be added to the culture system.
  • the wcaG, gmd, manA, and manB are single-copy inserted into the genome for overexpression; the manC and futC are multi-copy overexpression.
  • manA, manB, wcaG, gmd, lacY, manC and futC were overexpressed using P trc promoter.
  • the plasmid is selected from any one of pTrc99a, pSB4K5, pET28a or pET22b.
  • nucleotide sequence of setA is shown in SEQ ID NO: 1; the nucleotide sequence of lacZ is shown in SEQ ID NO: 2; the nucleotide sequence of wcaj is shown in SEQ ID NO: 3; the nucleotide sequence of nudd is shown in SEQ ID NO: 4; the nucleotide sequence of manA is shown in SEQ ID NO: 5; the nucleotide sequence of manB is shown in SEQ ID NO : 6; the nucleotide sequence of wcaG is shown in SEQ ID NO: 7; the nucleotide sequence of gmd is shown in SEQ ID NO: 8; the nucleotide sequence of lacY is shown in SEQ ID NO: 9; the nucleotide sequence of manC is shown in SEQ ID NO: 10; the nucleotide sequence of futC is shown in SEQ ID NO: 11; the nucleotide sequence of the P trc promoter The sequence is shown in SEQ ID NO: 1
  • the starting strain is selected from any one of E. coli K12 MG1655, E. coli BL21 (DE3), E. coli JM109, and E. coli BW25113.
  • the above strains have been widely used, and Escherichia coli strain K12MG1655 is one of the most famous and well-studied organisms in biology.
  • the second technical solution of the present invention provides a method for constructing the aforementioned recombinant strain, specifically a method for constructing Escherichia coli K12 MG1655 as the starting strain.
  • the construction method includes the following steps (in no particular order):
  • Plasmids overexpressing the mannose-1-phosphate guanine transferase encoding gene and the 2'-fucosyllactose synthase encoding gene are introduced into the starting strain; preferably, a plasmid containing the two genes in tandem is constructed and then introduced Recombinant strains were constructed from starting strains.
  • step S2 Overexpress manA and manB using the P trc promoter on the adhe site of the alcohol dehydrogenase encoding gene of the strain obtained in step S1;
  • step S4 Insert a constitutive promoter in front of setA of the strain obtained in step S3, and insert the promoter of the chloramphenicol resistance gene;
  • S5 Construct plasmid pTrc99a-P trc -futC-manC, the nucleotide sequence of the plasmid pTrc99a-P trc -futC-manC is shown in SEQ ID NO: 18;
  • step S6 Introduce the plasmid obtained in step S5 into the strain obtained in step S4 to construct a recombinant strain.
  • nucleotide sequence of the aforementioned constitutive promoter is public, and can be found in the public resources provided at http://parts.igem.org/Part:BBa_J23100.
  • setA in situ overexpression of setA can unexpectedly increase the fermentation yield of 2'-fucosyllactose compared to setA plasmid overexpression.
  • setA When setA is overexpressed in situ, it is combined with genes encoding enzymes related to the de novo synthesis pathway of 2'-fucosyllactose, genes encoding enzymes related to the 2'-fucosyllactose salvage pathway, the lactose lac operon sequence, and 2'-fucosyllactose.
  • beneficialal editing of genes encoding enzymes related to the degradation of fucosyllactose synthesis precursors will be more conducive to improving the fermentation yield of 2'-fucosyllactose.
  • E. coli strains of various genetic backgrounds are commercially available or publicly available from microbiological depositories. Therefore, it is useful for the art to utilize E. coli strains with a genetic background capable of synthesizing 2'-fucosyllactose, including lacZ knockout, overexpression of wcaG, gmd, lacY, manA, manB, manC, futC.
  • a genetic background capable of synthesizing 2'-fucosyllactose, including lacZ knockout, overexpression of wcaG, gmd, lacY, manA, manB, manC, futC.
  • lacI, lacZ, wcaj, nudd have been knocked out
  • wcaG, gmd, lacY, manA, manB, etc. have been inserted or overexpressed in situ to reduce the construction steps of recombinant strains, which is It goes without saying.
  • the third technical solution of the present invention provides the application of the aforementioned recombinant strain in fermentative production of 2'-fucosyllactose.
  • the carbon source of the fermentation medium of the recombinant strain is selected from at least one of glycerol and lactose; the organic nitrogen source of the fermentation medium of the recombinant strain is selected from at least one of yeast powder, tryptone, and beef extract. ;
  • the temperature of fermentation culture is 36°C ⁇ 40°C; the pH of fermentation culture is 6.8 ⁇ 7.8.
  • the present invention unexpectedly obtains a higher 2'-fucosyllactose yield than overexpression of the setA gene plasmid.
  • inserting a constitutive promoter in front of the setA gene and retaining the promoter of the chloramphenicol resistance gene is more conducive to in situ overexpression of the setA gene.
  • the recombinant strain further contains genes encoding enzymes related to the de novo synthesis pathway of E.
  • genes encoding enzymes related to the 2'-fucosyllactose salvage pathway, lactose lac operon sequences, 2'- Higher 2'-fucosylactose yields can be achieved when beneficial gene editing of genes encoding enzymes related to the degradation of fucosyllactose synthesis precursors.
  • the writing order of the primer sequences in the following examples is from 5' end to 3' end.
  • Escherichia coli W2 E.coli K12 MG1655 ⁇ lacIZ::P trc -wcaG-gmd-lacy, ⁇ adhE::P trc -manB-manA
  • UDP on the genome is knocked out -
  • the gene wcaj encoding glucose lipid carrier transferase and the gene nudd encoding GDP-mannose hydrolase were used to construct strain TKYW1.
  • E. coli W2 is briefly described based on the content of patent document CN112501106A, and the construction method of E. coli W2 is introduced into this embodiment.
  • E. coli W2 in CN112501106A is constructed using Escherichia coli K12 MG1655 as the starting strain.
  • the P lac promoter sequence and regulatory genes lacI and lacZ in the lactose lac operon sequence of the starting strain are deleted, and the original lacZ site is Then, the P trc promoter was used to overexpress wcaG, gmd, and lacY to obtain the W1 strain, and then the P trc promoter was used to overexpress manA and manB at the adhe site of the alcohol dehydrogenase encoding gene to obtain the W2 strain.
  • coli W2 is shown in SEQ ID NO: 2;
  • the nucleotide sequence of manA is shown in SEQ ID NO: 5;
  • the nucleotide sequence of manB is shown in SEQ ID NO: 6;
  • the nucleotide sequence of wcaG is shown in SEQ ID NO: 7;
  • the nucleotide sequence of gmd The sequence is shown in SEQ ID NO: 8;
  • the nucleotide sequence of lacY is shown in SEQ ID NO: 9;
  • the nucleotide sequence of P trc promoter is shown in SEQ ID NO: 12;
  • the nucleotide sequence of lacI is shown in SEQ ID NO: 13 is shown,
  • CRISPR/Cas9 technology was used to knock out wcaj (SEQ ID NO: 3).
  • the CRISPR/Cas9 technology used in the experiment refers to previous research reports [Zhao D, et al. CRISPR/Cas9-assisted gRNA-free one-step genome editing with no sequence limitations and improved targeting efficiency.Sci Rep 7,16624].
  • the first step homologous recombination fragment including the upstream and downstream homology arms, the chloramphenicol resistance gene cat and the universal N20+NGG sequence (tagtccatcgaaccgaagtaagg), and introduce the first step homologous recombination fragment into pCAGO containing In the W2 strain of the plasmid, the first step of recombination is performed.
  • the pCAGO plasmid contains the recombinase gene, as well as cas9 and gRNA genes [Zhao D, et al. CRISPR/Cas9-assisted gRNA-free one-step genome editing with no sequence limitations and improved targeting efficiency.Scientific Reports,7,16624].
  • the first step is to construct homologous recombination fragments.
  • E.coli strain E.coli K12 MG1655 genome GeneBank accession NO.NC_000913
  • the primers up-1 and up-2, and the primers down-1 and down-2 in Table 1, respectively the same results were obtained by PCR amplification.
  • the upstream and downstream homology arms of source recombination Take the genome of a strain with the chloramphenicol resistance gene cat stored in the laboratory (the nucleotide sequence is shown in SEQ ID NO: 17.
  • SEQ ID NO: 17 contains the nucleotide sequence of the cat gene and its promoter ) as a template, use primers cat-1 and cat20-2 to perform PCR amplification, and obtain a fragment with cat-N20 sequence.
  • the upper and lower homology arms have fragments with cat-N20 sequences. These three fragments are used as templates.
  • Primers up-1 and down-2 are used for overlapping PCR amplification to obtain the first step of homologous recombination fragments.
  • the first step of homologous recombination The pCAGO plasmid was transformed into strain W2 using conventional plasmid transformation methods to obtain strain W2 (pCAGO).
  • the transformed bacterial solution is coated with 100mg/L ampicillin. Penicillin, 25 mg/L chloramphenicol, and 1% glucose were cultured on LB plates at 30°C. Select transformants for colony PCR identification to obtain the correct homologous recombination strain in the first step.
  • the second step of homologous recombination Inoculate the homologous recombination strain in the first step into a solution containing 100mg/L ampicillin, 0.1mM In the LB liquid medium with IPTG and 2g/L arabinose, culture it at 30°C for more than 6 hours, isolate single colonies by streaking on the plate, and screen out those that can grow on the LB plate containing 100mg/L ampicillin, but cannot grow on the LB plate containing 25mg/L ampicillin. Clones that cannot grow on LB plates with L-chloramphenicol. Sequencing verified that the correct clone that had undergone the second homologous recombination was further cultured at 37°C to lose the pCAGO plasmid, thus obtaining strain W2 ⁇ wcaj.
  • strain W2 ⁇ wcaj On the basis of E. coli strain W2 ⁇ wcaj, the same method as the above-mentioned CRISPR/Cas9 technology was used to knock out the nudd gene (SEQ ID NO: 4) on the genome, and the strain W2 ⁇ wcaj ⁇ nudd was constructed, named ZKYW1. The specific methods are described in detail below:
  • the first step is to construct homologous recombination fragments.
  • PCR amplification was performed to obtain the upstream and downstream homologs of homologous recombination. arm.
  • PCR amplification was performed using the primers Ncat-1 and Ncat20-2 in Table 2 to obtain a new fragment with cat-N20 sequence.
  • Primers Nup-1 and Ndown-2 are used for overlapping PCR amplification to obtain the first step of homologous recombination fragments.
  • the first step of homologous recombination The pCAGO plasmid was transformed into strain W2 ⁇ wcaj using conventional plasmid transformation methods to obtain strain W2 ⁇ wcaj (pCAGO).
  • Use LB medium containing 1% glucose and IPTG (isopropyl- ⁇ -D-thiogalactopyranoside) at a concentration of 0.1mM to prepare W2 ⁇ wcaj (pCAGO) competent cells, and use electroporation to introduce the first homologous Source the recombinant fragment, and spread the transformed bacterial solution on an LB plate containing 100 mg/L ampicillin, 25 mg/L chloramphenicol, and 1% glucose, and culture at 30°C. Select transformants for colony PCR identification to obtain the correct homologous recombination strain in the first step.
  • the second step of homologous recombination is the same as the above-mentioned knockout of wcaj gene. Sequencing verified that the correct clone that had undergone the second homologous recombination was obtained, and it was further cultured at 37°C to lose pCAGO. plasmid, thereby obtaining strain W2 ⁇ wcaj ⁇ nudd, named TKYW1.
  • strain TKYW1 On the basis of strain TKYW1, using the same method as the above-mentioned CRISPR/Cas9 technology, three constitutive starts of different strengths were inserted in front of the sugar efflux transporter gene setA (the nucleotide sequence is shown in SEQ ID NO: 1). P J23108 , P J23110 , P J23119 (http://parts.igem.org/Part:BBa_J23100), the promoter sequence is shown in Table 3. At the same time, when using CRISPR/Cas9 for the second recombination, the promoter of the chloramphenicol resistance gene was retained (the nucleotide sequence is shown in SEQ ID NO: 19) to achieve in situ recombination of setA with dual promoters. After overexpression, the obtained strains were named TKYW2-1, TKYW2-2, and TKYW2-3 respectively.
  • PCR amplification was performed to obtain homologous recombination upper and lower genes. Downstream homology arm.
  • PCR amplification was performed using the primers Scm-1 and Scm-2 in Table 4 to obtain a fragment with cat gene sequence.
  • PCR amplification was performed to obtain 3 genes with P J23108 , P J23110 , and P J23119 promoters respectively. gene fragment.
  • Sup-1 and Sdown-2 as primers, the upstream homology obtained by the above PCR amplification was used.
  • the pCAGO plasmid was transformed into strain TKYW1 using conventional plasmid transformation methods to obtain strain TKYW1 (pCAGO).
  • Use LB medium containing 1% glucose and IPTG at a concentration of 0.1mM to prepare TKYW1 (pCAGO) competent cells, and use the electrotransformation method to introduce the above-mentioned three types of homologous recombination for the first time, respectively, with P J23108 , P J23110 , Different fragments of the P J23119 promoter were spread on LB plates containing 100 mg/L ampicillin, 25 mg/L chloramphenicol, and 1% glucose and cultured at 30°C. Transformants were selected for colony PCR identification, and three strains carrying P J23108 , P J23110 , and P J23119 promoters were obtained after the correct first step of homologous recombination.
  • the second step of homologous recombination is the same as the above-mentioned knockout of wcaj gene. Sequencing verified that the correct clone that had undergone the second homologous recombination was obtained, and it was further cultured at 37°C to lose the pCAGO plasmid, thereby obtaining three strains with P J23108 , P J23110 , and P J23119 promoters respectively. They are named TKYW2-1, TKYW2-2, and TKYW2-3 respectively.
  • the plasmid pTrc99a-futC-manC described in the patent document CN112501106A was used as the template (plasmid
  • the nucleotide sequence of the P trc promoter involved in the construction of pTrc99a-futC-manC is shown in SEQ ID NO: 12; the nucleotide sequence of the arabinose-inducible promoter P ara promoter is shown in SEQ ID NO: 15;
  • the nucleotide sequence of the mannose-1-phosphate guanine transferase encoding gene manC is shown in SEQ ID NO: 10; the nucleotide sequence of the 2'-fucosyllactose synthase encoding gene futC is shown in SEQ ID NO: 11), use Darac-F and Darac-R in Table 5 as primers for PCR amplification.
  • promoters P trc , P J23108 , P J23110 , and P J23119 were used to overexpress setA, and plasmid pTrc99a-P trc -futC-manC-P trc -setA was constructed, pTrc99a-P trc -futC-manC-P J23108 -setA, pTrc99a-P trc -futC-manC-P J23110 -setA and pTrc99a-P trc -futC-manC-P J23119 -setA.
  • PCR amplification was performed using plasmid pTrc99a-P trc -futC-manC as a template and T-AZT-F and AZT-R in Table 6 as primers to obtain a linear vector fragment containing futC and manC genes.
  • the E. coli MG1655 genome was used as the template, AF and A-R1 were used as primers for PCR amplification, and a fragment containing the setA gene was obtained.
  • the plasmid pTrc99a-P trc -futC-manC was used as the template, and TF and TR were used as primers for PCR. Amplify and obtain the gene fragment with P trc promoter.
  • the primers were used for PCR amplification, and three linear vector fragments containing futC and manC genes were obtained.
  • seamless cloning enzyme was used to connect the three linear vector fragments containing futC and manC genes to the fragments containing setA gene, and then transformed into E.coli JM109 competent cells respectively. Cultivate on LB plates containing 100 mg/L ampicillin, select transformants and sequence them for verification, and obtain the correct recombinant plasmids, which are named pTrc99a-P trc -futC-manC-P J23108 -setA, pTrc99a-P trc -futC- manC-P J23110 -setA and pTrc99a-P trc -futC-manC-P J23119 -setA.
  • the plasmid pTrc99a-P trc -futC-manC was introduced into strains TKYW1, TKYW2-1, TKYW2-2 and TKYW2-3, respectively, to construct strain TKYW1 (pTrc99a-P trc -futC-manC).
  • TKYW2-1 pTrc99a-P trc -futC-manC
  • TKYW2-2 pTrc99a-P trc -futC-manC
  • TKYW2-3 pTrc99a-P trc -futC-manC.
  • the plasmids pTrc99a-P trc -futC-manC-P trc -setA, pTrc99a-P trc -futC-manC-P J23108 -setA, pTrc99a-P trc -futC-manC-P J23110 -setA and pTrc99a-P trc -futC -manC-P J23119 -setA were introduced into strain TKYW1 to construct strain TKYW1 (pTrc99a-P trc -futC-manC-P trc -setA), TKYW1 (pTrc99a-P trc -futC-manC-P J23108 -setA), TKYW1 (pTrc99a-P trc -futC-manC-P J23110
  • the level of fermentation production of 2'-fucosyllactose by the above strains was tested.
  • the medium used was: LB medium: NaCl 10g/L, yeast powder 5g/L, peptone 10g/L, pH 7.0.
  • Fermentation medium KH 2 PO 4 3g/L, yeast powder 8g/L, (NH 4 ) 2 SO 4 4g/L, citric acid 1.7g/L, MgSO 4 ⁇ 7H 2 O 2g/L, thiamine 10mg /L, glycerin 10g/L, lactose 5g/L, 1ml/L trace elements (FeCl 3 ⁇ 6H 2 O 25g/L, MnCl 2 ⁇ 4H 2 O 9.8g/L, CoCl 2 ⁇ 6H 2 O 1.6g/L , CuCl 2 ⁇ H 2 O 1g/L, H 3 BO 3 1.9g/L, ZnCl 2 2.6g/L, Na 2 MOO 4 ⁇ 2H
  • the fermentation test process is:
  • the chromatographic column used for HPLC analysis was Carbohydrate ES 5u250mm*4.6mm, the detector was an evaporative light detector, the mobile phase was 70% acetonitrile (acetonitrile: water), and the flow rate was 0.8 mL/min, the column temperature was set to 30°C, and the injection volume was 5 ⁇ L. Sample concentrations were quantified using 2'-fucosyllactose standards. The yield of 2’-fucosyllactose is shown in Table 8.

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Abstract

本发明涉及一种生产2'-岩藻糖基乳糖的重组菌株及构建方法和应用，属于基因工程技术领域。本发明提供的重组菌株及其构建方法的特点在于通过基因编辑对setA基因进行原位过表达。本发明提供的重组菌株也可进一步包含对大肠杆菌2'-岩藻糖基乳糖从头合成途径相关酶的编码基因、2'-岩藻糖基乳糖补救途径相关酶的编码基因、乳糖lac操纵子序列、2'-岩藻糖基乳糖合成前体降解相关酶的编码基因的有益基因编辑。本发明提供的重组菌株通过setA基因原位过表达获得了比setA基因质粒过表达更高2'-岩藻糖基乳糖产率。

Description

一种生产2′-岩藻糖基乳糖的重组菌株及构建方法和应用 技术领域
本发明属于基因工程技术领域，具体涉及一种生产2'-岩藻糖基乳糖的重组菌株及构建方法和应用。
背景技术
2'-岩藻糖基乳糖(2'-fucosyllactose，2'-FL)是母乳低聚糖(human milk oligosaccharides，HMOs)的重要成分之一。作为母乳成分之一，2'-岩藻糖基乳糖占母乳低聚糖的比例可达30％，并具有较高的营养和药用价值。目前生产2'-岩藻糖基乳糖的方法包括化学合成法、酶法、发酵法等。其中发酵法生产2'-岩藻糖基乳糖具有成本低、环境友好等优势。大肠杆菌细胞具有高代谢活性和高繁殖率的优点，因此各种遗传背景的大肠杆菌是分子生物学和生物技术领域中最常用的生物之一。
本领域已对大肠杆菌发酵生产2'-岩藻糖基乳糖进行了广泛研究，以期提高2'-岩藻糖基乳糖的产率。其中对大肠杆菌合成2'-岩藻糖基乳糖的从头合成途径和补救途径以及相关的关键酶的研究较为深入(Bych K,et al.Production of HMOs using microbial hosts-from cell engineering to large scale production[J].Current Opinion in Biotechnology,2019,56:130-137；CN112501106A；CN114276971A等)。从头合成途径和补救途径中涉及的关键酶包括：磷酸甘露糖异构酶ManA、磷酸甘露糖变位酶ManB、甘露糖-1-磷酸鸟嘌呤转移酶ManC、GDP-甘露糖-4,6-脱水酶Gmd、GDP-岩藻糖合成酶WcaG、2'-岩藻糖基乳糖合成酶FutC等。其中补救途径发酵生产2'-岩藻糖基乳糖所需碳源成本较高，而从头合成途径发酵生产2'-岩藻糖基乳糖的产率较低。乳糖lac操纵子序列中，β-半乳糖苷透性酶编码基因lacY的编码蛋白可将半乳糖苷运入到细胞中，β-半乳糖苷酶编码基因lacZ的编码蛋白则可切断乳糖的半乳糖苷键，调节基因lacI的产物称为lac阻遏物。UDP-葡萄糖脂质载体转移酶编码基因wcaj及GDP-甘露糖水解酶编码基因nudd的编码蛋白则是参与前体GDP-L-岩藻糖和GDP-甘露糖降解的酶类(Ni ZJ,et al.Multi-Path Optimization for Efficient Production of 2′-Fucosyllactose in an Engineered Escherichia coli C41(DE3)Derivative[J].Frontiers in Bioengineering and Biotechnology,2020,8)。
糖外排转运体(Sugar efflux transporter，Set)是1999年在大肠杆菌中发现的转运蛋白家族，包括SetA、SetB和SetC。该族转运蛋白可将葡萄糖、乳糖、某些单糖和二糖以及诱 导分子例如异丙基-β-D-硫代半乳糖苷(Isopropy-β-D-thiogalactoside，IPTG)外排。其中SetA具有广泛的底物特异性，偏好用于具有烷基或芳基取代基的糖苷或半乳糖苷。但常规条件下setA基因表达水平较低且对庚糖或三糖等较大的寡糖转运活性较低(Liu JY,et al.Functional and Biochemical Characterization of Escherichia coli Sugar Efflux Transporters[J].Journal of Biological Chemistry,1999,274(33):22977-22984；US8652808B2)。SetA被发现具有外排2'-岩藻糖基乳糖的作用，从而可能通过降低胞内2'-岩藻糖基乳糖提高胞外2'-岩藻糖基乳糖水平，然而SetA同时对碳源乳糖具有外排作用，且导致菌株细胞生长率降低(Hollands K,et al.Engineering two species of yeast as cell factories for 2′-fucosyllactose[J].Metabolic Engineering,52(2019)-232-242)。由于setA基因转录调控机制不完全清楚，其基因过表达对2'-岩藻糖基乳糖生产的影响尚难以预测。因此，目前的研究仅限于质粒过表达setA基因或其他糖外排转运体对大肠杆菌发酵生产2'-岩藻糖基乳糖或其他HMOs的影响(ParschatK,et al.High-Titer De Novo Biosynthesis of the Predominant Human Milk Oligosaccharide 2′-Fucosyllactose from Sucrose in Escherichia coli[J].ACS Synthetic Biology,2020,9(10)；US8652808B2)。
对大肠杆菌合成2'-岩藻糖基乳糖的从头合成途径和补救途径的关键酶进行编辑以及质粒过表达setA基因等技术手段虽然提高了2'-岩藻糖基乳糖产率，但其产率仍然较低。进一步对菌株进行改良，以提高2'-岩藻糖基乳糖产率，仍是目前需要解决的问题。
发明内容
为了解决上述技术问题，本发明采用基因编辑技术，对大肠杆菌的糖外排转运体A编码基因setA进行了编辑。发现setA基因的原位过表达出乎预料地提高了2'-岩藻糖基乳糖的产率，其效果优于setA基因的质粒过表达，从而有利于提高2'-岩藻糖基乳糖的生产水平。
在利用原核细胞等细胞体系发酵生产特定物质时，出于提高目的物质产率的目的，往往需要利用基因工程技术对相关基因的表达水平进行上调。使目的基因表达量明显提高的技术，称为基因过表达，包括原位过表达、质粒过表达等。其中质粒过表达是使用具有不同转录或翻译强度的表达调控元件，离位(ex situ)构建质粒文库，并将质粒转化进入微生物细胞内。伴随质粒在细胞质内的自我复制，可快速实现目的基因的表达。而原位过表达或称为染色体原位过表达，是本领域约定俗称的术语，是指在原位(in situ)进行过表达，即通过分子生物学手段，比如启动子、核糖体结合位点及转录调控因子改造或密码子优化等，对位于染色体原位(in situ)的目的基因进行调控，提高基因转录翻译水平。
用于基因过表达(包括原位过表达、质粒过表达)的额外插入的启动子可以是组成型启动子和/或诱导型启动子。毫无疑问，此处所述的启动子，包括组成型启动子和诱导型启动 子，是指适用于原核表达系统的启动子，尤其是适用于大肠杆菌表达系统的启动子，包括天然启动子及人工构建的启动子。
本发明的首先提供一种生产2'-岩藻糖基乳糖的重组菌株，相较于setA基因质粒过表达具有更高的2'-岩藻糖基乳糖的产率。该重组菌株以大肠杆菌(学名:Escherichiacoli，通常简写为E.coli)为出发菌株经过基因编辑获得，出发菌株可以是各种遗传背景的大肠杆菌。相较于糖外排转运体A编码基因setA的质粒过表达而言，其主要特点是该重组菌株基因组的糖外排转运体A编码基因setA在原位进行过表达。即通过插入额外的启动子提高setA的染色体原位表达水平，或者通过本领域技术人员比较容易想到的其它方式提高原位表达水平，比如改造setA基因前面的小分子调控RNA基因sgrS的启动子活性，或者在sgrS基因前插入额外的启动子。
进一步地，所述重组菌株基因组的setA前面插入组成型启动子，从而使setA原位过表达。优选的，所述组成型启动子选自PJ23102、PJ23104、PJ23105、PJ23108、PJ23100、PJ23110、PJ23111、PJ23113、PJ23119、P637、P699中的任一种。进一步优选的，所述组成型启动子选自PJ23108、PJ23110、PJ23119中的任一种。
进一步地，所述重组菌株基因组的setA前插入氯霉素抗性基因的启动子。利用氯霉素抗性基因的启动子使setA原位过表达。或setA基因前插入氯霉素抗性基因的启动子和一种组成型启动子进行原位过表达。当setA前面插入组成型启动子，例如PJ23102、PJ23104、PJ23105、PJ23108、PJ23100、PJ23110、PJ23111、PJ23113、PJ23119、P637、P699中的一种，并插入氯霉素抗性基因的启动子时，可利用双启动子对setA进行原位过表达。优选的，所述氯霉素抗性基因的启动子的核苷酸序列如SEQ ID NO：19所示。
进一步地，所述所述出发菌株是进行过2'-岩藻糖基乳糖从头合成途径相关酶的编码基因的基因改造的重组菌株；优选地，其包括敲除大肠杆菌基因组上的β-半乳糖苷酶编码基因lacZ，并过表达磷酸甘露糖异构酶编码基因manA、磷酸甘露糖变位酶编码基因manB、GDP-岩藻糖合成酶编码基因wcaG、GDP-甘露糖-4,6-脱水酶编码基因gmd、β-半乳糖苷透性酶编码基因lacY、甘露糖-1-磷酸鸟嘌呤转移酶编码基因manC；更优选地，还包括2'-岩藻糖基乳糖补救途径相关酶的编码基因、乳糖lac操纵子序列(具体是敲除出发菌株的乳糖lac操纵子序列中的Plac启动子序列及调节基因lacI和lacZ)、敲除2'-岩藻糖基乳糖合成前体降解相关酶的编码基因的基因改造；进一步优选地，
M1：敲除大肠杆菌基因组上的β-半乳糖苷酶编码基因lacZ；
M2：敲除大肠杆菌基因组上的UDP-葡萄糖脂质载体转移酶编码基因wcaj；
M3：敲除大肠杆菌基因组上的GDP-甘露糖水解酶编码基因nudd；
M4：在大肠杆菌基因组上插入磷酸甘露糖异构酶编码基因manA；
M5：在大肠杆菌基因组上插入磷酸甘露糖变位酶编码基因manB；
M6：在大肠杆菌基因组上插入GDP-岩藻糖合成酶编码基因wcaG；
M7：在大肠杆菌基因组上插入GDP-甘露糖-4,6-脱水酶编码基因gmd；
M8：原位过表达β-半乳糖苷透性酶编码基因lacY；
M9：在质粒上过表达甘露糖-1-磷酸鸟嘌呤转移酶编码基因manC；
M10：在质粒上过表达2'-岩藻糖基乳糖合成酶编码基因futC。
其中，补救途径的相关酶的编码基因改造包括对涉及岩藻糖分解的基因采用敲除手段，如Fuc I、Fuc k、araA、rhaA；和/或对岩藻糖磷酸化相关的基因如fkp、fuc T2进行过表达。其中，核心是L-岩藻糖激酶/GDP-岩藻糖焦磷酸化酶(FKP)，为一种双功能酶，它可以通过GDP-岩藻糖补救合成途径独立完成从L-岩藻糖合成GDP-岩藻糖的过程。因此利用补救途径时，只不过需要在培养体系中添加昂贵的L-岩藻糖。
更进一步地，所述wcaG、gmd、manA、manB为单拷贝插入基因组过表达；所述manC、futC为多拷贝过表达。
进一步地，所述manA、manB、wcaG、gmd、lacY、manC和futC使用Ptrc启动子过表达。
进一步地，所述质粒选自pTrc99a、pSB4K5、pET28a或pET22b中的任一种。
进一步地，所述setA的核苷酸序列如SEQ ID NO：1所示；所述lacZ的核苷酸序列如SEQ ID NO：2所示；所述wcaj的核苷酸序列如SEQ ID NO：3所示；所述nudd的核苷酸序列如SEQ ID NO：4所示；所述manA的核苷酸序列如SEQ ID NO：5所示；所述manB的核苷酸序列如SEQ ID NO：6所示；所述wcaG的核苷酸序列如SEQ ID NO：7所示；所述gmd的核苷酸序列如SEQ ID NO：8所示；所述lacY的核苷酸序列如SEQ ID NO：9所示；所述manC的核苷酸序列如SEQ ID NO：10所示；所述futC的核苷酸序列如SEQ ID NO：11所示；所述Ptrc启动子的核苷酸序列如SEQ ID NO：12所示。
进一步地，所述出发菌株选自大肠杆菌K12 MG1655、大肠杆菌BL21(DE3)、大肠杆菌JM109、大肠杆菌BW25113中的任一种。上述菌株已得到大量应用，大肠杆菌K12 MG1655(Escherichia coli strain K12MG1655)是生物学中最著名和研究最充分的生物体之一。其中大 肠杆菌K12 MG1655在美国典型培养物保护藏中心已有保藏(保藏编号ATCC 53103、ATCC 47076、ATCC 700926)；大肠杆菌BL21(DE3)在BCCM genecorner已有保藏(保藏编号LMBP 1455)；大肠杆菌JM109在美国典型培养物保护藏中心已有保藏(保藏编号ATCC68635、ATCC68868)；大肠杆菌BW25113在大肠杆菌遗传学保藏中心(Coli Genetics Stock Center)已有保藏(保藏编号CGSC#7636)。作为本领域技术人员常用的出发菌株，本领域技术人员有能力获知上述菌株的来源和购买渠道。
本发明的技术方案之二，提供了一种前述重组菌株的构建方法，具体来说是以大肠杆菌K12 MG1655为出发菌株的构建方法，所述构建方法包括以下步骤(不分先后)：
敲除出发菌株的乳糖lac操纵子序列中的Plac启动子序列及调节基因lacI和β-半乳糖苷酶编码基因，在原β-半乳糖苷酶编码基因位点之后以Ptrc启动子过表达GDP-岩藻糖合成酶编码基因、GDP-甘露糖-4,6-脱水酶编码基因和β-半乳糖苷透性酶编码基因；
在出发菌株的乙醇脱氢酶编码基因adhe位点上以Ptrc启动子过表达磷酸甘露糖异构酶编码基因和磷酸甘露糖变位酶编码基因；
敲除出发菌株的UDP-葡萄糖脂质载体转移酶编码基因和GDP-甘露糖水解酶编码基因；
在出发菌株的setA前面插入组成型启动子，并插入氯霉素抗性基因的启动子；
在出发菌株中导入过表达甘露糖-1-磷酸鸟嘌呤转移酶编码基因和2'-岩藻糖基乳糖合成酶编码基因的质粒；优选地，通过构建含有该两基因串联的质粒，再导入出发菌株中构建重组菌株。
在一个具体实例中，包括如下步骤：
S1：敲除出发菌株的乳糖lac操纵子序列中的Plac启动子序列及调节基因lacI和lacZ，在原lacZ位点之后以Ptrc启动子过表达wcaG、gmd和lacY；
S2：在步骤S1所得菌株的乙醇脱氢酶编码基因adhe位点上以Ptrc启动子过表达manA和manB；
S3：敲除步骤S2所得菌株的wcaj和nudd；
S4：在步骤S3所得菌株的setA前面插入组成型启动子，并插入氯霉素抗性基因的启动子；
S5：构建质粒pTrc99a-Ptrc-futC-manC，所述质粒pTrc99a-Ptrc-futC-manC的核苷酸序列如SEQ ID NO：18所示；
S6：将步骤S5所得质粒导入步骤S4所得菌株构建重组菌株。
前述组成型启动子的核苷酸序列是公开的，可参见http://parts.igem.org/Part:BBa_J23100提供的公开资源。
本发明发现setA原位过表达相对于setA质粒过表达可出乎预料的提高2'-岩藻糖基乳糖的发酵产率。当setA原位过表达配合2'-岩藻糖基乳糖从头合成途径相关酶的编码基因、2'-岩藻糖基乳糖补救途径相关酶的编码基因、乳糖lac操纵子序列、2'-岩藻糖基乳糖合成前体降解相关酶的编码基因的有益编辑时，更有利于提高2'-岩藻糖基乳糖的发酵产率。有多种遗传背景的大肠杆菌菌株可在市场上购买或由微生物保藏机构对外公开提供。因此，对于本领域而言，利用能合成2'-岩藻糖基乳糖的遗传背景的大肠杆菌菌株，包括已敲除lacZ，过表达wcaG、gmd、lacY、manA、manB，manC，futC。当然，更优选地包括已敲除lacI、lacZ、wcaj、nudd的菌株和/或wcaG、gmd、lacY、manA、manB等已插入或原位过表达的菌株可减少重组菌株的构建步骤，这是不言而喻的。
前述重组菌株及其构建方法涉及的基因编辑技术以及实现前述基因的原位过表达或质粒过表达常用的额外插入的启动子(如组成型启动子P406、P479、P535等；诱导表达型启动子Ptac等)是本领域技公知的，可参见彭秀玲等编著的《基因工程实验技术》(长沙：湖南科学技术出版社1998年第2版)、袁婺洲编著的《基因工程》(北京：化学工业出版社，2019年第2版)韦宇拓编著的《基因工程原理与技术》(北京：北京大学出版社，2017年第1版)、曹卫军编著的《微生物工程》(科学出版社2007年第2版)等。在此前公开的技术文献中，也有充分的公开和报道，例如但不限于以下文献：
(1)Chen D,et al.Development of a DNA double-strand break-free base editing tool in Corynebacterium glutamicum for genome editing and metabolic engineering-ScienceDirect.Metabolic Engineering Communications,2020,11,e00135.
(2)刘洋,等.微生物细胞工厂的代谢调控.生物工程学报,2021,37(5):1541-1563.
(3)Liang ST,et al.Activities of constitutive promoters in Escherichia coli.Journal of Molecular Biology,1999,292(1):19-37.
(4)Zhou L,et al.Chromosome engineering of Escherichia coli for constitutive production of salvianic acid A.Microbial Cell Factories,2017,16(1):84.
(5)Zhao,D,et al.CRISPR/Cas9-assisted gRNA-free one-step genome editing with no sequence limitations and improved targeting efficiency.Scientific Reports,2017,7(1):16624.
(6)Guo,M,et al.Using the promoters of MerR family proteins as"rheostats"to engineer whole-cell heavy metal biosensors with adjustable sensitivity[J].Journal of Biological Engineering,2019,13(1):1-9(PMID:31452678).
本发明的技术方案之三，提供了前述重组菌株在发酵生产2'-岩藻糖基乳糖中的应用。
进一步地，所述重组菌株的发酵培养基碳源选自甘油、乳糖中的至少一种；所述重组菌株的发酵培养基有机氮源选自酵母粉、胰蛋白胨、牛肉膏中的至少一种；发酵培养的温度为36℃～40℃；发酵培养的pH为6.8～7.8。
本发明的有益效果：
本发明通过对大肠杆菌的setA基因进行原位过表达，出乎预料的获得了比setA基因质粒过表达更高的2'-岩藻糖基乳糖产率。尤其是setA基因前面插入组成型启动子并保留氯霉素抗性基因的启动子更有利于setA基因的原位过表达。当重组菌株进一步包含对大肠杆菌2'-岩藻糖基乳糖从头合成途径相关酶的编码基因、2'-岩藻糖基乳糖补救途径相关酶的编码基因、乳糖lac操纵子序列、2'-岩藻糖基乳糖合成前体降解相关酶的编码基因的有益基因编辑时，可达到更高的2'-岩藻糖基乳糖产率。
具体实施方式
下面通过具体的实施方案叙述本发明。除非特别说明，本发明中所用的技术手段均为本领域技术人员所公知的方法。另外，实施方案应理解为说明性的，而非限制本发明的范围，本发明的实质和范围仅由权利要求书所限定。对于本领域技术人员而言，在不背离本发明实质和范围的前提下，对这些实施方案中的物料成分和用量进行的各种改变或改动也属于本发明的保护范围。
以下实施例中引物序列的书写顺序均为5’端至3’端。
实施例1构建菌株TKYW1
在专利文献CN112501106A中所述大肠杆菌W2(E.coli K12 MG1655△lacIZ::Ptrc-wcaG-gmd-lacy,△adhE::Ptrc-manB-manA)的基础上，敲除基因组上的UDP-葡萄糖脂质载体转移酶编码基因wcaj和GDP-甘露糖水解酶编码基因nudd，构建出菌株TKYW1。
在此基于专利文献CN112501106A的内容对大肠杆菌W2的构建进行简要描述，将大肠杆菌W2构建方法引入本实施例。其中CN112501106A中大肠杆菌W2是以大肠杆菌K12MG1655(Escherichia coli K12 MG1655)为出发菌株构建，敲除出发菌株的乳糖lac操纵子序列中的Plac启动子序列及调节基因lacI和lacZ，在原lacZ位点之后以Ptrc启动子过表达wcaG、gmd和lacY得到W1菌株，进而在乙醇脱氢酶编码基因adhe位点上以Ptrc启动子过表达manA和manB得到W2菌株。大肠杆菌W2构建涉及的lacZ的核苷酸序列如SEQ ID NO：2所示； manA的核苷酸序列如SEQ ID NO：5所示；manB的核苷酸序列如SEQ ID NO：6所示；wcaG的核苷酸序列如SEQ ID NO：7所示；gmd的核苷酸序列如SEQ ID NO：8所示；lacY的核苷酸序列如SEQ ID NO：9所示；Ptrc启动子的核苷酸序列如SEQ ID NO：12所示；lacI的核苷酸序列如SEQ ID NO：13所示、Plac启动子的核苷酸序列如SEQ ID NO：14所示，adhE的核苷酸序列如SEQ ID NO：16所示。
1.构建菌株W2△wcaj
使用菌株W2作为出发菌株，利用CRISPR/Cas9技术敲除wcaj(SEQ ID NO：3)。实验中所用的CRISPR/Cas9技术参考前期的研究报道【Zhao D,et al.CRISPR/Cas9-assisted gRNA-free one-step genome editing with no sequence limitations and improved targeting efficiency.Sci Rep 7,16624】。首先，构建第一步同源重组片段，包含上下游同源臂、氯霉素抗性基因cat和通用的N20+NGG序列(tagtccatcgaaccgaagtaagg)，将第一步同源重组片段通过电转化导入含有pCAGO质粒的W2菌株中，进行第一步重组，pCAGO质粒含有重组酶基因，以及cas9和gRNA基因等【Zhao D,et al.CRISPR/Cas9-assisted gRNA-free one-step genome editing with no sequence limitations and improved targeting efficiency.Scientific Reports,7,16624】。挑选正确克隆，进行第二次同源重组。挑取第二次同源重组后的正确克隆，传代丢失pCAGO质粒，从而获得敲除wcaj基因的W2△wcaj菌株。
以下详细描述具体方法：
(1)第一步同源重组片段的构建。以大肠杆菌菌株E.coli K12 MG1655基因组(GeneBank accession NO.NC_000913)为模板，分别利用表1中的引物up-1和up-2，以及引物down-1和down-2，PCR扩增得到同源重组的上、下游同源臂。以实验室保存的一株带有氯霉素抗性基因cat的菌株基因组(核苷酸序列如SEQ ID NO：17所示。SEQ ID NO：17含有cat基因及其启动子的核苷酸序列)为模板，利用引物cat-1和cat20-2进行PCR扩增，获得带有cat-N20序列的片段。以上、下游同源臂，带有cat-N20序列的片段，这3个片段为模板，利用引物up-1和down-2进行重叠PCR扩增，得到第一步同源重组片段。
(2)第一步同源重组。利用常规的质粒转化法将pCAGO质粒转化到菌株W2中，获得菌株W2(pCAGO)。利用含有1％葡萄糖以及浓度为0.1mM的IPTG的LB培养基制备W2(pCAGO)感受态，利用电转化方法导入第一次同源重组片段，转化后的菌液涂布于含100mg/L氨苄青霉素和25mg/L氯霉素，以及1％葡萄糖的LB平板上，30℃培养。挑取转化子进行菌落PCR鉴定，获得正确的第一步同源重组菌株。
(3)第二步同源重组。将第一步同源重组菌株接种到含有100mg/L氨苄青霉素、0.1mM 的IPTG以及2g/L阿拉伯糖的LB液体培养基中，在30℃培养6h以上，平板划线分离单菌落，筛选出能够在含有100mg/L氨苄青霉素的LB平板上生长，但是在含有25mg/L氯霉素的LB平板上不能够生长的克隆。测序验证发生第二次同源重组的正确克隆，进一步将其在37℃条件下培养，丢失其中的pCAGO质粒，从而获得菌株W2△wcaj。
表1敲除wcaj基因所用引物
2.构建菌株W2△wcaj△nudd
在大肠杆菌菌株W2△wcaj的基础上，利用与上述CRISPR/Cas9技术同样的方法，敲除基因组上的nudd基因(SEQ ID NO：4)，构建出菌株W2△wcaj△nudd，命名为ZKYW1。以下详细描述具体方法：
(1)第一步同源重组片段的构建。以菌株E.coli K12 MG1655基因组为模板，分别利用表2中的引物对Nup-1和Nup-2，引物对Ndown-1和Ndown-2，PCR扩增得到同源重组的上、下游同源臂。以构建菌株W2△wcaj时获得的带有cat-N20序列的片段为模板，利用表2中的引物Ncat-1和Ncat20-2进行PCR扩增，获得新的带有cat-N20序列的片段。以上、下游同源臂，新的带有cat-N20序列的片段，这3个片段为模板，利用引物Nup-1和Ndown-2进行重叠PCR扩增，得到第一步同源重组片段。
(2)第一步同源重组。利用常规的质粒转化法将pCAGO质粒转化到菌株W2△wcaj中，获得菌株W2△wcaj(pCAGO)。利用含有1％葡萄糖以及浓度为0.1mM的IPTG(异丙基-β-D-硫代半乳糖苷)的LB培养基制备W2△wcaj(pCAGO)感受态，利用电转化方法导入第一次同源重组片段，转化后的菌液涂布于含100mg/L氨苄青霉素和25mg/L氯霉素，以及1％葡萄糖的LB平板上，30℃培养。挑取转化子进行菌落PCR鉴定，获得正确的第一步同源重组菌株。
(3)第二步同源重组。与上述敲除wcaj基因时第二步同源重组的步骤相同。测序验证获得发生第二次同源重组的正确克隆，进一步将其在37℃条件下培养，丢失其中的pCAGO 质粒，从而获得菌株W2△wcaj△nudd，命名为TKYW1。
表2敲除nudd基因所用引物
实施例2构建菌株TKYW2-1、TKYW2-2、TKYW2-3
在菌株TKYW1的基础上，利用与上述CRISPR/Cas9技术同样的方法，在糖流出转运蛋白基因setA(核苷酸序列如SEQ ID NO：1所示)前面分别插入3种不同强度的组成型启动子PJ23108、PJ23110、PJ23119(http://parts.igem.org/Part:BBa_J23100)，启动子序列见表3。同时，在利用CRISPR/Cas9进行第二次重组时，保留了氯霉素抗性基因的启动子(核苷酸序列如SEQ ID NO：19所示)，以实现双启动子对setA进行原位过表达，所获菌株分别命名为TKYW2-1、TKYW2-2、TKYW2-3。
表3在基因组上过表达setA基因所用启动子
1.第一步同源重组片段的构建
以菌株E.coli K12 MG1655基因组为模板，分别利用表4中的引物对Sup-1和Sup-2，引物对Sdown-1和Sdown-2为引物，分别PCR扩增得到同源重组的上、下游同源臂。以构建菌株W2△wcaj时获得的带有cat-N20序列的片段为模板，利用表4中的引物Scm-1和Scm-2进行PCR扩增，获得带有cat基因序列的片段。以N20-1为上游引物，分别以108-2，110-2，119-2为下游引物，不使用模板，进行PCR扩增，获得分别带有PJ23108、PJ23110、PJ23119启动子的3个基因片段。以Sup-1和Sdown-2为引物，利用上述PCR扩增得到的上游同源 臂、带有cat基因序列的片段、带有PJ23108(或PJ23110或PJ23119)启动子的基因片段、以及下游同源臂，共4个片段为模板，进行重叠PCR扩增，得到第一步同源重组用的3种分别带有PJ23108、PJ23110或PJ23119启动子的不同片段。
表4构建在基因组上过表达setA基因的菌株所用引物
2.第一步同源重组
利用常规的质粒转化法将pCAGO质粒转化到菌株TKYW1中，获得菌株TKYW1(pCAGO)。利用含有1％葡萄糖和浓度为0.1mM的IPTG的LB培养基制备TKYW1(pCAGO)感受态，利用电转化方法分别导入上述第一次同源重组用的3种分别带有PJ23108、PJ23110、PJ23119启动子的不同片段，转化后的菌液分别涂布于含100mg/L氨苄青霉素和25mg/L氯霉素，以及1％葡萄糖的LB平板上，30℃培养。挑取转化子进行菌落PCR鉴定，获得正确的第一步同源重组后的3种分别带有PJ23108、PJ23110、PJ23119启动子的菌株。
3.第二步同源重组
与上述敲除wcaj基因时第二步同源重组的步骤相同。测序验证获得发生第二次同源重组的正确克隆，进一步将其在37℃条件下培养，丢失其中的pCAGO质粒，从而获得3种分别带有PJ23108，PJ23110，PJ23119启动子的菌株，分别命名为TKYW2-1，TKYW2-2，TKYW2-3。实施例3质粒pTrc99a-Ptrc-futC-manC构建
以专利文献CN112501106A所述的质粒pTrc99a-futC-manC为模板(质粒 pTrc99a-futC-manC构建涉及的Ptrc启动子的核苷酸序列如SEQ ID NO：12所示；阿拉伯糖诱导型启动子Para启动子的核苷酸序列如SEQ ID NO：15所示；甘露糖-1-磷酸鸟嘌呤转移酶编码基因manC的核苷酸序列如SEQ ID NO：10所示；2'-岩藻糖基乳糖合成酶编码基因futC的核苷酸序列如SEQ ID NO：11所示)，以表5中的Darac-F和Darac-R为引物进行PCR扩增，将PCR产物进行纯化和回收后，使用无缝克隆酶(pEASY-Uni SeamlessCloning and Assembly Kit，北京全式金生物技术有限公司)自接，转化到大肠杆菌E.coli JM109感受态细胞中，在含有100mg/L氨苄青霉素的LB平板上培养，挑取转化子测序验证，获得正确的重组质粒，命名为质粒pTrc99a-Ptrc-futC-manC，其序列如SEQ ID NO：18所示。
表5构建质粒pTrc99a-Ptrc-futC-manC所用引物
实施例4在质粒pTrc99a-Ptrc-futC-manC上过表达setA基因的质粒构建
在质粒pTrc99a-Ptrc-futC-manC的基础上，分别利用启动子Ptrc、PJ23108、PJ23110、PJ23119过表达setA，构建出质粒pTrc99a-Ptrc-futC-manC-Ptrc-setA,pTrc99a-Ptrc-futC-manC-PJ23108-setA,pTrc99a-Ptrc-futC-manC-PJ23110-setA和pTrc99a-Ptrc-futC-manC-PJ23119-setA。
1.质粒pTrc99a-Ptrc-futC-manC-Ptrc-setA的构建
以质粒pTrc99a-Ptrc-futC-manC为模板，以表6中的T-AZT-F和AZT-R为引物进行PCR扩增，得到含有futC和manC基因的线性载体片段。以大肠杆菌MG1655基因组为模板，以A-F和A-R1为引物进行PCR扩增，获得带有setA基因的片段，以质粒pTrc99a-Ptrc-futC-manC为模板，以T-F和T-R为引物进行PCR扩增，获得带有Ptrc启动子的基因片段，将上述三种PCR产物进行纯化和回收后，使用无缝克隆酶(pEASY-Uni SeamlessCloning and Assembly Kit，北京全式金生物技术有限公司)连接，转化到E.coli JM109感受态细胞中，在含有100mg/L氨苄青霉素的LB平板上培养，挑取转化子测序验证，获得正确的重组质粒，命名为pTrc99a-Ptrc-futC-manC-Ptrc-setA。
表6构建质粒pTrc99a-Ptrc-futC-manC-Ptrc-setA所用引物

2.利用启动子PJ23108，PJ23110和PJ23119过表达setA的质粒构建
以质粒pTrc99a-Ptrc-futC-manC为模板，分别以表7中的108-AZT-F，110-AZT-F和119-AZT-F为上游引物，以表6中的AZT-R为下游引物进行PCR扩增，得到3种含有futC和manC基因的线性载体片段。以大肠杆菌MG1655基因组为模板，以表6中的A-F和表7中的A-R为引物进行PCR扩增，获得带有setA基因的片段。将上述PCR产物进行纯化和回收后，使用无缝克隆酶，将3种含有futC和manC基因的线性载体片段分别与带有setA基因的片段连接，分别转化到E.coli JM109感受态细胞中，在含有100mg/L氨苄青霉素的LB平板上培养，挑取转化子测序验证，获得正确的重组质粒，分别命名为pTrc99a-Ptrc-futC-manC-PJ23108-setA,pTrc99a-Ptrc-futC-manC-PJ23110-setA和pTrc99a-Ptrc-futC-manC-PJ23119-setA。
表7构建利用pTrc99a-Ptrc-futC-manC过表达setA的质粒所用引物
实施例52'-岩藻糖基乳糖生产菌株的构建和发酵测试
利用电转化的方法，将质粒pTrc99a-Ptrc-futC-manC分别导入到菌株TKYW1，TKYW2-1，TKYW2-2和TKYW2-3中，构建出菌株TKYW1(pTrc99a-Ptrc-futC-manC)，TKYW2-1(pTrc99a-Ptrc-futC-manC)，TKYW2-2(pTrc99a-Ptrc-futC-manC)和TKYW2-3(pTrc99a-Ptrc-futC-manC)。将质粒pTrc99a-Ptrc-futC-manC-Ptrc-setA,pTrc99a-Ptrc-futC-manC-PJ23108-setA,pTrc99a-Ptrc-futC-manC-PJ23110-setA和pTrc99a-Ptrc-futC-manC-PJ23119-setA分别导入菌株TKYW1中，构建出菌株TKYW1 (pTrc99a-Ptrc-futC-manC-Ptrc-setA)，TKYW1(pTrc99a-Ptrc-futC-manC-PJ23108-setA)，TKYW1(pTrc99a-Ptrc-futC-manC-PJ23110-setA)和TKYW1(pTrc99a-Ptrc-futC-manC-PJ23119-setA)。
测试上述菌株发酵生产2'-岩藻糖基乳糖的水平，所用培养基为：LB培养基：NaCl 10g/L，酵母粉5g/L，蛋白胨10g/L，pH为7.0。发酵培养基：KH2PO4 3g/L，酵母粉8g/L，(NH4)2SO44g/L，柠檬酸1.7g/L，MgSO4·7H2O 2g/L，硫胺素10mg/L，甘油10g/L，乳糖5g/L，1ml/L微量元素(FeCl3·6H2O 25g/L，MnCl2·4H2O 9.8g/L，CoCl2·6H2O 1.6g/L，CuCl2·H2O 1g/L，H3BO3 1.9g/L，ZnCl2 2.6g/L，Na2MOO4·2H2O 1.1g/L，Na2SeO3 1.5g/L，NiSO4·6H2O 1.5g/l)，利用氨水调pH为7.2。
发酵测试过程为：
挑取2'-岩藻糖基乳糖生产菌株单菌落，转接到含有50mg/L氨苄青霉素的LB液体培养基中，进行摇瓶培养，摇床温度为37℃、转速为220转/min，培养过夜。取10微升培养液作为种子，转接到每个孔中含有1mL发酵培养基的24深孔板中，发酵培养基中含有50mg/L氨苄青霉素以及0.1mM的IPTG，在孔板震荡培养箱中培养，温度为37℃，转速为800转/min。每个菌株平行培养3个样品。培养48h后取样0.5ml，利用超声破碎仪破碎细胞，离心收集上清，煮沸十分钟，加入等体积的乙腈，再次离心收集上清，然后利用0.22μm滤膜过滤。利用HPLC检测2'-岩藻糖基乳糖的浓度，HPLC分析所用色谱柱为Carbohydrate ES 5u250mm*4.6mm，检测器为蒸发光检测器，流动相为70％乙腈(乙腈：水)，流速为0.8mL/min，柱温设定为30℃，进样量为5μL。利用2'-岩藻糖基乳糖标准品对样品浓度进行定量。2’-岩藻糖基乳糖产量见表8。
表8不同菌株生产2'-岩藻糖基乳糖测试结果
从表8可以看出，与对照菌株TKYW1(pTrc99a-Ptrc-futC-manC)相比，所有过表达setA 基因的测试菌株均能够提高2'-FL产量，利用基因组原位过表达setA基因提高2'-FL产量的效果明显优于利用质粒过表达setA基因的效果。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种提高2'-岩藻糖基乳糖生产能力的重组菌株，所述重组菌株的出发菌株为大肠杆菌，其特征在于，所述重组菌株原位过表达糖外排转运体A编码基因，且所述出发菌株为在体内能够合成2'-岩藻糖基乳糖的大肠杆菌。
  2. 根据权利要求1所述的重组菌株，其特征在于，所述重组菌株基因组的原位过表达糖外排转运体A编码基因前面插入组成型启动子和/或氯霉素抗性基因的启动子，以使得原位过表达糖外排转运体A编码基因。
  3. 根据权利要求2所述的重组菌株，其特征在于，所述重组菌株基因组的糖外排转运体A编码基因前插入氯霉素抗性基因的启动子和一种组成型启动子进行原位过表达。
  4. 根据权利要求2所述的重组菌株，其特征在于，所述组成型启动子选自PJ23102、PJ23104、PJ23105、PJ23108、PJ23100、PJ23110、PJ23111、PJ23113、PJ23119、P637、P699中的任一种。
  5. 根据权利要求1所述的重组菌株，其特征在于，所述出发菌株是进行过2'-岩藻糖基乳糖从头合成途径相关酶的编码基因的基因改造的重组菌株；
    优选地，其包括敲除大肠杆菌基因组上的β-半乳糖苷酶编码基因，并过表达磷酸甘露糖异构酶编码基因、磷酸甘露糖变位酶编码基因、GDP-岩藻糖合成酶编码基因、GDP-甘露糖-4,6-脱水酶编码基因、β-半乳糖苷透性酶编码基因、甘露糖-1-磷酸鸟嘌呤转移酶编码基因；
    还优选地，括敲除大肠杆菌基因组上的lacI、UDP-葡萄糖脂质载体转移酶编码基因、GDP-甘露糖水解酶编码基因；
    更优选地，还包括2'-岩藻糖基乳糖补救途径相关酶的编码基因、乳糖lac操纵子序列(具体是敲除出发菌株的乳糖lac操纵子序列中的Plac启动子序列及调节基因lacI和lacZ)、敲除2'-岩藻糖基乳糖合成前体降解相关酶的编码基因的基因改造；更优选地，包括下述基因改造：
    敲除大肠杆菌基因组上的β-半乳糖苷酶编码基因；
    敲除大肠杆菌基因组上的UDP-葡萄糖脂质载体转移酶编码基因；
    敲除大肠杆菌基因组上的GDP-甘露糖水解酶编码基因；
    在大肠杆菌基因组上插入磷酸甘露糖异构酶编码基因；
    在大肠杆菌基因组上插入磷酸甘露糖变位酶编码基因；
    在大肠杆菌基因组上插入GDP-岩藻糖合成酶编码基因；
    在大肠杆菌基因组上插入GDP-甘露糖-4,6-脱水酶编码基因；
    原位过表达β-半乳糖苷透性酶编码基因；
    在质粒上过表达甘露糖-1-磷酸鸟嘌呤转移酶编码基因；
    在质粒上过表达2'-岩藻糖基乳糖合成酶编码基因。
  6. 根据权利要求5所述的重组菌株，其特征在于，所述GDP-岩藻糖合成酶编码基因、GDP-甘露糖-4,6-脱水酶编码基因、磷酸甘露糖异构酶编码基因、磷酸甘露糖变位酶编码基因为单拷贝插入基因组过表达；所述甘露糖-1-磷酸鸟嘌呤转移酶编码基因、2'-岩藻糖基乳糖合成酶编码基因为多拷贝过表达。
  7. 根据权利要求5所述的重组菌株，其特征在于，所述磷酸甘露糖异构酶编码基因、磷酸甘露糖变位酶编码基因、GDP-岩藻糖合成酶编码基因、GDP-甘露糖-4,6-脱水酶编码基因、β-半乳糖苷透性酶编码基因、甘露糖-1-磷酸鸟嘌呤转移酶编码基因、2'-岩藻糖基乳糖合成酶编码基因futC使用Ptrc启动子过表达；所述质粒选自pTrc99a、pSB4K5、pET28a或pET22b中的任一种。
  8. 根据权利要求7所述的重组菌株，其特征在于，所述糖外排转运体A编码基因的核苷酸序列如SEQ ID NO.1所示；所述β-半乳糖苷酶编码基因的核苷酸序列如SEQ ID NO：2所示；所述UDP-葡萄糖脂质载体转移酶编码基因的核苷酸序列如SEQ ID NO：3所示；所述GDP-甘露糖水解酶编码基因的核苷酸序列如SEQ ID NO：4所示；所述磷酸甘露糖异构酶编码基因的核苷酸序列如SEQ ID NO：5所示；所述磷酸甘露糖变位酶编码基因的核苷酸序列如SEQ ID NO：6所示；所述GDP-岩藻糖合成酶编码基因的核苷酸序列如SEQ ID NO：7所示；所述GDP-甘露糖-4,6-脱水酶编码基因的核苷酸序列如SEQ ID NO：8所示；所述β-半乳糖苷透性酶编码基因的核苷酸序列如SEQ ID NO：9所示；所述甘露糖-1-磷酸鸟嘌呤转移酶编码基因的核苷酸序列如SEQ ID NO：10所示；所述2'-岩藻糖基乳糖合成酶编码基因的核苷酸序列如SEQ ID NO：11所示；所述Ptrc启动子的核苷酸序列如SEQ ID NO：12所示；
    另外优选地，所述出发菌株选自大肠杆菌K12 MG1655、大肠杆菌BL21(DE3)、大肠杆菌JM109、大肠杆菌BW25113中的任一种。
  9. 一种提高2'-岩藻糖基乳糖生产能力的重组菌株的构建方法，其特征在于，所述出发菌株为大肠杆菌，优选地选自大肠杆菌K12 MG1655，所述构建方法包括不分先后的如下步骤：
    敲除出发菌株的乳糖lac操纵子序列中的Plac启动子序列及调节基因lacI和β-半乳糖苷 酶编码基因，在原β-半乳糖苷酶编码基因位点之后以Ptrc启动子过表达GDP-岩藻糖合成酶编码基因、GDP-甘露糖-4,6-脱水酶编码基因和β-半乳糖苷透性酶编码基因；
    在出发菌株的乙醇脱氢酶编码基因adhe位点上以Ptrc启动子过表达磷酸甘露糖异构酶编码基因和磷酸甘露糖变位酶编码基因；
    敲除出发菌株的UDP-葡萄糖脂质载体转移酶编码基因和GDP-甘露糖水解酶编码基因；
    在出发菌株的setA前面插入组成型启动子，并插入氯霉素抗性基因的启动子；
    在出发菌株中导入过表达甘露糖-1-磷酸鸟嘌呤转移酶编码基因和2'-岩藻糖基乳糖合成酶编码基因的质粒；优选地，通过构建含有该两基因串联的质粒，再导入出发菌株中构建重组菌株。
  10. 权利要求1至8任一项所述的重组菌株或权利要求9所述方法构建的重组菌株在发酵生产2'-岩藻糖基乳糖中的应用；优选地，所述重组菌株的发酵培养基碳源选自甘油、乳糖中的至少一种；所述重组菌株的发酵培养基有机氮源选自酵母粉、胰蛋白胨、牛肉膏中的至少一种；发酵培养的温度为36℃～40℃；发酵培养的pH为6.8～7.8。
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