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WO2024130899A1 - 一种甲硫氨酸氨肽酶突变体、大肠杆菌工程菌及应用 - Google Patents

一种甲硫氨酸氨肽酶突变体、大肠杆菌工程菌及应用 Download PDF

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WO2024130899A1
WO2024130899A1 PCT/CN2023/087185 CN2023087185W WO2024130899A1 WO 2024130899 A1 WO2024130899 A1 WO 2024130899A1 CN 2023087185 W CN2023087185 W CN 2023087185W WO 2024130899 A1 WO2024130899 A1 WO 2024130899A1
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gene
encoding
seq
encoding gene
fucosyllactose
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PCT/CN2023/087185
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English (en)
French (fr)
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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 methionine aminopeptidase mutant, an Escherichia coli engineering bacterium and applications.
  • Human milk oligosaccharides are nutritional components of breast milk. They have a complex structure, with more than 200 known components. 2'-fucosyllactose (2'-FL) and 3-fucosyllactose (3-FL) are the main components of human milk oligosaccharides, which play important physiological functions in regulating intestinal flora and immunity, and have broad market prospects.
  • the European Commission has approved 2'-fucosyllactose and 3-fucosyllactose as new resource foods for sale in 2019 and 2021, respectively.
  • Microbial fermentation is currently the mainstream production method for 2'-fucosyllactose and 3-fucosyllactose.
  • Commonly used strains are Escherichia coli of various genetic backgrounds.
  • the fermentation of Escherichia coli to prepare various human milk oligosaccharides involves a variety of shared enzymes and transporters (such as the sugar efflux transporter SetA shown in WO2010142305A1).
  • the biosynthetic pathways of 2'-fucosyllactose and 3-fucosyllactose are very similar; in order to improve the fermentation yield of 2'-fucosyllactose and 3-fucosyllactose, the editing strategies of the relevant genes involved in the biosynthetic pathways of the two (such as knockout of specific genes, overexpression of specific genes, insertion of specific genes, etc.) are also very similar (Huang D, Yang KX, Liu J, et al. Metabolic engineering of Escherichia coli for the production of 2'-fucosyllactose and 3-fucosyllactose through modular pathway enhancement [J].
  • ⁇ -galactosidase encoding gene lacZ UDP-glucose lipid carrier transferase encoding gene wcaj, GDP-mannose hydrolase encoding gene nudd, regulatory gene lacI in the lactose lac operon sequence, L-fucose isomerase encoding gene fucI, L-fucokinase encoding gene fucK, L-fucose-1-phosphate aldolase encoding gene fucA, D-arabinose isomerase encoding gene ara A, L-rhamnose isomerase encoding gene rhaA; and/or one or more of the following genes were overexpressed or inserted: GDP-fucose synthase encoding gene wcaG, GDP-mannose-4,6-dehydratase encoding gene gmd, ⁇ -galactosidase permease encoding gene lac
  • the present invention uses gene editing technology to edit the coding genes of multiple enzymes or transporters of Escherichia coli.
  • a methionine aminopeptidase mutant that can increase the yield of 2'-fucosyllactose and 3-fucosyllactose is obtained through gene editing technology, and the corresponding Escherichia coli engineering bacteria are constructed.
  • One of the technical solutions of the present invention provides a methionine aminopeptidase mutant, whose amino acid sequence corresponds to the wild-type methionine aminopeptidase (Map) amino acid sequence shown in SEQ ID NO: 24, and the 44th amino acid position has a mutation: I>S, that is, the 44th isoleucine (Ile) is mutated to serine (Ser).
  • This mutation is beneficial to improving the fermentation yield of 2'-fucosyllactose and 3-fucosyllactose.
  • the methionine aminopeptidase mutant is identical to the wild-type methionine aminopeptidase amino acid shown in SEQ ID NO: 24. Sequence homology >90% is acceptable.
  • the amino acid sequence of the methionine aminopeptidase mutant is shown in SEQ ID NO: 15.
  • the methionine aminopeptidase mutant shown in SEQ ID NO: 15 has only the 44th amino acid position mutated from isoleucine to serine relative to the wild-type methionine aminopeptidase. Fermentation verification using Escherichia coli showed that the methionine aminopeptidase mutant shown in SEQ ID NO: 15 is beneficial to improving the fermentation yield of 2'-fucosyllactose and 3-fucosyllactose.
  • the second technical solution of the present invention provides a coding gene, which expresses the aforementioned methionine aminopeptidase mutant in cells.
  • the coding gene expresses the methionine aminopeptidase mutant shown in SEQ ID NO: 15 in the cell. Due to the degeneracy of codons, an amino acid sequence can be translated and expressed by countless different nucleic acid sequences. Therefore, the present invention further provides a coding gene, which carries at least a nucleotide fragment shown in SEQ ID NO: 22 or a nucleotide sequence fragment encoding the same amino acid sequence.
  • the nucleotide fragment shown in SEQ ID NO: 22 can encode and express the methionine aminopeptidase mutant shown in SEQ ID NO: 15 in the cell.
  • the third technical solution of the present invention provides an Escherichia coli genetically engineered bacterium that can be used for fermentation production of 2'-fucosyllactose and/or 3-fucosyllactose, and is beneficial to improving the fermentation yield of 2'-fucosyllactose and/or 3-fucosyllactose.
  • the genetically engineered bacterium is capable of synthesizing 2'-fucosyllactose and/or 3-fucosyllactose in vivo, and expresses the aforementioned methionine aminopeptidase mutant.
  • the genetically engineered bacterium expresses the methionine aminopeptidase mutant shown in SEQ ID NO: 15.
  • the genome of the genetically engineered bacterium carries at least the nucleotide fragment shown in SEQ ID NO: 22 or a nucleotide sequence fragment encoding the same amino acid sequence, wherein the nucleotide fragment shown in SEQ ID NO: 22 can express the methionine aminopeptidase mutant shown in SEQ ID NO: 15 after being introduced into the genetically engineered bacterium by site-directed mutation of the wild-type methionine aminopeptidase encoding gene.
  • the target gene (such as the nucleotide fragment shown in SEQ ID NO: 22) can be introduced into the genetically engineered bacteria by methods such as plasmid transformation.
  • the starting strain of the genetically engineered bacteria is selected from any one of Escherichia coli K12 MG1655, Escherichia coli BL21 (DE3), Escherichia coli JM109, and Escherichia coli BW25113. These strains have been widely used, among which Escherichia coli K12 MG1655 is one of the most famous and well-studied organisms in biology.
  • Escherichia coli K12 MG1655 has been deposited in the American Type Culture Collection (deposit number ATCC 53103, ATCC 47076, ATCC700926); Escherichia coli BL21 (DE3) has been deposited in BCCM genecorner (deposit number LMBP 1455); Escherichia coli JM109 has been deposited in the American Type Culture Collection (deposit number ATCC68635, ATCC68868); Escherichia coli BW25113 has been deposited in the Escherichia coli Genetic Stock Center (Coli The strain is deposited at the Genetics Stock Center (deposit number CGSC#7636). As a starting strain commonly used by those skilled in the art, those skilled in the art have the ability to learn the source and purchase channels of the above strains.
  • the coding genes of the related enzymes or transporters involved in the synthesis pathway of 2'-fucosyllactose and/or 3-fucosyllactose in the genetically engineered bacteria can also be further edited.
  • These gene edits can be to knock out at least one of the following genes on the Escherichia coli genome: ⁇ -galactosidase encoding gene lacZ, UDP-glucose lipid carrier transferase encoding gene wcaj, GDP-mannose hydrolase encoding gene nudd, regulatory gene lacI in the lactose lac operon sequence, L-fucose isomerase encoding gene fucI, L-fucokinase encoding gene fucK, L-fucose-1-phosphate aldolase encoding gene fucA, D-arabinose isomerase encoding gene ara A, L-rhamnose isomerase encoding gene rhaA.
  • At least one of the following genes may be overexpressed or inserted: GDP-fucose synthase encoding gene wcaG, GDP-mannose-4,6-dehydratase encoding gene gmd, ⁇ -galactosidase permease encoding gene lacY, phosphomannose isomerase encoding gene manA, phosphomannose mutase encoding gene manB, sugar efflux transporter A encoding gene setA, mannose-1-phosphate guanine transferase encoding gene manC, 2'-fucosyllactose synthase encoding gene futC, ⁇ -(1,3)-fucosyltransferase encoding gene futA, L-fucokinase/GDP-L-fucose pyrophosphorylase encoding gene fkp.
  • the above-mentioned multiple gene editing methods may be any of the above-mentioned multiple gene editing methods (gene knock
  • the gene editing of the coding genes of the relevant enzymes or transporters involved in the synthesis pathway of 2'-fucosyllactose and/or 3-fucosyllactose in the genetically engineered bacteria comprises:
  • mannose-1-phosphate guanylyltransferase encoding gene manC was overexpressed on a plasmid
  • the above-mentioned gene editing of lacZ, wcaG, gmd, lacY, manA, manB, wcaj, nudd, setA, and manC is beneficial to improving the fermentation yields of 2'-fucosyllactose and 3-fucosyllactose;
  • the above-mentioned gene editing of futC is beneficial to improving the fermentation yield of 2'-fucosyllactose, and the above-mentioned gene editing of futA is beneficial to improving the fermentation yield of 2'-fucosyllactose.
  • the wcaG, gmd, manA, and manB are single-copy inserted into the genome and overexpressed; and the manC, futA, and futC are multi-copy overexpressed.
  • the manA, manB, wcaG, gmd and lacY are overexpressed using the P trc promoter.
  • the plasmid is selected from any one of pTrc99a, pSB4K5, pET28a or pET22b.
  • nucleotide sequence of the aforementioned P trc promoter is shown in SEQ ID NO:4.
  • nucleotide sequence of lacZ is shown in SEQ ID NO: 3; the nucleotide sequence of wcaG is shown in SEQ ID NO: 5; the nucleotide sequence of gmd is shown in SEQ ID NO: 6; the nucleotide sequence of lacY is shown in SEQ ID NO: 7; the nucleotide sequence of manA is shown in SEQ ID NO: 9; the nucleotide sequence of manB is shown in SEQ ID NO: 10; the nucleotide sequence of wcaj is shown in SEQ ID NO: 11; the nucleotide sequence of nudd is shown in SEQ ID NO: 13; the nucleotide sequence of setA is shown in SEQ ID NO: 14; the nucleotide sequence of manC is shown in SEQ ID NO: 17; and the nucleotide sequence of futC is shown in SEQ ID NO: 18.
  • the nucleotide sequence of futA is shown in SEQ ID NO: 21.
  • the nucleotide sequence templates that can be used in plasmid construction are diverse and variable.
  • the gene fragment shown in SEQ ID NO: 20 is used as the template of futA in plasmid construction.
  • a constitutive promoter is inserted in front of setA for in situ overexpression and/or a promoter of a chloramphenicol resistance gene is inserted for in situ overexpression.
  • the nucleotide sequence of the promoter of the chloramphenicol resistance gene is as shown in SEQ ID NO: 23.
  • 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 .
  • in situ overexpression is a term opposite to overexpression on a plasmid, and is also a commonly used term in the art, which refers to overexpression in situ, that is, through molecular biological means, such as promoter, ribosome binding site and transcription regulatory factor modification or codon optimization, the target gene located in situ on the chromosome is regulated to improve the level of gene transcription and translation.
  • “In situ overexpression” of a gene means overexpression in situ on the chromosome, which can be achieved by inserting an additional promoter to increase the level of in situ expression on the chromosome, or by other methods that are more easily conceived by those skilled in the art.
  • the promoter activity of the small molecule regulatory RNA gene sgrS in front of the setA gene can also be modified, or an additional promoter can be inserted in front of the sgrS gene.
  • the additionally inserted promoter used for gene overexpression can be a constitutive promoter and/or an inducible promoter.
  • the constitutive promoter and inducible promoter described herein can be promoters suitable for prokaryotic expression systems, especially promoters suitable for Escherichia coli expression systems, including natural promoters and artificially constructed promoters.
  • the aforementioned genetically engineered bacteria, mutants, coding genes and gene editing technologies involved in their construction, as well as additionally inserted promoters commonly used to achieve in situ overexpression or plasmid overexpression of the aforementioned genes are well known in the art, and can be found in Genetic Engineering Experimental Technology compiled by Peng Xiuling et al.
  • a fourth technical solution of the present invention provides the use of the aforementioned genetically engineered bacteria in the fermentation production of 2'-fucosyllactose and/or 3-fucosyllactose.
  • the genetically engineered bacteria overexpressing futA on the plasmid are particularly suitable for improving the fermentation yield of 3-fucosyllactose
  • the genetically engineered bacteria overexpressing futC on the plasmid are particularly suitable for improving the fermentation yield of 2'-fucosyllactose.
  • the present invention obtains the corresponding methionine aminopeptidase by site-directed mutagenesis of Escherichia coli methionine aminopeptidase. It is found that the yield of 2'-fucosyllactose and 3-fucosyllactose of Escherichia coli expressing the methionine aminopeptidase is unexpectedly improved.
  • the Escherichia coli setA gene is additionally overexpressed in situ, and the Escherichia coli lacZ, wcaG, gmd, lacY, manA, manB, wcaj, nudd, futC, manC, futA are edited by beneficial genes such as in situ overexpression and plasmid overexpression, the yield of 2'-fucosyllactose and 3-fucosyllactose is higher.
  • Escherichia coli W2 is briefly described based on the content of patent document CN112501106A, and its construction method is introduced into this embodiment.
  • Escherichia coli W2 in CN112501106A is constructed with Escherichia coli K12MG1655 as the starting strain
  • the P lac promoter sequence and the regulatory genes lacI and lacZ in the lactose lac operon sequence of the starting strain are knocked out, and wcaG, gmd and lacY are overexpressed with the P trc promoter after the original lacZ site to obtain the W1 strain
  • manA and manB are overexpressed with the P trc promoter at the alcohol dehydrogenase encoding gene adhe site to obtain the W2 strain.
  • the nucleotide sequence of the P lac promoter involved in the construction of Escherichia coli W2 is shown in SEQ ID NO: 1; the nucleotide sequence of lacI is shown in SEQ ID NO: 2; the nucleotide sequence of lacZ is shown in SEQ ID NO: 3; the nucleotide sequence of the P trc promoter is shown in SEQ ID NO: 4; the nucleotide sequence of wcaG is shown in SEQ ID NO: 5; the nucleotide sequence of gmd is shown in SEQ ID NO: 6; the nucleotide sequence of lacY is shown in SEQ ID NO: 7; the nucleotide sequence of adhE is shown in SEQ ID NO: 8; the nucleotide sequence of manA is shown in SEQ ID NO: 9; and the nucleotide sequence of manB is shown in SEQ ID NO: 10.
  • CRISPR/Cas9 technology was used to knock out wcaj (nucleotide sequence is shown in SEQ ID NO: 11).
  • the CRISPR/Cas9 technology used in the experiment refers to the previous research report [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 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. Sci Rep 7, 16624].
  • SEQ ID NO: 12 is the nucleotide sequence of the cat gene and its promoter
  • primers cat-1 and cat20-2 were used for PCR amplification to obtain a fragment with the cat-N20 sequence.
  • primers up-1 and down-2 were used for overlapping PCR amplification to obtain the first step homologous recombination fragment.
  • the pCAGO plasmid was transformed into strain W2 using conventional plasmid transformation methods to obtain strain W2 (pCAGO).
  • W2 (pCAGO) competent cells were prepared using LB medium containing 1% glucose and 0.1 mM IPTG (isopropyl- ⁇ -D-thiogalactoside), and the first homologous recombination fragment was introduced using electroporation.
  • the transformed bacterial solution was spread on an LB plate containing 100 mg/L ampicillin and 25 mg/L chloramphenicol, as well as 1% glucose, and cultured at 30°C. Transformants were picked for colony PCR identification to obtain the correct first step homologous recombination strain.
  • Second step homologous recombination The first step homologous recombination strain was inoculated into LB liquid medium containing 100 mg/L ampicillin, 0.1 mM IPTG and 2 g/L arabinose, cultured at 30°C for more than 6 h, and single colonies were isolated by streaking. Clones that could grow on LB plates containing 100 mg/L ampicillin but could not grow on LB plates containing 25 mg/L chloramphenicol were screened. Sequencing confirmed the correct clone that underwent the second homologous recombination, and further cultured it at 37°C to lose the pCAGO plasmid, thereby obtaining strain W2 ⁇ wcaj.
  • the nudd gene on the genome was knocked out using the same method as the above CRISPR/Cas9 technology (nucleotide sequence as shown in SEQ ID NO: 13), and the strain W2 ⁇ wcaj ⁇ nudd was constructed and named ZKYW1.
  • the specific method is described in detail below:
  • Second step of homologous recombination The steps of the second step of homologous recombination were the same as those for knocking out the wcaj gene.
  • the correct clone that underwent the second homologous recombination was obtained by sequencing and further cultured at 37°C to lose the pCAGO plasmid, thereby obtaining the strain W2 ⁇ wcaj ⁇ nudd, which was named TKYW1.
  • the constitutive promoter P J23110 http://parts.igem.org/Part:BBa_J23100 was inserted in front of the sugar efflux transporter setA gene (nucleotide sequence as shown in SEQ ID NO: 14) using the same method as the above-mentioned CRISPR/Cas9 technology.
  • the sequence of the promoter P J23110 is: TTTACGGCTAGCTCAGTCCTAGGTACAATGCTAGC; at the same time, when CRISPR/Cas9 was used for the second recombination, the promoter of the chloramphenicol resistance gene (nucleotide sequence as shown in SEQ ID NO: 23) was retained to achieve in situ overexpression of setA by dual promoters.
  • the resulting strain was named TKYW2-2.
  • the first step is to construct the homologous recombination fragment.
  • the primer pairs Sup-1 and Sup-2 and the primer pairs Sdown-1 and Sdown-2 in Table 3 were used as primers to PCR amplify the upstream and downstream homologous arms of homologous recombination.
  • the primers Scm-1 and Scm-2 in Table 3 were used for PCR amplification to obtain a fragment with the cat gene sequence.
  • PCR amplification was performed without using a template to obtain a gene fragment with the P J23110 promoter.
  • Sup-1 and Sdown-2 as primers, the upstream homologous arm obtained by the above PCR amplification, the fragment with the cat gene sequence, the gene fragment with the P J23110 promoter, and the downstream homologous arm, a total of 4 fragments were used as templates for overlapping PCR amplification to obtain the fragments for the first step of homologous recombination.
  • the pCAGO plasmid was transformed into the strain TKYW1 using a conventional plasmid transformation method to obtain the strain TKYW1 (pCAGO).
  • the TKYW1 (pCAGO) competent state was prepared using LB medium containing 1% glucose and 0.1 mM IPTG, and the above-mentioned fragments for the first homologous recombination were introduced respectively using the electroporation method.
  • the transformed bacterial liquid was spread on LB plates containing 100 mg/L ampicillin and 25 mg/L chloramphenicol, as well as 1% glucose, and cultured at 30°C. The transformants were picked for colony PCR identification to obtain the correct first step homologous recombination strain.
  • Second step of homologous recombination The steps of the second step of homologous recombination were the same as those for knocking out the wcaj gene.
  • the correct clone that underwent the second homologous recombination was obtained by sequencing and further cultured at 37°C to lose the pCAGO plasmid, thereby obtaining a strain with the P J23110 promoter, named TKYW2-2.
  • the amino acid sequence of the wild-type methionine aminopeptidase of Escherichia coli K12 MG1655 is shown in SEQ ID NO: 24.
  • strain TKYW2-2 Based on strain TKYW2-2, the methionine aminopeptidase gene map on the genome was subjected to point mutation using the same method as the above-mentioned CRISPR/Cas9 technology.
  • the 44th isoleucine of its translated protein was mutated to serine (Map Ile44Ser ), and the amino acid sequence corresponding to the mutated methionine aminopeptidase is shown in SEQ ID NO: 15.
  • the constructed strain was named TKYW3.
  • a mutant strain of MG1655map T131G (map T131G nucleotide sequence is shown in SEQ ID NO: 22) with a point mutation in the map gene preserved in the laboratory was used as a template, and the primer pair Mup-1 and Mup-2, and the primer pair Mdown-1 and Mdown-2 in Table 4 were used as primers to PCR amplify the upstream and downstream homology arms of homologous recombination.
  • the fragment with the cat-N20 sequence obtained by PCR when constructing strain W2 ⁇ wcaj was used as a template, and primers Mcat-1 and Mcat20-2 were used to perform PCR amplification to obtain a new fragment with the cat-N20 sequence.
  • the secondary homologous recombination fragment contains a point mutation in the map gene, that is, the 131st base of the wild-type map gene changes from T to G.
  • the pCAGO plasmid was transformed into the strain TKYW2-2 using a conventional plasmid transformation method to obtain the strain TKYW2-2 (pCAGO).
  • the TKYW2-2 (pCAGO) competent state was prepared using LB medium containing 1% glucose and 0.1 mM IPTG, and the first homologous recombination fragment was introduced using an electroporation method.
  • the transformed bacterial solution was spread on an LB plate containing 100 mg/L ampicillin and 25 mg/L chloramphenicol, as well as 1% glucose, and cultured at 30°C. The transformants were picked for colony PCR identification to obtain the correct first step homologous recombination strain.
  • the steps of the second homologous recombination were the same as those for knocking out the wcaj gene.
  • the correct clone that underwent the second homologous recombination was obtained by sequencing and further cultured at 37°C to lose the pCAGO plasmid, thereby obtaining the strain TKYW2-2map T131G , which was named TKYW3.
  • the plasmid pTrc99a-futC-manC described in patent document CN112501106A was used as a template (the nucleotide sequence of the P trc promoter involved in the construction of the plasmid pTrc99a-futC-manC is shown in SEQ ID NO: 4; the nucleotide sequence of the arabinose-inducible promoter P ara promoter is shown in SEQ ID NO: 16; the nucleotide sequence of the mannose-1-phosphate guanine transferase encoding gene manC is shown in SEQ ID NO: 17; the nucleotide sequence of the 2'-fucosyllactose synthase encoding gene futC is shown in SEQ ID NO: 18), and PCR amplification was performed using Darac-F and Darac-R in Table 5 as primers.
  • the seamless cloning enzyme pEASY-Uni Seamless Cloning and Assembly Kit, Beijing Quanshijin Biotechnology Co., Ltd.
  • Escherichia coli JM109 competent cells cultured on LB plates containing 100 mg/L ampicillin, and the transformants were picked for sequencing verification to obtain the correct recombinant plasmid, named plasmid pTrc99a-P trc -futC-manC, whose nucleotide sequence is shown in SEQ ID NO: 19.
  • Plasmid pTrc99a-P trc -futA-manC was constructed by replacing futC in plasmid pTrc99a-P trc -futC-manC with the ⁇ -(1,3)-fucosyltransferase gene futA from Helicobacter pylori NCTC 11637. FutA can catalyze the production of 3-fucosyllactose using GDP-fucose and lactose as substrates.
  • Plasmid pTrc99a-P trc -futA-manC was used as a template, and FUTA-ZT-F and FUTA-ZT-R in Table 6 were used as primers for PCR amplification to obtain a linear vector fragment with futC removed.
  • the vector containing the artificially synthesized futA gene fragment (nucleotide sequence as shown in SEQ ID NO: 20) was used as a template (the gene fragment contains the futA gene as shown in SEQ ID NO: 21), and FUTA-F and FUTA-R were used as primers for PCR amplification to obtain a fragment with the futA gene.
  • plasmids pTrc99a-P trc -futC-manC and pTrc99a-P trc -futA-manC were introduced into TKYW2-2 and TKYW3, respectively, to construct 2'-fucosyllactose producing strains TKYW2-2 (pTrc99a-P trc -futC-manC) and TKYW3 (pTrc99a-P trc -futC-manC), and 3-fucosyllactose producing strains TKYW2-2 (pTrc99a-P trc -futA-manC) and TKYW3 (pTrc99a-P trc -futA-manC), respectively.
  • the fermentation production level of the above strains was tested, and the culture medium used was: LB medium: NaCl 10g/L, yeast powder 5g/L, peptone 10g/L, pH 7.0. Fermentation medium: KH2PO4 3g /L, yeast powder 8g/L, ( NH4 ) 2SO4 4g /L, citric acid 1.7g/L, MgSO4 ⁇ 7H2O 2g/L, thiamine 10mg/L, glycerol 10g/L, lactose 5g/L, 1ml/L trace elements (FeCl 3 ⁇ 6H 2 O 25 g/L, MnCl 2 ⁇ 4H 2 O 9.8 g/L, CoCl 2 ⁇ 6H 2 O 1.6 g/L, CuCl 2 ⁇ H 2 O 1 g/L, H 3 BO 3 1.9 g/L, ZnCl 2 2.6 g/L, Na 2 MOO 4 ⁇ 2H 2 O 1.1 g/
  • the fermentation test process is:
  • the column used for HPLC analysis was Carbohydrate ES 5u 250mm*4.6mm, the detector was an evaporative light detector, the mobile phase was 70% acetonitrile (acetonitrile: water), the flow rate was 0.8mL/min, the column temperature was set at 30°C, and the injection volume was 5 ⁇ L.
  • the sample concentration was quantified using 2'-fucosyllactose or 3-fucosyllactose standards.

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Abstract

提供一种甲硫氨酸氨肽酶突变体、大肠杆菌工程菌及应用。提供的甲硫氨酸氨肽酶突变体对野生型甲硫氨酸氨肽酶进行了点突变，表达该甲硫氨酸氨肽酶突变体的大肠杆菌工程菌的2'-岩藻糖基乳糖和3-岩藻糖基乳糖的发酵产率得到出乎预料地提高。进一步对基因工程菌的wcaG、gmd、lacY、manA、manB、setA基因进行过表达，敲除lacZ、wcaj、nudd基因，质粒上多拷贝过表达futC或futA及manC基因更有利于提高2'-岩藻糖基乳糖和3-岩藻糖基乳糖的产率。

Description

一种甲硫氨酸氨肽酶突变体、大肠杆菌工程菌及应用 技术领域
本发明属于基因工程技术领域，具体涉及一种甲硫氨酸氨肽酶突变体、大肠杆菌工程菌及应用。
背景技术
母乳低聚糖(human milk oligosaccharides，HMOs)是母乳中的营养组成成分。其构成复杂，已知成分已达200多种。2'-岩藻糖基乳糖(2'-fucosyllactose，2'-FL)和3-岩藻糖基乳糖(3-fucosyllactose，3-FL)是母乳低聚糖的主要组成成分，在调节肠道菌群、调节免疫等方面发挥重要生理功能，具有广阔的市场前景。欧盟委员会已分别于2019年和2021年批准2'-岩藻糖基乳糖和3-岩藻糖基乳糖作为新资源食品上市销售。
微生物发酵法是目前2'-岩藻糖基乳糖和3-岩藻糖基乳糖的主流生产方法。常用的菌株为各种遗传背景的大肠杆菌。大肠杆菌发酵制备各种母乳低聚糖涉及到多种共用的酶和转运体(如WO2010142305A1所展示的糖外排转运体SetA)。其中2'-岩藻糖基乳糖和3-岩藻糖基乳糖的生物合成路径极为相似；为提高2'-岩藻糖基乳糖和3-岩藻糖基乳糖的发酵产率，对二者生物合成路径所涉及的相关基因的编辑策略(如特定基因的敲除、特定基因的过表达、特定基因的插入等)也极为相似(Huang D,Yang KX,Liu J,et al.Metabolic engineering of Escherichia coli for the production of 2’-fucosyllactoseand 3-fucosyllactose through modular pathway enhancement[J].Metab Eng,2017,41:23-38；徐铮,李娜,陈盈利,等.人乳寡糖2'-FL和3-FL的生物制备研究进展[J].生物工程学报,2020,36(12):12.)。
通过大肠杆菌发酵生产2'-岩藻糖基乳糖的研究，本领域对2'-岩藻糖基乳糖的从头合成途径和补救途径的关键酶已有深入了解，包括参与合成前体降解的相关酶、糖外排转运体等。并通过对相关酶或转运体编码基因的基因编辑，考察了其对2'-岩藻糖基乳糖、3-岩藻糖基乳糖发酵产率的影响。根据报道，敲除以下基因中的一种或多种：β-半乳糖苷酶编码基因lacZ、UDP-葡萄糖脂质载体转移酶编码基因wcaj、GDP-甘露糖水解酶编码基因nudd、乳糖lac操纵子序列中的调节基因lacI、L-岩藻糖异构酶编码基因fucI、L-墨角藻糖激酶编码基因fucK、L-墨角藻糖-1-磷酸醛缩酶编码基因fucA、D-阿拉伯糖异构酶编码基因ara A、L-鼠李糖异构酶编码基因rhaA；和/或过表达或插入以下基因的一种或多种：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、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、糖外排转运体A编码基因setA、甘露糖-1-磷酸鸟嘌呤转移酶编码基因manC、2'-岩藻糖基乳糖合成酶编码基因futC、L-岩藻糖激酶/GDP-L-岩藻糖焦磷酸化酶编码基因fkp有利于提高2'-岩藻糖基乳糖和3-岩藻糖基乳糖的发酵产率，其中fucI、fucK、fucA、ara A、rhaA、fkp等参与2'-岩藻糖基乳糖的补救合成途径。由于α-(1,3)-岩藻糖基转移酶(FutA)的低活性和不溶性表达(insoluble expression)，与2'-岩藻糖基乳糖相比，3-岩藻糖基乳糖的发酵产率较低。因此，进一步过表达或插入α-(1,3)-岩藻糖基转移酶编码基因futA或对α-(1,3)-岩藻糖基转移酶编码基因futA进行有益突变，更有利于提高3-岩藻糖基乳糖的发酵产率(Yun HC,Park BS,Seo J,et al.Biosynthesis of the human milk oligosaccharide 3‐fucosyllactosein metabolically engineered Escherichia coli via the salvage pathway through increasing GTP synthesis andβ‐galactosidasemodification[J].Biotechnology and Bioengineering,2019.)。
目前的公开出版物中，对2'-岩藻糖基乳糖和3-岩藻糖基乳糖发酵生产工程菌的构建大部分涉及上述酶及转运体的编码基因的编辑(例如CN112662604A、CN112501106A、CN114276971A、CN113195509A、CN113151211A等)。然而，工业生产中2'-岩藻糖基乳糖和3-岩藻糖基乳糖的发酵生产的产率仍然较低，导致其成本较高影响了下游产业发展。且关于甲硫氨酸氨肽酶对大肠杆菌发酵生产2'-岩藻糖基乳糖和3-岩藻糖基乳糖的影响目前缺乏研究。
发明内容
为解决上述技术问题，本发明采用基因编辑技术，对大肠杆菌多种酶或转运体的编码基因进行编辑。通过基因编辑技术获得了一种可提高2'-岩藻糖基乳糖和3-岩藻糖基乳糖产率的甲硫氨酸氨肽酶突变体，并构建了相应的大肠杆菌工程菌。
本发明的技术方案之一，提供了一种甲硫氨酸氨肽酶突变体，其氨基酸序列对应于SEQ ID NO：24所示的野生型甲硫氨酸氨肽酶(Map)氨基酸序列的第44位氨基酸位点具有突变：I>S，即第44位的异亮氨酸(Ile)突变为丝氨酸(Ser)。该突变有利于提高2'-岩藻糖基乳糖和3-岩藻糖基乳糖的发酵产率。
本领域技术人员公知的是，对酶关键位点的突变才会引起酶功能的变化。换言之，对酶非关键位点的突变不导致酶功能的变化。故此，除对应于野生型甲硫氨酸氨肽酶的第44位氨基酸位点的突变外，在不影响酶功能的前提下，对其他非关键位点的突变也是可以接受的。因而，所述甲硫氨酸氨肽酶突变体与SEQ ID NO：24所示的野生型甲硫氨酸氨肽酶氨基酸 序列的同源性>90％是可以接受的。
进一步优选地，所述甲硫氨酸氨肽酶突变体的氨基酸序列如SEQ ID NO：15所示。SEQ ID NO：15所示的甲硫氨酸氨肽酶突变体相对于野生型甲硫氨酸氨肽酶仅第44位氨基酸位点由异亮氨酸突变为丝氨酸。采用大肠杆菌的发酵验证表明，SEQ ID NO：15所示的甲硫氨酸氨肽酶突变体有利于提高2'-岩藻糖基乳糖和3-岩藻糖基乳糖的发酵产率。
在此基础上，本发明的技术方案之二，提供了一种编码基因，所述编码基因在细胞内表达前述甲硫氨酸氨肽酶突变体。
优选地，所述编码基因在细胞内表达SEQ ID NO：15所示的甲硫氨酸氨肽酶突变体。由于密码子的简并性，一种氨基酸序列可以被无数种不同的核酸序列翻译表达出来。因此，本发明进一步提供一种编码基因，所述编码基因至少携带SEQ ID NO：22所示的核苷酸片段或编码相同氨基酸序列的核苷酸序列片段。SEQID NO：22所示的核苷酸片段可在细胞内编码表达SEQ ID NO：15所示的甲硫氨酸氨肽酶突变体。
本发明的技术方案之三，提供了可用于发酵生产2'-岩藻糖基乳糖和/或3-岩藻糖基乳糖，并有利于提高2'-岩藻糖基乳糖和/或3-岩藻糖基乳糖发酵产率的大肠杆菌基因工程菌。该基因工程菌在体内能够合成2'-岩藻糖基乳糖和/或3-岩藻糖基乳糖的大肠杆菌，且表达前述甲硫氨酸氨肽酶突变体。优选地，所述基因工程菌表达SEQ ID NO：15所示的甲硫氨酸氨肽酶突变体。进一步优选地，所述基因工程菌的基因组至少携带SEQ ID NO：22所示的核苷酸片段或编码相同氨基酸序列的核苷酸序列片段，其中SEQID NO：22所示的核苷酸片段通过对野生型甲硫氨酸氨肽酶编码基因的定点突变，在导入基因工程菌内后可表达SEQ ID NO：15所示的甲硫氨酸氨肽酶突变体。
又进一步优选地，可采用质粒转化法等方法将目的基因(如SEQ ID NO：22所示的核苷酸片段)导入基因工程菌。
更进一步优选地，所述基因工程菌的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。这些菌株已得到大量应用，其中大肠杆菌K12 MG1655(Escherichia colistrain K12 MG1655)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在美国模式培养集存库已有保藏(保藏编号ATCC 53103、ATCC 47076、ATCC700926)；大肠杆菌BL21(DE3)在BCCM genecorner已有保藏(保藏编号LMBP 1455)；大肠杆菌JM109在美国模式培养集存库已有保藏(保藏编号ATCC68635、ATCC68868)；大肠杆菌BW25113在大肠杆菌遗传学库存中心(Coli  GeneticsStock 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。
显然，为进一步提高2'-岩藻糖基乳糖和/或3-岩藻糖基乳糖的发酵产率，也可以对所述基因工程菌中参与2'-岩藻糖基乳糖和/或3-岩藻糖基乳糖合成途径的相关酶或转运体的编码基因进行进一步的基因编辑。这些基因编辑可以是，敲除大肠杆菌基因组上以下基因的至少一种：β-半乳糖苷酶编码基因lacZ、UDP-葡萄糖脂质载体转移酶编码基因wcaj、GDP-甘露糖水解酶编码基因nudd、乳糖lac操纵子序列中的调节基因lacI、L-岩藻糖异构酶编码基因fucI、L-墨角藻糖激酶编码基因fucK、L-墨角藻糖-1-磷酸醛缩酶编码基因fucA、D-阿拉伯糖异构酶编码基因ara A、L-鼠李糖异构酶编码基因rhaA。也可以是，过表达或插入以下基因的至少一种：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、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、糖外排转运体A编码基因setA、甘露糖-1-磷酸鸟嘌呤转移酶编码基因manC、2'-岩藻糖基乳糖合成酶编码基因futC、α-(1,3)-岩藻糖基转移酶编码基因futA、L-岩藻糖激酶/GDP-L-岩藻糖焦磷酸化酶编码基因fkp。也可以是上述多种(基因敲除、基因插入、基因过表达)基因编辑手段的结合，例如，敲除lacZ并过表达manC。
再进一步优选地，为进一步提高2'-岩藻糖基乳糖和/或3-岩藻糖基乳糖的发酵产率，对所述基因工程菌中参与2’-岩藻糖基乳糖和/或3-岩藻糖基乳糖合成途径的相关酶或转运体的编码基因的基因编辑包括：
敲除大肠杆菌基因组上的β-半乳糖苷酶编码基因lacZ；
在大肠杆菌基因组上插入GDP-岩藻糖合成酶编码基因wcaG；
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；
原位过表达β-半乳糖苷透性酶编码基因lacY；
在大肠杆菌基因组上插入磷酸甘露糖异构酶编码基因manA；
在大肠杆菌基因组上插入磷酸甘露糖变位酶编码基因manB；
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；
敲除大肠杆菌基因组上的GDP-甘露糖水解酶编码基因nudd；
原位过表达糖外排转运体A编码基因setA；
在质粒上过表达甘露糖-1-磷酸鸟嘌呤转移酶编码基因manC；
在质粒上过表达2’-岩藻糖基乳糖合成酶编码基因futC或在质粒上过表达α-(1,3)-岩藻糖 基转移酶编码基因futA。
其中对lacZ、wcaG、gmd、lacY、manA、manB、wcaj、nudd、setA、manC的上述基因编辑有利于提高2'-岩藻糖基乳糖和3-岩藻糖基乳糖的发酵产率；对futC的上述基因编辑有利于提高2'-岩藻糖基乳糖发酵产率，对futA的上述基因编辑有利于提高2'-岩藻糖基乳糖发酵产率。
进一步地，前述基因编辑中，所述wcaG、gmd、manA、manB为单拷贝插入基因组过表达；所述manC、futA、futC为多拷贝过表达。
进一步地，前述基因编辑中，所述manA、manB、wcaG、gmd和lacY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，前述所述Ptrc启动子的核苷酸序列如SEQ ID NO：4所示。
进一步地，前述基因编辑中，lacZ的核苷酸序列如SEQ ID NO：3所示；wcaG的核苷酸序列如SEQ ID NO：5所示；gmd的核苷酸序列如SEQ ID NO：6所示；lacY的核苷酸序列如SEQ ID NO：7所示；manA的核苷酸序列如SEQ ID NO：9所示；manB的核苷酸序列如SEQ ID NO：10所示；wcaj的核苷酸序列如SEQ ID NO：11所示；nudd的核苷酸序列如SEQ ID NO：13所示；setA的核苷酸序列如SEQ ID NO：14所示；manC的核苷酸序列如SEQ ID NO：17所示；futC的核苷酸序列如SEQ ID NO：18所示。
进一步地，前述基因编辑中，所述futA的核苷酸序列如SEQ ID NO：21所示。在质粒构建中可采用的核苷酸序列模板是多样的、可变化的。优选地，在质粒构建中采用SEQID NO：20所示的基因片段为futA的模板。
进一步地，前述基因编辑中，所述setA前面插入组成型启动子进行原位过表达和/或插入氯霉素抗性基因的启动子进行原位过表达。优选地，所述氯霉素抗性基因的启动子的核苷酸序列如SEQ ID NO：23所示。优选地，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。
前述术语“原位过表达”是与在质粒上进行过表达相对的术语，也是本领域约定俗成的术语，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。基因的“原位过表达”即在染色体原位的过表达，可通过插入额外的启动子提高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：以setA基因为例，也可以改造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。用于基因过表达(包括在原位进行过表达及在质粒上进行过表达)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。此处所述组成型启动子和诱导型启动子，可以是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。
前述基因工程菌、突变体、编码基因及构建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 inCorynebacterium glutamicumfor 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 inEscherichia coli.Journal of Molecular Biology,1999,292(1):19-37.
(4)Zhou L,et al.Chromosome engineering ofEscherichia colifor 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 withadjustable sensitivity[J].Journal of BiologicalEngineering,2019,13(1):1-9(PMID:31452678).
本发明的技术方案之四，提供了前述基因工程菌在发酵生产2'-岩藻糖基乳糖和/或3-岩藻糖基乳糖中的应用。其中在质粒上过表达futA的基因工程菌尤其适于提高3-岩藻糖基乳糖的发酵产率，在质粒上过表达futC的基因工程菌尤其适于提高2'-岩藻糖基乳糖的发酵产率。
本发明的有益效果：
本发明通过对大肠杆菌甲硫氨酸氨肽酶进行定点突变，获得了相应的甲硫氨酸氨肽酶体。经研究发现，表达所述甲硫氨酸氨肽酶体的大肠杆菌2'-岩藻糖基乳糖和3-岩藻糖基乳糖的产率得到出乎预料地提高。当额外对大肠杆菌setA基因进行原位过表达，并对大肠杆菌lacZ、wcaG、gmd、lacY、manA、manB、wcaj、nudd、futC、manC、futA进行原位过表达、质粒过表达等有益基因编辑时，2'-岩藻糖基乳糖和3-岩藻糖基乳糖的产率更高。
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TKYW2-2
在专利文献CN112501106A中所述大肠杆菌W2(E.coliK12MG1655△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，进一步在基因组原位过表达糖流出转运蛋白基因setA，构建出菌株TKYW2-2。
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，将其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 coliK12 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Plac启动子的核苷酸序列如SEQ ID NO：1所示；lacI的核苷酸序列如SEQ ID NO：2所示；lacZ的核苷酸序列如SEQ ID NO：3所示；Ptrc启动子的核苷酸序列如SEQ ID NO：4所示；wcaG的核苷酸序列如SEQ ID NO：5所示；gmd的核苷酸序列如SEQ ID NO：6所示；lacY的核苷酸序列如SEQ ID NO：7所示；adhE的核苷酸序列如SEQ ID NO：8所示；manA的核苷酸序列如SEQ ID NO：9所示；manB的核苷酸序列如SEQ ID NO：10所示。
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(核苷酸序列如SEQ ID NO：11所示)。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 targetingefficiency.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 sequencelimitations and improved targeting efficiency.Sci Rep 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.coliK12 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(核苷酸序列如SEQ ID NO：12所示，SEQ ID NO：12为cat基因及其启动子的核苷酸序列)的菌株基因组为模板，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(异丙基-β-D-硫代半乳糖苷)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(核苷酸序列如SEQ ID NO：13所示)，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.coliK12 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基因时第二部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
3.构建菌株TKYW2-2
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setA基因(核苷酸序列如SEQ ID NO：14所示)前面插入组成型启动子PJ23110(http://parts.igem.org/Part:BBa_J23100)，启动子PJ23110序列为：TTTACGGCTAGCTCAGTCCTAGGTACAATGCTAGC；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：23所示)，以实现双启动子对setA进行原位过表达，所获菌株命名为TKYW2-2。
(1)第一步同源重组片段的构建。以菌株E.coliK12 MG1655基因组为模板，分别利用表3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3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，以110-2为下游引物，不使用模板，进行PCR扩增，获得带有PJ23110启动子的基因片段。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PJ23110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片段。
表3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的菌株。
(3)第二步同源重组。与上述敲除wcaj基因时第二部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，从而获得带有PJ23110启动子的菌株，命名为TKYW2-2。
实施例2构建菌株TKYW3
大肠杆菌K12 MG1655的野生型甲硫氨酸氨肽酶氨基酸序列如SEQ ID NO：24所示。在菌株TKYW2-2的基础上，利用与上述CRISPR/Cas9技术同样的方法，对基因组上的甲硫氨酸氨肽酶基因map进行点突变。将其翻译蛋白的第44位异亮氨酸突变为丝氨酸(MapIle44Ser)，对应甲硫氨酸氨肽酶突变的氨基酸序列如SEQ ID NO：15所示，构建出的菌株命名为TKYW3。
1.第一步同源重组片段的构建
以实验室保存的一株带有map基因点突变的MG1655突变株MG1655mapT131G(mapT131G核苷酸序列如SEQ ID NO：22所示)为模板，分别利用表4中的引物对Mup-1和Mup-2，引物对Mdown-1和Mdown-2为引物，PCR扩增得到同源重组的上、下游同源臂。以构建菌株W2△wcaj时PCR获得的带有cat-N20序列的片段为模板，利用引物Mcat-1和Mcat20-2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Mup-1和Mdown-2进行重叠PCR，得到第一 次同源重组片段，该片段含有map基因的点突变，即野生型map基因的第131位碱基由T变为G。
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TKYW2-2中，获得菌株TKYW2-2(pCAGO)。利用含有1％葡萄糖和浓度为0.1mM的IPTG的LB培养基制备TKYW2-2(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基因时第二部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TKYW2-2mapT131G，命名为TKYW3。
表4构建map基因点突变菌株所用引物
实施例3构建质粒pTrc99a-Ptrc-futC-manC
以专利文献CN112501106A中所述质粒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：4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：16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：17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：18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.coliJM109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，其核苷酸序列如SEQ ID NO：19所示。
表5构建质粒pTrc99a-Ptrc-futC-manC所用引物
实施例4质粒pTrc99a-Ptrc-futA-manC构建
利用幽门螺杆菌(Helicobacter pylori)NCTC 11637来源的α-(1,3)-岩藻糖基转移酶基因futA替换质粒pTrc99a-Ptrc-futC-manC中的futC替换，构建出质粒pTrc99a-Ptrc-futA-manC。FutA能够以GDP-岩藻糖与乳糖为底物催化产生3-岩藻糖基乳糖。
以质粒pTrc99a-Ptrc-futA-manC为模板，以表6中的FUTA-ZT-F和FUTA-ZT-R为引物进行PCR扩增，得到去除futC的线性载体片段。以人工合成的含有futA基因片段(核苷酸序列如SEQ ID NO：20所示)的载体为模板(该基因片段包含如SEQ ID NO：21所示的futA基因)，以FUTA-F和FUTA-R为引物进行PCR扩增，获得带有futA基因的片段。将上述两种PCR产物进行纯化和回收后，使用无缝克隆酶连接，转化到E.coliJM109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-futA-manC。
表6构建质粒pTrc99a-Ptrc-futA-manC所用引物
实施例5 2'-岩藻糖基乳糖和3-岩藻糖基乳糖生产菌种的构建和发酵测试
利用电转化的方法，将质粒pTrc99a-Ptrc-futC-manC和pTrc99a-Ptrc-futA-manC分别导入TKYW2-2和TKYW3，分别构建出2'-岩藻糖基乳糖生产菌株TKYW2-2(pTrc99a-Ptrc-futC-manC)和TKYW3(pTrc99a-Ptrc-futC-manC)，以及3-岩藻糖基乳糖生产菌株TKYW2-2(pTrc99a-Ptrc-futA-manC)和TKYW3(pTrc99a-Ptrc-futA-manC)。测试上述菌株发酵生产水平，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：KH2PO43g/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，H3BO31.9g/L，ZnCl22.6g/L，Na2MOO4·2H2O 1.1g/L，Na2SeO31.5g/L，NiSO4·6H2O 1.5g/l)，利用氨水调pH为7.2。
发酵测试过程为：
分别挑取2'-岩藻糖基乳糖生产菌株和3-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0.1mmol/L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检测样品中2'-岩藻糖基乳糖或3-岩藻糖基乳糖的浓度，HPLC分析所用色谱柱为Carbohydrate ES 5u 250mm*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'-岩藻糖基乳糖或3-岩藻糖基乳糖标准品对样品浓度进行定量。
表7 2'-岩藻糖基乳糖生产菌株发酵测试结果
表8 3-岩藻糖基乳糖生产菌株发酵测试结果
由表7和表8可以看出，Map蛋白第44位异亮氨酸突变为丝氨酸后，能够大幅提高菌株的2'-岩藻糖基乳糖或3-岩藻糖基乳糖产量。
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 (17)

  1. 一种甲硫氨酸氨肽酶突变体，其特征在于，所述甲硫氨酸氨肽酶突变体的氨基酸序列对应于SEQ ID NO：24所示的野生型甲硫氨酸氨肽酶的第44位氨基酸位点具有突变：I>S；且所述甲硫氨酸氨肽酶突变体与SEQ ID NO：24所示的野生型甲硫氨酸氨肽酶氨基酸序列的同源性>90％。
  2. 根据权利要求1所述的甲硫氨酸氨肽酶突变体，其特征在于，所述甲硫氨酸氨肽酶突变体的氨基酸序列如SEQ ID NO：15所示。
  3. 一种编码基因，其特征在于，所述编码基因至少携带编码权利要求1或2所述的甲硫氨酸氨肽酶突变体的核苷酸序列片段。
  4. 根据权利要求3所述的编码基因，其特征在于，所述编码基因至少携带编码权利要求2所述的甲硫氨酸氨肽酶突变体的核苷酸序列片段。
  5. 根据权利要求4所述的编码基因，其特征在于，所述编码基因至少携带SEQ ID NO：22所示的核苷酸片段。
  6. 一种大肠杆菌基因工程菌，其特征在于，所述基因工程菌表达权利要求1或2所述的甲硫氨酸氨肽酶突变体，且所述基因工程菌是在体内能够合成2'-岩藻糖基乳糖和/或3-岩藻糖基乳糖的大肠杆菌。
  7. 根据权利要求6所述的基因工程菌，其特征在于，所述基因工程菌表达权利要求2述的甲硫氨酸氨肽酶突变体。
  8. 根据权利要求7所述的基因工程菌，其特征在于，所述基因工程菌通过SEQ ID NO：22所示的编码基因表达甲硫氨酸氨肽酶突变体。
  9. 根据权利要求6所述的大基因工程菌，其特征在于，所述基因工程菌的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。
  10. 根据权利要求6所述的基因工程菌，其特征在于，所述基因工程菌包括对2'-岩藻糖基乳糖和/或3-岩藻糖基乳糖合成途径相关酶或转运体的编码基因的基因编辑；
    优选地，其包括敲除大肠杆菌基因组上以下基因的至少一种：β-半乳糖苷酶编码基因、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、乳糖lac操纵子序列中的调节基因、L-岩藻糖异构酶编码基因、L-墨角藻糖激酶编码基因、L-墨角藻糖-1-磷酸醛缩酶编码基因、D-阿拉伯糖异构酶编码基因、L-鼠李糖异构酶编码基因；和/或其包括过表达或插入以下基因的至少一种：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、磷酸甘露糖异构酶编码基因、磷酸甘露糖变位酶编码基因、糖外排转运体A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、α-(1,3)-岩藻糖基转移酶编码基因、L-岩藻糖激酶/GDP-L-岩藻糖焦磷酸化酶编码基因；
    更有选地，所述基因工程菌包括下述基因编辑：
    敲除大肠杆菌基因组上的β-半乳糖苷酶编码基因；
    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；
    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；
    原位过表达糖外排转运体A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或在质粒上过表达α-(1,3)-岩藻糖基转移酶编码基因。
  11. 根据权利要求10所述的基因工程菌，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、α-(1,3)-岩藻糖基转移酶编码基因、2'-岩藻糖基乳糖合成酶编码基因为多拷贝过表达。
  12. 根据权利要求10所述的基因工程菌，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-脱水酶编码基因和β-半乳糖苷透性酶编码基因使用Ptrc启动子过表达；所述质粒选自pTrc99a、pSB4K5、pET28a或pET22b中的任一种；所述Ptrc启动子的核苷酸序列如SEQ ID NO：4所示。
  13. 根据权利要求10所述的基因工程菌，其特征在于，所述β-半乳糖苷酶编码基因的核苷酸序列如SEQ ID NO：3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：5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：6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：7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：9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：10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：11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：13所示；所述糖外排转运体A编码基因的核苷酸序列如SEQ ID NO：14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：17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：18所示；所述α-(1,3)-岩藻糖基转移酶编码基因的核苷酸序列如SEQ ID NO：21所示。
  14. 根据权利要求10所述的基因工程菌，其特征在于，所述糖外排转运体A编码基因前面插入组成型启动子进行原位过表达和/或插入氯霉素抗性基因的启动子进行原位过表达；所述氯霉素抗性基因的启动子的核苷酸序列如SEQ ID NO：23所示。
  15. 根据权利要求14所述的基因工程菌，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。
  16. 权利要求10至15任一项所述的基因工程菌在发酵生产2'-岩藻糖基乳糖中的应用，其特征在于，所述基因工程菌在质粒上过表达2'-岩藻糖基乳糖合成酶编码基因。
  17. 权利要求10至15任一项所述的基因工程菌在发酵生产3-岩藻糖基乳糖中的应用，其特征在于，所述基因工程菌在质粒上过表达α-(1,3)-岩藻糖基转移酶编码基因。
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