WO2021037165A1 - 基于大肠杆菌的重组菌株及其构建方法与应用 - Google Patents

基于大肠杆菌的重组菌株及其构建方法与应用 Download PDF

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WO2021037165A1
WO2021037165A1 PCT/CN2020/111840 CN2020111840W WO2021037165A1 WO 2021037165 A1 WO2021037165 A1 WO 2021037165A1 CN 2020111840 W CN2020111840 W CN 2020111840W WO 2021037165 A1 WO2021037165 A1 WO 2021037165A1
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strain
nucleotide sequence
seq
recombinant
sequence
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PCT/CN2020/111840
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English (en)
French (fr)
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孟刚
魏爱英
贾慧萍
赵春光
周晓群
马风勇
郭小炜
田斌
苏厚波
杨立鹏
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内蒙古伊品生物科技有限公司
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Priority claimed from CN201910804035.3A external-priority patent/CN110592084B/zh
Priority claimed from CN201910927600.5A external-priority patent/CN110846333B/zh
Application filed by 内蒙古伊品生物科技有限公司 filed Critical 内蒙古伊品生物科技有限公司
Priority to JP2022513936A priority Critical patent/JP7461463B2/ja
Priority to US17/753,367 priority patent/US20220315962A1/en
Priority to EP23180520.1A priority patent/EP4253570A3/en
Priority to KR1020227005078A priority patent/KR20220034220A/ko
Priority to EP20857632.2A priority patent/EP3992294A4/en
Priority to CA3148183A priority patent/CA3148183A1/en
Publication of WO2021037165A1 publication Critical patent/WO2021037165A1/zh

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12P13/00—Preparation of nitrogen-containing organic compounds
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli

Definitions

  • the invention belongs to the field of genetic engineering and microbial technology, and specifically relates to a recombinant strain based on Escherichia coli and a construction method and application thereof.
  • L-threonine is one of the eight essential amino acids, and it is an amino acid that humans and animals cannot synthesize by themselves. L-threonine can strengthen the absorption of grains, regulate the balance of metabolism in the body, and promote the growth and development of the body. It is widely used in feed, medicine and food industries.
  • L-threonine mainly includes chemical synthesis, proteolysis and microbial fermentation. Among them, microbial fermentation has low production cost, high production intensity, and low environmental pollution. Therefore, it has become the current application of industrial production of L-threonine. The most extensive method.
  • a variety of bacteria can be used for the microbial fermentation production of L-threonine, such as wild-type induced mutant strains of Escherichia coli, Corynebacterium spp., Serratia spp. as production strains. Specific examples include anti-amino acid analog mutant strains or various auxotrophs such as methionine, threonine, and isoleucine.
  • Escherichia coli as a host for foreign gene expression, has a clear genetic background, simple technical operation and culture conditions, and large-scale fermentation economical. It is highly valued by genetic engineering experts.
  • the genomic DNA of E. coli is a circular molecule in the nucleus, and there can be multiple circular plasmid DNAs at the same time.
  • the nucleus of E. coli cells has 1 DNA molecule with a length of about 4,700,000 base pairs. There are about 4,400 genes distributed on the DNA molecule, and the average length of each gene is about 1,000 base pairs.
  • the E. coli strains commonly used in molecular biology, with a few exceptions, most of the strains used in DNA recombination experiments are E. coli strain K12 and its derivatives.
  • the present invention provides a recombinant strain based on Escherichia coli strain K12 or its derivatives, its recombinant construction method, and its application in the fermentation and production of amino acids.
  • the present invention focuses on the wild-type deoB gene in E. coli K12 strain and its derivative strains (such as MG1655, W3110, etc.) (the ORF sequence is shown in the sequence 3902352-3903575 in Genbank accession number CP032667.1), wild-type rhtA gene Promoter sequence PrhtA (as shown in the sequence 850520-850871 in Genbank accession number AP009048.1), it is found that the mutant gene obtained after site-directed mutation of the gene and the recombinant strain containing the mutant gene can be used for L-threonine Compared with the unmutated wild-type strain, the obtained strain can greatly increase the production of L-threonine, and the strain has good stability. As an L-threonine production strain, the production cost is reduced and the production is improved. effectiveness.
  • the first part provides a nucleotide sequence which includes a sequence formed by a mutation at the 1049th base of the wild-type deoB gene coding sequence shown in SEQ ID NO:1.
  • the mutation refers to a change in the base/nucleotide of the site
  • the mutation method can be selected from at least one of methods such as mutagenesis, PCR site-directed mutagenesis, and/or homologous recombination.
  • methods such as mutagenesis, PCR site-directed mutagenesis, and/or homologous recombination.
  • the mutation is that the 1049th base in SEQ ID NO: 1 is mutated from guanine (G) to adenine (A); specifically, the mutated nucleotide sequence is as SEQ ID NO: 2 shown.
  • the present invention also provides a recombinant protein encoded by the nucleotide sequence as described above.
  • the recombinant protein according to the present invention includes the amino acid sequence shown in SEQ ID NO:4.
  • the present invention also provides a recombinant vector including the above-mentioned nucleotide sequence.
  • the recombinant vector according to the present invention is constructed by introducing the above-mentioned nucleotide sequence into a plasmid; as an embodiment, the plasmid is a pKOV plasmid.
  • the nucleotide sequence and the plasmid can be digested with endonuclease to form complementary sticky ends, and the two can be connected to construct a recombinant vector.
  • the present invention also provides a recombinant strain, which contains a deoB gene encoding nucleotide sequence with a point mutation in the encoding sequence.
  • the recombinant strain according to the present invention contains the mutated nucleotide sequence as described above.
  • the present invention contains the nucleotide sequence shown in SEQ ID NO: 2.
  • it contains the amino acid sequence shown in SEQ ID NO:4.
  • the recombinant strain according to the present invention is formed by introducing the recombinant vector as described above into a host strain;
  • the host strain is not particularly limited, and can be selected from L-threonine-producing strains known in the art that retain the deoB gene , For example selected from Escherichia coli.
  • the host strain is E. coli K12 (W3110) strain, E. coli CGMCC 7.232 strain.
  • the pKOV plasmid is used as the vector.
  • the present invention also provides a method for constructing a recombinant strain, which includes the following steps:
  • the nucleotide sequence of the open reading frame region of the wild-type deoB gene as shown in SEQ ID NO:1 was modified to make the 1049th base mutated to obtain an L-threonine-producing recombinant strain containing the mutant deoB encoding gene.
  • the modification includes at least one of methods such as mutagenesis, PCR site-directed mutagenesis, and/or homologous recombination.
  • the mutation refers to the mutation of base 1049 in SEQ ID NO:1 from guanine (G) to adenine (A); specifically, the nucleotide sequence after the mutation is as SEQ ID NO: 2 is shown.
  • the construction method includes the following steps:
  • the step (1) includes: point mutation deoB gene coding region construction, that is, according to the deoB gene coding sequence, two pairs of primers for amplifying the deoB gene coding region fragment are synthesized, and the PCR site-directed mutagenesis method A point mutation was introduced into the coding region of the wild-type deoB gene (SEQ ID NO: 1), and the nucleotide sequence of the coding region of the point mutation deoB gene (SEQ ID NO: 2) was obtained, denoted as deoB (G1049A) .
  • the primer is:
  • P2 5'GATCGTAACCGTGGTCAG 3'(SEQ ID NO: 6)
  • P3 5'CTGACCACGGTTACGATC 3'(SEQ ID NO: 7)
  • the step (1) includes: using E. coli K12 as a template, and using primers P1/P2 and P3/P4 to perform PCR amplification, to obtain two separations containing deoB gene coding regions The size is 836bp and 890bp DNA fragments (deoB Up and deoB Down). After the above two DNA fragments were separated and purified by agarose gel electrophoresis, the above two DNA fragments were used as templates, and P1 and P4 were used as primers to amplify by overlap PCR (Overlap PCR) to obtain deoB (G1049A) -Up -Down.
  • the size of the deoB (G1049A)-Up -Down nucleotide sequence is 1726 bp.
  • the PCR amplification is performed as follows: denaturation at 94°C for 30s, annealing at 52°C for 30s, and extension at 72°C for 30s (30 cycles).
  • the overlap PCR amplification is performed as follows: denaturation at 94°C for 30 s, annealing at 52°C for 30 s, and extension at 72°C for 60 s (30 cycles).
  • the step (2) includes the construction of a recombinant vector, the above-mentioned deoB (G1049A) -Up -Down fragment is separated and purified by agarose gel electrophoresis, and then it and the pKOV plasmid are respectively used BamH I/ Not I double enzyme digestion, the digested deoB (G1049A)-Up -Down fragment and pKOV plasmid were separated and purified by agarose gel electrophoresis and ligated to obtain the recombinant vector pKOV-deoB (G1049A) .
  • the step (3) includes the construction of a recombinant strain: the recombinant vector pKOV-deoB (G1049A) is transformed into a host strain to obtain a recombinant strain.
  • the transformation in step (3) is an electrotransformation method; illustratively, in step (3), a recombinant vector is introduced into the host strain.
  • the construction method according to the present invention further includes the step of screening recombinant strains; illustratively, chloramphenicol medium is used for screening.
  • the present invention also provides a recombinant strain obtained by the above-mentioned construction method.
  • the present invention also provides the application of the above-mentioned recombinant strain in the preparation of L-threonine or increasing the fermentation amount of L-threonine.
  • the application of the recombinant strain according to the present invention in the preparation of L-threonine includes using the recombinant strain for fermentation to prepare L-threonine.
  • the present invention provides a promoter, which includes a nucleotide sequence formed by mutating the -67th base upstream of the nucleotide sequence shown in SEQ ID NO: 13.
  • the mutation refers to a change in the base of the site
  • the mutation method can be selected from at least one of methods such as mutagenesis, PCR site-directed mutagenesis, and/or homologous recombination.
  • the mutation is that the -67 base in SEQ ID NO: 13 is mutated from adenine (A) to guanine (G); specifically, the nucleotide sequence of the mutated promoter is as SEQ ID NO: 14 is shown.
  • the present invention provides an expression cassette comprising the above-mentioned promoter and the coding nucleotide sequence of rhtA gene.
  • the promoter is located 5'upstream of the nucleotide sequence encoding the rhtA gene to form an expression cassette.
  • the encoding nucleotide sequence of the rhtA gene includes the nucleotide sequence shown in SEQ ID NO: 15, and the nucleotide sequence encoding includes the amino acid sequence shown in SEQ ID NO: 16.
  • the present invention provides a recombinant vector including the above-mentioned promoter.
  • the recombinant vector according to the present invention is constructed by introducing the nucleotide sequence including the above-mentioned promoter into a plasmid; as an embodiment, the plasmid is a pKOV plasmid.
  • the nucleotide sequence including the promoter and the plasmid can be digested with endonuclease to form complementary sticky ends, and the two can be connected to construct a recombinant vector.
  • the present invention also provides a recombinant strain comprising the above-mentioned promoter.
  • the recombinant strain according to the present invention includes the promoter nucleotide sequence shown in SEQ ID NO: 14; further, the recombinant strain includes the expression cassette as described above.
  • the recombinant strain according to the present invention is formed by introducing the aforementioned recombinant vector into a host strain;
  • the host strain is not particularly limited, and can be selected from L-threonine-producing strains known in the art that retain the rhtA gene, for example, From Escherichia coli.
  • the host strain is E. coli K12, or its derivative strain E. coli K12 (W3110) strain, E. coli CGMCC 7.232 strain.
  • the pKOV plasmid is used as the vector.
  • the present invention also provides a method for constructing a recombinant strain, which includes the following steps:
  • the promoter region shown in SEQ ID NO: 13 is modified to make the -67 base mutated to obtain a recombinant strain containing a point mutation promoter.
  • the modification includes at least one of methods such as mutagenesis, PCR site-directed mutagenesis, and/or homologous recombination.
  • the mutation refers to that the -67 base in SEQ ID NO: 13 is mutated from adenine (A) to guanine (G); specifically, the rhtA gene containing the point mutation is activated
  • the nucleotide sequence of the child is shown in SEQ ID NO: 14.
  • construction method includes the following steps:
  • the method for mutating the base includes mutagenesis, PCR site-directed mutagenesis or homologous recombination, preferably PCR site-directed mutagenesis.
  • the step (1) includes:
  • the primer is:
  • the step (1) includes: using E. coli K12 as a template, and using primers P1 and P2, P3 and P4, respectively, to perform PCR amplification to obtain two promoter regions containing rhtA gene
  • the isolated DNA fragments are 690bp and 640bp in size, namely PrhtA (A(-67)G) -Up and PrhtA (A(-67)G) -Down fragments; using the above two DNA fragments as templates, using P1 and P4 As primers, they were amplified by Overlap PCR, which was performed as follows: denaturation at 94°C for 30s, annealing at 52°C for 30s, and extension at 72°C for 60s (30 cycles) to obtain PrhtA (A( -67)G) -Up-Down fragment.
  • the step (2) includes: performing agarose gel electrophoresis and separation and purification on the PrhtA (A(-67)G) -Up-Down fragment, and after the fragment is digested with BamH I/Not I double enzyme , Ligate with the plasmid after EcoR I/Sph I double enzyme digestion to obtain a recombinant vector with allelic replacement.
  • the conversion of step (3) is an electroconversion method.
  • the present invention also provides a recombinant strain obtained by the above-mentioned construction method.
  • the present invention provides the application of the above-mentioned recombinant strain in the preparation of L-threonine.
  • the application of the recombinant strain according to the present invention in the preparation of L-threonine includes using the recombinant strain for fermentation to prepare L-threonine.
  • Pentose phosphate mutase is encoded by the deoB gene.
  • E.coli K12 strain and its derivative strains such as MG1655, etc.
  • the ORF sequence of the wild-type deoB gene is shown in the sequence 3902352-3903575 in Genbank accession number CP032667.1 .
  • the primer design is as follows (synthesized by Shanghai invitrogen company):
  • P2 5'GATCGTAACCGTGGTCAG 3'(SEQ ID NO: 6)
  • P3 5'CTGACCACGGTTACGATC 3'(SEQ ID NO: 7)
  • the construction method is: using the wild-type strain E.coli K12 genome as a template, PCR amplification is carried out with primers P1 and P2, P3 and P4 respectively, and two DNA fragments (deoB) containing point mutations with lengths of 836bp and 890bp are obtained. (G1049A) -Up and deoB (G1049A) -Down fragments).
  • PCR system 10 ⁇ Ex Taq Buffer 5 ⁇ L, dNTP Mixture (2.5mM each) 4 ⁇ L, MgCl 2 (25mM) 4 ⁇ L, primers (10pm) each 2 ⁇ L, Template 1 ⁇ L, Ex Taq (5U/ ⁇ l) 0.25 ⁇ L, total volume 50 ⁇ L, The PCR amplification was performed as follows: pre-denaturation at 94°C for 5 min, (denaturation at 94°C for 30 s, annealing at 52°C for 30 s, and extension at 72°C for 90 s, 30 cycles), and over extension at 72°C for 10 min.
  • Overlap PCR system 10 ⁇ Ex Taq Buffer 5 ⁇ L, dNTP Mixture (2.5mM each) 4 ⁇ L, MgCl 2 (25mM) 4 ⁇ L, primers (10pm) each 2 ⁇ L, Template 1 ⁇ L, Ex Taq (5U/ ⁇ l) 0.25 ⁇ L, total volume 50 ⁇ L
  • the PCR amplification is carried out as follows: 94°C pre-denaturation for 5 minutes, (94°C denaturation for 30s, 52°C annealing for 30s, and 72°C extension for 90s, 30 cycles), 72°C over extension for 10 minutes.
  • the above-mentioned deoB (G1049A) -Up -Down fragment was separated and purified by agarose gel electrophoresis, and then it and pKOV plasmid (purchased from Addgene) were digested with BamH I/Not I, and the digested deoB ( G1049A)-Up -Down fragment and pKOV plasmid were separated, purified and connected by agarose gel electrophoresis to obtain the vector pKOV-deoB (G1049A) .
  • the vector pKOV-deoB (G1049A) was sent to a sequencing company for sequencing and identification, and the result is shown in SEQ ID NO: 11. Save the vector pKOV-deoB (G1049A) containing the correct point mutation (deoB (G1049A)) for future use.
  • the wild-type E. coli strain E. coli K12 (W3110) and the high-producing L-threonine strain E. coli CGMCC 7.232 both retain the wild-type deoB gene on their chromosomes.
  • the constructed plasmid pKOV-deoB (G1049A) was transformed into E. coli K12 (W3110) and E. coli CGMCC 7.232 respectively, and the deoB gene sequence in the chromosomes of these two strains was compared with SEQ ID NO by allele replacement. :1
  • the 1049th base G is changed to A.
  • the plasmid pKOV-deoB (G1049A) is transformed into competent cells of the host bacteria by electric shock, and then 0.5mL of SOC liquid medium is added; resuscitated in a shaker at 30°C and 100rpm for 2h; 100 ⁇ L of culture medium is used for coating Cultivate in LB solid medium with a chloramphenicol content of 34 mg/mL at 30°C for 18 hours; select the grown monoclonal colonies and inoculate them in 10 mL of LB liquid medium, culture at 37°C and 200 rpm for 8 hours; take 100 ⁇ L of the culture solution for coating Cultivate in LB solid medium with a chloramphenicol content of 34 mg/mL at 42°C for 12 hours; select 1-5 single colonies to inoculate 1 mL of LB liquid medium, and incubate at 37°C and 200 rpm for 4 hours; LB solid medium containing 10% sucrose, cultured at 30°C for 24
  • the PCR amplified products were subjected to SSCP (Single-Strand Conformation Polymorphism) electrophoresis, the amplified fragment of plasmid pKOV-deoB (G1049A) was used as a positive control, and the amplified fragment of wild-type E. coli was used as a negative control. As a blank control.
  • SSCP electrophoresis single-stranded oligonucleotide chains with the same length and different sequence arrangement have different spatial structures formed in an ice bath, and their mobility during electrophoresis will also be different. Therefore, the fragment electrophoresis position is inconsistent with the negative control fragment position, and the strain that is consistent with the positive control fragment position is the strain with successful allelic replacement.
  • the target fragment was amplified by PCR again with primers P5 and P6, and the target fragment was ligated to the pMD19-T vector and sequenced. Through the sequence comparison of the sequencing result, the sequencing result is as shown in SEQ ID NO: 12.
  • the recombinant strain whose base G at position 1049 in the coding region sequence of the deoB gene is changed to A is the successfully modified strain.
  • the recombinant from E. coli K12 (W3110) was named YPThr09
  • the recombinant from E. coli CGMCC 7.232 was named YPThr10.
  • the E. coli K12 (W3110) strain, the E. coli CGMCC 7.232 strain, and the mutant strains YPThr09 and YPThr10 were respectively inoculated into 25 mL of the liquid medium described in Table 1, and cultured at 37° C. and 200 rpm for 12 hours. Then, 1 mL of the culture of each strain was inoculated into 25 mL of the liquid medium described in Table 1, and fermented and cultured at 37° C. and 200 rpm for 36 hours. The content of L-threonine was determined by HPLC, and each strain was made in three parallels, and the average value was calculated. The test results are shown in Table 2.
  • Threonine and homoserine efflux protein are encoded by rhtA gene.
  • the wild-type rhtA gene promoter sequence PrhtA such as Genbank accession number is AP009048 Shown in the sequence 850520-850871 in .1.
  • two pairs of primers for amplifying the promoter PrhtA were designed and synthesized, and a vector was constructed to convert the base sequence (SEQ ID NO: 13) upstream of the -67th base A of the PrhtA promoter in the starting strain to G( SEQ ID NO: 14).
  • the primer design is as follows (synthesized by Shanghai invitrogen company):
  • the construction method was as follows: using the wild-type strain E.coli K12 genome as a template, PCR amplification was carried out with primers P1 and P2, P3 and P4, and two DNA fragments (PrhtA) containing point mutations with lengths of 690bp and 640bp were obtained. (A (- 67) G) -Up and PrhtA (A (-67) G) -Down fragment).
  • PCR system 10 ⁇ Ex Taq Buffer 5 ⁇ L, dNTP Mixture (2.5mM each) 4 ⁇ L, Mg 2+ (25mM) 4 ⁇ L, primers (10pM) each 2 ⁇ L, Ex Taq (5U/ ⁇ L) 0.25 ⁇ L, total volume 50 ⁇ L, the PCR amplification was carried out as follows: pre-denaturation at 94°C for 5 min, (denaturation at 94°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 30 s, 30 cycles), and over extension at 72°C for 10 min.
  • PCR system 10 ⁇ Ex Taq Buffer 5 ⁇ L, dNTP Mixture (2.5mM each) 4 ⁇ L, Mg 2+ (25mM) 4 ⁇ L, primers (10pM) each 2 ⁇ L, Ex Taq (5U/ ⁇ L) 0.25 ⁇ L, total volume 50 ⁇ L, the Overlap PCR was performed as follows: denaturation at 94°C for 30s, annealing at 52°C for 30s, and extension at 72°C for 60s (30 cycles).
  • PrhtA (A(-67)G) -Up-Down fragment was separated and purified by agarose gel electrophoresis, and then it and the pKOV plasmid (purchased from Addgene) were digested with BamH I/Not I.
  • the cut PrhtA (A(-67)G) -Up-Down fragment and pKOV plasmid were separated, purified and connected by agarose gel electrophoresis to obtain the vector pKOV-PrhtA (A(-67)G) .
  • the vector pKOV-PrhtA (A(-67)G) was sent to a sequencing company for sequencing and identification, and the vector pKOV-PrhtA (A(-67)G) containing the correct point mutation (PrhtA (A(-67)G) ) Save it for later use.
  • the wild-type E. coli strain E. coli K12 (W3110) and the high-producing L-threonine strain E. coli CGMCC 7.232 retain the wild-type PrhtA promoter on the chromosomes. .
  • the constructed plasmid pKOV-PrhtA (A(-67)G) was transformed into E.coli K12(W3110) and E.coli CGMCC 7.232 respectively, and the PrhtA promoter in the chromosomes of these two strains was replaced by alleles
  • the base A at the -67th position upstream of the base sequence is changed to G.
  • the specific process is as follows: After the plasmid pKOV-PrhtA (A(-67)G) is transformed into competent cells of the host bacteria by electric shock, 0.5mL of SOC liquid medium is added; and it is resuscitated in a shaker at 30°C and 100rpm for 2h; Take 100 ⁇ L of culture solution and spread it on LB solid medium with a chloramphenicol content of 34 ⁇ g/mL, culture at 30°C for 18h; select the grown monoclonal colonies, inoculate it in 10mL LB liquid medium, culture at 37°C, 200rpm for 8h; Take 100 ⁇ L of culture solution and spread it on LB solid medium with a chloramphenicol content of 34 ⁇ g/mL, and incubate at 42°C for 12h; select 1-5 single colonies to inoculate in 1mL LB liquid medium, and culture at 37°C and 200rpm for 4h; take Spread 100 ⁇ L of culture medium on LB solid medium containing 10%
  • PCR amplification uses the following primers (synthesized by Shanghai invitrogen company):
  • the above PCR system 10 ⁇ Ex Taq Buffer 5 ⁇ L, dNTP Mixture (2.5mM each) 4 ⁇ L, Mg 2+ (25mM) 4 ⁇ L, primers (10pM) 2 ⁇ L each, Ex Taq (5U/ ⁇ L) 0.25 ⁇ L, total volume 50 ⁇ L,
  • the PCR amplification was performed as follows: 94°C pre-denaturation for 5 minutes, (94°C denaturation for 30s, 52°C annealing for 30s, 72°C extension for 30s, 30 cycles), 72°C over extension for 10 minutes.
  • the PCR amplified product was subjected to SSCP (Single-Strand Conformation Polymorphism) electrophoresis.
  • SSCP Single-Strand Conformation Polymorphism
  • the amplified fragment of plasmid pKOV-PrhtA (A(-67)G) was used as a positive control, and the amplified fragment of wild-type E. coli was used as Negative control, water as a blank control.
  • SSCP electrophoresis single-stranded oligonucleotide chains with the same length and different sequence arrangement have different spatial structures in the ice bath, and their mobility during electrophoresis will also be different. Therefore, the electrophoresis position of the fragment is inconsistent with the position of the negative control fragment, and the strain that is consistent with the position of the positive control fragment is the strain with successful allelic replacement.
  • the target fragment was amplified by PCR again with primers P5 and P6, and the target fragment was ligated to the pMD19-T vector and sequenced.
  • the recombinant strain whose base A changes from base A to G at position -67 upstream of the base sequence of the PrhtA promoter is the successfully modified strain.
  • the recombinant from E. coli K12 (W3110) was named YPThr01
  • the recombinant from E. coli CGMCC 7.232 was named YPThr02.
  • the E. coli K12 (W3110) strain, the E. coli CGMCC 7.232 strain, and the mutant strains YPThr01 and YPThr02 were respectively inoculated into 25 mL of the liquid medium described in Table 1, and cultured at 37° C. and 200 rpm for 12 hours. Then, 1 mL of the culture of each strain was inoculated into 25 mL of the liquid medium described in Table 1, and fermented and cultured at 37° C. and 200 rpm for 36 hours. The content of L-threonine was determined by HPLC. Three parallel experiments were performed for each strain, and the average value was calculated. The test results are shown in Table 2.

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Abstract

提供了一种核苷酸序列，该序列是SEQ ID NO:1所示的野生型deoB基因编码序列第1049位碱基发生突变而形成的序列或SEQ ID NO:13所示的核苷酸序列上游第-67位碱基发生突变而形成的启动子核苷酸序列。还提供了表达盒，重组蛋白，重组载体，重组菌株及构建方法，以及它们在发酵制备L-苏氨酸中的应用。所获得的菌株与未突变的野生型菌株相比，可生产更高浓度的L-苏氨酸，且菌株稳定性好，作为L-苏氨酸生产菌株降低了生产成本。

Description

基于大肠杆菌的重组菌株及其构建方法与应用
本申请要求2019年09月27日向中国国家知识产权局提交的专利申请号为2019109276005的在先申请的优先权，要求2019年08月28日向中国国家知识产权局提交的专利申请号为2019108040353的在先申请的优先权，上述在先申请的全文通过引用的方式结合于本申请中。
技术领域
本发明属于基因工程和微生物技术领域，具体涉及基于大肠杆菌中的重组菌株及其构建方法与应用。
背景技术
L-苏氨酸是八大必需氨基酸之一，且是人和动物自身不能合成的氨基酸。L-苏氨酸可以强化谷物吸收，调节体内代谢平衡，促进机体生长发育，被广泛应用于饲料、医药及食品工业中。
目前，L-苏氨酸生产主要有化学合成法、蛋白水解法和微生物发酵法，其中微生物发酵法生产成本低、生产强度高、对环境污染小，因而成为目前工业生产L-苏氨酸应用最广泛的方法。多种细菌可用于L-苏氨酸的微生物发酵生产，如大肠杆菌、棒状杆菌属、沙雷氏菌属等的野生型诱导获得的突变株作为生产菌株。具体实例包括抗氨基酸类似物突变株或甲硫氨酸、苏氨酸、异亮氨酸等多种营养缺陷型。然而，传统诱变育种由于随机突变造成菌株生长缓慢及产生较多副产物，不易获得高产菌株。因此，运用代谢工程手段构建重组大肠杆菌是生产L-苏氨酸的有效途径。目前，利用表达质粒介导的氨基酸合成途径和竞争途径中关键性酶基因的过表达或弱化是对大肠杆菌进行基因改造的主要手段。但是仍然存在对开发以高产率更经济地生产L-苏氨酸的方法的需要。
大肠杆菌作为外源基因表达的宿主，遗传背景清楚，技术操作和培养条件简单，大规模发酵经济，倍受遗传工程专家的重视。大肠杆菌的基因组DNA为拟核中的一个环状分子，同时可以有多个环状质粒DNA。大肠杆菌细胞的拟核有1个DNA分子，长度约为4700000个碱基对，在DNA分子上分布着大约4400个基因，每个基因的平均长度约为1000个碱基对。分子生物学中常用的大肠杆菌菌株，除了少数几个例外，在DNA重组实 验中所用的菌株大多数都是大肠杆菌菌株K12和其衍生物。
发明内容
本发明提供基于大肠杆菌菌株K12或其衍生物的重组菌株，其重组构建方法，及在发酵生产氨基酸中的应用。
本发明着眼于E.coli K12菌株及其衍生菌株(如MG1655、W3110等)中的野生型deoB基因(ORF序列如Genbank登录号为CP032667.1中序列3902352-3903575所示)、野生型rhtA基因启动子序列PrhtA(如Genbank登录号为AP009048.1中序列850520-850871所示)，发现所述基因经定点突变后获得的突变基因及包含所述突变基因的重组菌株可用于L-苏氨酸的生产，所获得的菌株与未突变的野生型菌株相比，可大幅提高L-苏氨酸的产量，且菌株稳定性好，作为L-苏氨酸生产菌株降低了生产成本，提高了生产效率。
基于上述发明，本发明提供如下二部分技术方案：
第一部分，提供一种核苷酸序列，所述核苷酸序列包括SEQ ID NO:1所示的野生型deoB基因编码序列第1049位碱基发生突变而形成的序列。
根据本发明，所述突变是指所述位点的碱基/核苷酸发生变化，所述突变方法可以选自诱变、PCR定点突变法、和/或同源重组等方法中的至少一种。
根据本发明，所述突变为SEQ ID NO:1中第1049位碱基由鸟嘌呤(G)突变为腺嘌呤(A)；具体地，所述突变后的核苷酸序列如SEQ ID NO:2所示。
本发明还提供如上所述的核苷酸序列编码的重组蛋白。
根据本发明的重组蛋白，其包括如SEQ ID NO:4所示的氨基酸序列。
本发明还提供包括上述核苷酸序列的重组载体。
根据本发明的重组载体，是将上述核苷酸序列导入质粒构建而成；作为一个实施方案，所述质粒为pKOV质粒。具体地，可以将所述核苷酸序列和所述质粒通过内切酶进行酶切，形成互补的粘性末端，将二者连接构建成重组载体。
本发明还提供一种重组菌株，其含有编码序列发生点突变的deoB基因编码核苷酸序列。
根据本发明的重组菌株，其含有如上所述的突变后的核苷酸序列。
作为本发明的一个实施方案，其含有如SEQ ID NO:2所示的核苷酸序列。
作为本发明的一个实施方案，其含有如SEQ ID NO:4所示的氨基酸序列。
根据本发明的重组菌株，是将如上所述的重组载体导入宿主菌株中重组形成；所述宿主菌株没有特别的限定，可以选自本领域已知的保留deoB基因的产L-苏氨酸菌株，例如选自大肠杆菌。作为本发明的一个实施方案，所述宿主菌株为E.coli K12(W3110)菌株、E.coli CGMCC 7.232菌株。
根据本发明的重组菌株，是以pKOV质粒为载体。
根据本发明的重组菌株，其可以进一步包括其他改造。
本发明还提供一种重组菌株的构建方法，包括如下步骤：
改造如SEQ ID NO:1所示的野生型deoB基因开放阅读框区域的核苷酸序列，使其第1049位碱基发生突变，得到包含突变deoB编码基因的产L-苏氨酸重组菌株。
根据本发明的构建方法，所述改造包括诱变、PCR定点突变法、和/或同源重组等方法中的至少一种。
根据本发明的构建方法，所述突变是指SEQ ID NO:1中第1049位碱基由鸟嘌呤(G)突变为腺嘌呤(A)；具体地，所述突变后的核苷酸序列如SEQ ID NO:2所示。
示例性的，所述构建方法包括如下步骤：
(1)改造如SEQ ID NO:1所示的野生型deoB基因开放阅读框区域的核苷酸序列，使其第1049位碱基发生突变，得到突变的deoB基因开放阅读框区域的核苷酸序列；
(2)将所述突变的核苷酸序列与质粒连接，构建重组载体；
(3)将所述重组载体导入宿主菌株，得到所述包含点突变的产L-苏氨酸重组菌株。
根据本发明的构建方法，所述步骤(1)包括：点突变的deoB基因编码区构建，即根据deoB基因编码序列，合成两对扩增deoB基因编码区片段的引物，通过PCR定点突变法在野生型deoB基因编码区(SEQ ID NO:1)中引入点突变，得到点突变的deoB基因编码区核苷酸序列(SEQ ID NO:2)，记为deoB (G1049A)。
在本发明的一个实施方案中，所述步骤(1)中，所述引物为：
P1:5'CG GGATCCATGGACGGCAACGCTGAAG 3'(划线部分为限制性内切酶酶切位点BamH I)(SEQ ID NO:5)
P2:5'GATCGTAACCGTGGTCAG 3'(SEQ ID NO:6)
P3:5'CTGACCACGGTTACGATC 3'(SEQ ID NO:7)
P4:5'AAGGAAAAAA GCGGCCGCGCTCGTGAGTGCGGATGT 3'(划线部分为限制性内切酶酶切位点Not I)(SEQ ID NO:8)；
在本发明的一个实施方案中，所述步骤(1)包括：以E.coli K12为模板，分别以引物P1/P2及P3/P4，进行PCR扩增，获得两条含有deoB基因编码区分离的大小为836bp和890bp DNA片段(deoB Up和deoB Down)。将上述两条DNA片段经琼脂糖凝胶电泳分离纯化后，再以上述两条DNA片段为模板，以P1和P4为引物，通过重叠PCR扩增(Overlap PCR)，获得deoB (G1049A)-Up-Down。
在本发明的一个实施方案中，所述deoB (G1049A)-Up-Down核苷酸序列大小为1726bp。
在本发明的一个实施方案中，所述PCR扩增按如下方式进行：94℃变性30s，52℃退火30s，以及72℃延伸30s(30个循环)。
在本发明的一个实施方案中，所述重叠PCR扩增按如下方式进行：94℃变性30s，52℃退火30s，以及72℃延伸60s(30个循环)。
根据本发明的构建方法，所述步骤(2)包括重组载体的构建，将上述deoB (G1049A)-Up-Down片段经琼脂糖凝胶电泳分离纯化，然后将其和pKOⅤ质粒分别用BamH I/Not I双酶切，将酶切后的deoB (G1049A)-Up-Down片段和pKOⅤ质粒经琼脂糖凝胶电泳分离纯化并连接，获得重组载体pKOⅤ-deoB (G1049A)。
根据本发明的构建方法，所述步骤(3)包括重组菌株的构建：将重组载体pKOⅤ-deoB (G1049A)转化宿主菌株，得到重组菌株。
在本发明的一个实施方案中，所述步骤(3)的转化为电转化法；示例性地，所述步骤(3)中，是将重组载体导入至所述宿主菌株。
根据本发明的构建方法，还进一步包括筛选重组菌株的步骤；示例性地，采用氯霉素培养基进行筛选。
本发明还提供如上所述的构建方法所获得的重组菌株。
本发明还提供如上所述的重组菌株在L-苏氨酸制备或者提高L-苏氨酸发酵量中的应用。
根据本发明所述的重组菌株在L-苏氨酸制备中的应用，包括采用所述重组菌株进行发酵，制备得到L-苏氨酸。
第二部分，本发明提供一种启动子，其包括SEQ ID NO:13所示的核苷酸序列上游第-67位碱基发生突变而形成的核苷酸序列。
根据本发明，所述突变是指所述位点的碱基发生变化，所述突变方法可以选自诱变、PCR定点突变法、和/或同源重组等方法中的至少一种。
根据本发明，所述突变为SEQ ID NO:13中第-67位碱基由腺嘌呤(A)突变为鸟嘌呤(G)；具体地，所述突变后的启动子的核苷酸序列如SEQ ID NO:14所示。
本发明提供一种表达盒，包括上述启动子和rhtA基因的编码核苷酸序列。作为本发明的一个实施方案，所述启动子位于rhtA基因编码核苷酸序列的5’上游，组成表达盒。
根据本发明的表达盒，所述rhtA基因的编码核苷酸序列包含SEQ ID NO:15所示的核苷酸序列，所述核苷酸序列编码包含SEQ ID NO:16所示的氨基酸序列。
本发明提供一种重组载体，包括上述启动子。
根据本发明的重组载体，是将包括上述启动子的核苷酸序列导入质粒构建而成；作为一个实施方案，所述质粒为pKOV质粒。具体地，可以将包括所述启动子的核苷酸序列和所述质粒通过内切酶进行酶切，形成互补的粘性末端，将二者连接构建成重组载体。
本发明还提供一种重组菌株，包含上述启动子。
根据本发明的重组菌株，其包含SEQ ID NO:14所示的启动子核苷酸序列；进一步地，所述重组菌株包含如上所述的表达盒。
根据本发明的重组菌株，是将上述重组载体导入宿主菌株中重组形成；所述宿主菌株没有特别的限定，可以选自本领域已知的保留rhtA基因的产L-苏氨酸菌株，例如选自大肠杆菌。作为本发明的一个实施方案，所述宿主菌株为E.coli K12、或其衍生菌株E.coli K12(W3110)菌株、E.coli CGMCC 7.232菌株。
根据本发明的重组菌株，是以pKOV质粒为载体。
根据本发明的重组菌株，其可以进一步包括或者不包括其他改造。
本发明还提供一种重组菌株的构建方法，包括如下步骤：
改造如SEQ ID NO:13所示的启动子区域，使其第-67位碱基发生突变，得到包含点突变的启动子的重组菌株。
根据本发明的构建方法，所述改造包括诱变、PCR定点突变法、和/或同源重组等方法中的至少一种。
根据本发明的构建方法，所述突变是指SEQ ID NO:13中第-67位碱基由腺嘌呤(A)突变为鸟嘌呤(G)；具体地，所述包含点突变的rhtA基因启动子的核苷酸序列如SEQ ID NO:14所示。
进一步地，所述构建方法包括如下步骤：
(1)改造如SEQ ID NO:13所示的rhtA基因的野生型启动子区域，使其-67位碱基发生突变，得到突变的启动子区域核苷酸序列；
(2)将所述突变的启动子区域核苷酸序列与质粒连接，构建重组载体；
(3)将所述重组载体导入宿主菌株，得到包含突变的启动子区域的重组菌株。
根据本发明，所述步骤(1)中，使所述碱基发生突变的方法包括诱变、PCR定点突变或同源重组，优选PCR定点突变法。
根据本发明，所述步骤(1)包括：
根据genebank中野生型rhtA基因启动子序列，合成两对扩增rhtA基因启动子区片段的引物，通过等位基因置换在宿主菌株背景中的rhtA基因启动子区。
在本发明的一个实施方案中，所述引物为：
P1:5'CG GGATCCTCGCTGGTGTCGTGTTTGTAGG 3'(划线部分为限制性内切酶酶切位点BamH I)(SEQ ID NO:17)
P2:5'TATACCCAATGCTGGTCGAG 3'(SEQ ID NO:18)
P3:5'CGACCAGCATTGGGTATATC 3'(SEQ ID NO:19)
P4:5'AAGGAAAAAA GCGGCCGCCGAAAATTAACGCTGCAATCAAC 3'(划线部分为限制性内切酶酶切位点Not I)(SEQ ID NO:20)。
在本发明的一个实施方案中，所述步骤(1)包括：以E.coli K12为模板，分别以引物P1和P2、P3和P4，进行PCR扩增，获得两条含有rhtA基因启动子区分离的DNA片段，大小为690bp及640bp，即PrhtA (A(-67)G)-Up和PrhtA (A(-67)G)-Down片段；以上述两条DNA片段为模板，以P1和P4为引物，通过重叠PCR扩增(Overlap PCR)，所述重叠PCR扩增按如下方式进行：94℃变性30s，52℃退火30s，以及72℃延伸60s(30个循环)，获得PrhtA (A(-67)G)-Up-Down片段。
根据本发明，所述步骤(2)包括：对于PrhtA (A(-67)G)-Up-Down片段进行琼脂糖凝胶电泳和分离纯化，将上述片段用BamH I/Not I双酶切后，与EcoR I/Sph I双酶切后的质粒相连接，获得等位替换的重组载体。
在本发明的一个实施方案中，所述步骤(3)的转化为电转化法。
本发明还提供如上所述的构建方法所获得的重组菌株。
本发明提供如上所述的重组菌株在L-苏氨酸制备中的应用。
根据本发明所述的重组菌株在L-苏氨酸制备中的应用，包括采用所述重组菌株进行发酵，制备得到L-苏氨酸。
具体实施方式
下文通过对本发明实施例的描述，更加详细地对本发明的上述及其他特性和优势进 行解释和说明。应当理解，下列实施例旨在对本发明的技术方案进行示例性的说明，而并非旨在对由权利要求及其等价方案所限定的本发明保护范围进行任何限制。
除非另有说明，本文中的材料和试剂均为市售商品，或可由本领域技术人员根据现有技术制备。
实施例1
(1)构建deoB基因编码区定点突变(G1049A)的质粒pKOⅤ-deoB (G1049A)(对应编码蛋白的氨基酸序列SEQ ID NO:3中第350位半胱氨酸被酪氨酸取代(C350Y)，取代后的氨基酸序列为SEQ ID NO:4)
磷酸戊糖变位酶由deoB基因编码，在E.coli K12菌株及其衍生菌株(如MG1655等)中，野生型的deoB基因ORF序列如Genbank登录号为CP032667.1中序列3902352-3903575所示。依据该序列设计并合成两对扩增deoB的引物，构建载体用于将出发菌株中deoB基因编码区序列(SEQ ID NO:1)的第1049位碱基G突变为A(获得核苷酸序列SEQ ID NO:2)。引物设计如下(由上海invitrogen公司合成)：
P1:5'CG GGATCCATGGACGGCAACGCTGAAG 3'(划线部分为限制性内切酶酶切位点BamH I)(SEQ ID NO:5)
P2:5'GATCGTAACCGTGGTCAG 3'(SEQ ID NO:6)
P3:5'CTGACCACGGTTACGATC 3'(SEQ ID NO:7)
P4:5'AAGGAAAAAA GCGGCCGCGCTCGTGAGTGCGGATGT 3'(划线部分为限制性内切酶酶切位点Not I)(SEQ ID NO:8)
构建方法为：以野生型菌株E.coli K12基因组为模板，分别以引物P1和P2，P3和P4进行PCR扩增，获得含有点突变的、长度分别为836bp和890bp的两条DNA片段(deoB (G1049A)-Up和deoB (G1049A)-Down片段)。PCR体系：10×Ex Taq Buffer 5μL，dNTP Mixture(各2.5mM)4μL，MgCl 2(25mM)4μL，引物(10pm)各2μL，Template 1μL，Ex Taq(5U/μl)0.25μL，总体积50μL，所述PCR扩增按如下方式进行：94℃预变性5min，(94℃变性30s、52℃退火30s、以及72℃延伸90s，30个循环)，72℃过度延伸10min。将上述两条DNA片段经琼脂糖凝胶电泳分离纯化后，再以纯化后的两条DNA片段为模板，以P1和P4为引物，通过Overlap PCR扩增出长度约为1726bp的片段(deoB (G1049A)-Up-Down片段)。Overlap PCR体系：10×Ex Taq Buffer 5μL，dNTP Mixture(各2.5mM)4μL，MgCl 2(25mM)4μL，引物(10pm)各2μL，Template 1μL，Ex Taq(5U/μl)0.25μL， 总体积50μL，所述PCR扩增按如下方式进行：94℃预变性5min，(94℃变性30s、52℃退火30s、以及72℃延伸90s，30个循环)，72℃过度延伸10min。将上述deoB (G1049A)-Up-Down片段经琼脂糖凝胶电泳分离纯化，然后将其和pKOⅤ质粒(购自Addgene公司)分别用BamH I/Not I双酶切，将酶切后的deoB (G1049A)-Up-Down片段和pKOⅤ质粒经琼脂糖凝胶电泳分离纯化并连接，获得载体pKOⅤ-deoB (G1049A)。将载体pKOⅤ-deoB (G1049A)送测序公司进行测序鉴定，结果如SEQ ID NO:11所示。将含有正确的点突变(deoB (G1049A))的载体pKOⅤ-deoB (G1049A)保存备用。
(2)包含点突变基因deoB (G1049A)的工程菌株的构建
野生型大肠杆菌菌株E.coli K12(W3110)和高产L-苏氨酸的菌株E.coli CGMCC 7.232(保藏至中国普通微生物菌种保藏管理中心)的染色体上均保留了野生型的deoB基因。将构建好的质粒pKOⅤ-deoB (G1049A)分别转入E.coli K12(W3110)和E.coli CGMCC 7.232，通过等位基因置换，将这两个菌株染色体中的deoB基因序列相对于SEQ ID NO:1的第1049位碱基G变为A。
具体过程为：将质粒pKOⅤ-deoB (G1049A)通过电击转化转入宿主菌感受态细胞后加入0.5mL的SOC液体培养基；在30℃、100rpm的摇床中复苏2h；取100μL培养液涂布于氯霉素含量为34mg/mL的LB固体培养基，30℃培养18h；挑选长出的单克隆菌落，接种于10mL LB液体培养基中，37℃、200rpm培养8h；取100μL培养液涂布于氯霉素含量为34mg/mL的LB固体培养基，42℃培养12h；挑选1-5个单菌落接种于1mL LB液体培养基中，37℃、200rpm培养4h；取100μL培养液涂布于含有10％蔗糖的LB固体培养基，30℃培养24h；挑选单克隆，并一一对应划线于LB固体培养基和氯霉素含量为34mg/mL的LB固体培养基；挑选在LB固体培养基上生长，同时在氯霉素含量为34mg/mL的LB固体培养基不能生长的对应菌株进行PCR扩增鉴定。PCR扩增采用如下引物(上海invitrogen公司合成)：
P5：5'TGACGCCACCATCAAAGAGA 3'(SEQ ID NO:9)
P6：5'GTCAACGCTCCGCCCAAAT 3'(SEQ ID NO:10)
上述PCR扩增产物进行SSCP(单链构象多态性，Single-Strand Conformation Polymorphism)电泳，以质粒pKOⅤ-deoB(G1049A)扩增片段为阳性对照，野生型大肠杆菌扩增片段为阴性对照，水作为空白对照。在SSCP电泳中，长度相同而序列排列不同的单链寡核苷酸链在冰浴中形成的空间结构不同，电泳时迁移率也会有所差异。所以， 片段电泳位置与阴性对照片段位置不一致，且与阳性对照片段位置一致的菌株即为等位替换成功的菌株。以等位替换成功的菌株为模板，用引物P5和P6再次通过PCR扩增目的片段，并将目的片段连接到pMD19-T载体，测序。通过测序结果序列比对，测序结果如SEQ ID NO:12所示，deoB基因编码区序列第1049位碱基G变为A的重组子即为改造成功的菌株。将来自E.coli K12(W3110)的重组子命名为YPThr09，将来自E.coli CGMCC 7.232的重组子命名为YPThr10。
(3)苏氨酸发酵实验
将E.coli K12(W3110)菌株、E.coli CGMCC 7.232菌株以及突变菌株YPThr09、YPThr10分别接种在25mL表1所述的液体培养基中，于37℃、200rpm培养12h。然后，分别取1mL各菌株的培养物接种在25mL表1所述的液体培养基中，于37℃、200rpm发酵培养36h。通过HPLC测定L-苏氨酸的含量，每株菌做三个平行，计算平均值，检测结果见表2。
表1培养基配方
成分 配方g/L
葡萄糖 40
硫酸铵 12
磷酸二氢钾 0.8
七水硫酸镁 0.8
七水硫酸亚铁 0.01
一水硫酸锰 0.01
酵母提取物 1.5
碳酸钙 0.5
L-甲硫氨酸 0.5
氢氧化钾调节pH值 pH 7.0
表2苏氨酸发酵实验结果
Figure PCTCN2020111840-appb-000001
Figure PCTCN2020111840-appb-000002
由表2结果所示，无论对于高产还是低产L-苏氨酸的原始菌株，deoB基因的氨基酸序列第350位半胱氨酸被酪氨酸取代后，都有助于L-苏氨酸产量的提高。
实施例2
(1)包含点突变的rhtA基因启动子的转化载体pKOV-PrhtA (A(-67)G)的构建
苏氨酸和高丝氨酸外排蛋白(RHTA酶)由rhtA基因编码，在E.coli K12菌株及其衍生菌株(如W3110等)中，野生型的rhtA基因启动子序列PrhtA如Genbank登录号为AP009048.1中序列850520-850871所示。依据该序列设计并合成两对扩增启动子PrhtA的引物，构建载体用于将出发菌株中PrhtA启动子的碱基序列(SEQ ID NO:13)上游第-67位碱基A变为G(SEQ ID NO:14)。引物设计如下(由上海invitrogen公司合成)：
P1:5'CG GGATCCTCGCTGGTGTCGTGTTTGTAGG 3'(划线部分为限制性内切酶酶切位点BamH I)(SEQ ID NO:17)
P2:5'TATACCCAATGCTGGTCGAG 3'(SEQ ID NO:18)
P3:5'CGACCAGCATTGGGTATATC 3'(SEQ ID NO:19)
P4:5'AAGGAAAAAA GCGGCCGCCGAAAATTAACGCTGCAATCAAC 3'(划线部分为限制性内切酶酶切位点Not I)(SEQ ID NO:20)。
构建方法为：以野生型菌株E.coli K12基因组为模板，分别以引物P1和P2，P3和P4进行PCR扩增，获得含有点突变的、长度分别为690bp和640bp的两条DNA片段(PrhtA (A(- 67)G)-Up和PrhtA (A(-67)G)-Down片段)。PCR体系：10×Ex Taq Buffer 5μL，dNTP Mixture(各2.5mM)4μL，Mg 2+(25mM)4μL，引物(10pM)各2μL，Ex Taq(5U/μL)0.25μL，总体积50μL，所述PCR扩增按如下方式进行：94℃预变性5min，(94℃变性30s、52℃退火30s、72℃延伸30s，30个循环)，72℃过度延伸10min。
将上述两条DNA片段经琼脂糖凝胶电泳分离纯化后，再以纯化后的两条DNA片段为模板，以P1和P4为引物，通过Overlap PCR扩增出长度约为1310bp的片段(PrhtA (A(-67)G)-Up-Down片段)。
PCR体系：10×Ex Taq Buffer 5μL，dNTP Mixture(各2.5mM)4μL，Mg 2+(25mM)4μL，引物(10pM)各2μL，Ex Taq(5U/μL)0.25μL，总体积50μL，所述Overlap PCR按如下方式进行：94℃变性30s，52℃退火30s，以及72℃延伸60s(30个循环)。
将上述PrhtA (A(-67)G)-Up-Down片段经琼脂糖凝胶电泳分离纯化，然后将其和pKOV质粒(购自Addgene公司)分别用BamH I/Not I双酶切，将酶切后的PrhtA (A(-67)G)-Up-Down片段和pKOV质粒经琼脂糖凝胶电泳分离纯化并连接，获得载体pKOV-PrhtA (A(-67)G)。将载体pKOV-PrhtA (A(-67)G)送测序公司进行测序鉴定，将含有正确的点突变(PrhtA (A(-67)G))的载体pKOV-PrhtA (A(-67)G)保存备用。
(2)包含点突变点PrhtA (A(-67)G)的工程菌株的构建
野生型大肠杆菌菌株E.coli K12(W3110)和高产L-苏氨酸的菌株E.coli CGMCC 7.232(保藏至中国普通微生物菌种保藏管理中心)的染色体上均保留了野生型的PrhtA启动子。将构建好的质粒pKOV-PrhtA (A(-67)G)分别转入E.coli K12(W3110)和E.coli CGMCC 7.232，通过等位基因置换，将这两个菌株染色体中的PrhtA启动子的碱基序列上游第-67位碱基A变为G。具体过程为：将质粒pKOV-PrhtA (A(-67)G)通过电击转化转入宿主菌感受态细胞后，加入0.5mL的SOC液体培养基；在30℃、100rpm的摇床中复苏2h；取100μL培养液涂布于氯霉素含量为34μg/mL的LB固体培养基，30℃培养18h；挑选长出的单克隆菌落，接种于10mL LB液体培养基中，37℃、200rpm培养8h；取100μL培养液涂布于氯霉素含量为34μg/mL的LB固体培养基，42℃培养12h；挑选1-5个单菌落接种于1mL LB液体培养基中，37℃、200rpm培养4h；取100μL培养液涂布于含有10％蔗糖的LB固体培养基，30℃培养24h；挑选单克隆，并一一对应划线于LB固体培养基和氯霉素含量为34μg/mL的LB固体培养基；挑选在LB固体培养基上生长，同时在氯霉素含量为34μg/mL的LB固体培养基不能生长的对应菌株进行PCR扩增鉴定。PCR扩增采用如下引物(上海invitrogen公司合成)：
P5:5'ATACACCGCTATCCATCT 3'(SEQ ID NO:21)
P6:5'AACCAGGCATCCTTTCTC 3'(SEQ ID NO:22)
上述PCR体系：10×Ex Taq Buffer 5μL，dNTP Mixture(各2.5mM)4μL，Mg 2+(25mM)4μL，引物(10pM)各2μL，Ex Taq(5U/μL)0.25μL，总体积50μL，所述PCR扩增按如下方式进行： 94℃预变性5min，(94℃变性30s、52℃退火30s、72℃延伸30s，30个循环)，72℃过度延伸10min。PCR扩增产物进行SSCP(单链构象多态性，Single-Strand Conformation Polymorphism)电泳，以质粒pKOV-PrhtA (A(-67)G)扩增片段为阳性对照，野生型大肠杆菌扩增片段为阴性对照，水作为空白对照。在SSCP电泳中，长度相同而序列排列不同的单链寡核苷酸链在冰浴中形成的空间结构不同，电泳时迁移率也会有所差异。所以，片段的电泳位置与阴性对照片段位置不一致，且与阳性对照片段位置一致的菌株即为等位替换成功的菌株。以等位替换成功的菌株为模板，用引物P5和P6再次通过PCR扩增目的片段，并将目的片段连接到pMD19-T载体，测序。通过测序结果序列比对，PrhtA启动子的碱基序列上游第-67位碱基A变为G的重组子即为改造成功的菌株。将来自E.coli K12(W3110)的重组子命名为YPThr01，将来自E.coli CGMCC 7.232的重组子命名为YPThr02。
(3)苏氨酸发酵实验
将E.coli K12(W3110)菌株、E.coli CGMCC 7.232菌株以及突变菌株YPThr01、YPThr02分别接种在25mL表1所述的液体培养基中，于37℃、200rpm培养12h。然后，分别取1mL各菌株的培养物接种在25mL表1所述的液体培养基中，于37℃、200rpm发酵培养36h。通过HPLC测定L-苏氨酸的含量，每株菌做三个平行实验，计算平均值，检测结果见表2。
表1培养基配方
成分 配方g/L
葡萄糖 40
硫酸铵 12
磷酸二氢钾 0.8
七水硫酸镁 0.8
七水硫酸亚铁 0.01
一水硫酸锰 0.01
酵母提取物 1.5
碳酸钙 0.5
L-甲硫氨酸 0.5
氢氧化钾调节pH值 pH 7.0
表2苏氨酸发酵实验结果
Figure PCTCN2020111840-appb-000003
Figure PCTCN2020111840-appb-000004
由表2结果所示，无论对于高产还是低产L-苏氨酸的原始菌株，rhtA基因的启动子序列PrhtA的第-67位碱基A突变为G，都有助于L-苏氨酸产量的提高。
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，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. 一种核苷酸序列，其包括选自如下的序列：
    i.SEQ ID NO:1所示的野生型deoB基因编码序列第1049位碱基发生突变而形成的序列；或者
    ii.SEQ ID NO:13所示的核苷酸序列上游第-67位碱基发生突变而形成的启动子核苷酸序列。
  2. 根据权利要求1的核苷酸序列，
    i.所述突变为SEQ ID NO:1中第1049位碱基由鸟嘌呤(G)突变为腺嘌呤(A)，优选的，所述突变后的核苷酸序列如SEQ ID NO:2所示；或者
    ii.所述突变为SEQ ID NO:13中第-67位碱基由腺嘌呤(A)突变为鸟嘌呤(G)，优选的，所述突变后的核苷酸序列如SEQ ID NO:14所示。
  3. 表达盒，包含权利要求1中的启动子核苷酸序列ii，和rhtA基因的编码核苷酸序列。
    优选的，所述rhtA基因的编码核苷酸序列包含SEQ ID NO:15所示的核苷酸序列。
  4. 重组蛋白，包括如SEQ ID NO:4所示的氨基酸序列；优选的，其由权利要求1中的核苷酸序列i编码。
  5. 重组载体，其含有如权利要求1所述的核苷酸序列。
  6. 根据权利要求5所述的重组载体，所述重组载体是将所述核苷酸序列导入质粒构建而成。
  7. 重组菌株，其含有权利要求1的核苷酸序列。
  8. 根据权利要求7的重组菌株，所述重组菌株是将权利要求5的重组载体导入宿主菌株中重组形成；所述宿主菌株选自大肠杆菌；例如，所述宿主菌株为E.coli K12、其衍生菌株E.coli K12(W3110)、或E.coli CGMCC 7.232菌株。
  9. 如权利要求7所述的重组菌株的构建方法，包括如下步骤：
    (1)改造如SEQ ID NO:1或者SEQ ID NO:13所示的野生型基因的核苷酸序列，得到如SEQ ID NO:2或者SEQ ID NO:14所示的突变后的核苷酸序列；
    (2)将所述突变后的核苷酸序列与质粒连接，构建重组载体；优选的，所述质粒为pKOV质粒；
    (3)将所述重组载体导入宿主菌株，得到所述重组菌株。
  10. 权利要求1的核苷酸序列，权利要求3的表达盒，权利要求4的重组蛋白，权利要求5的重组载体，或者权利要求7的重组菌株在发酵制备L-苏氨酸中的应用。
PCT/CN2020/111840 2019-08-28 2020-08-27 基于大肠杆菌的重组菌株及其构建方法与应用 WO2021037165A1 (zh)

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