WO2023097668A1 - 一种2-氮己环酮的生物合成代谢通路基因的表达载体和2-氮己环酮的合成方法 - Google Patents

一种2-氮己环酮的生物合成代谢通路基因的表达载体和2-氮己环酮的合成方法 Download PDF

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WO2023097668A1
WO2023097668A1 PCT/CN2021/135394 CN2021135394W WO2023097668A1 WO 2023097668 A1 WO2023097668 A1 WO 2023097668A1 CN 2021135394 W CN2021135394 W CN 2021135394W WO 2023097668 A1 WO2023097668 A1 WO 2023097668A1
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gene
promoter
mutant
expression vector
chnr
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罗小舟
赵西西
冯庭叶
卢欢
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中国科学院深圳先进技术研究院
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
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Definitions

  • the invention belongs to the technical field of microorganisms, and relates to the optimization of key gene expression of the biosynthetic metabolic pathway of 2-azicyclone and the high-efficiency biosynthesis method of 2-azecyclone, in particular to a method for the biosynthesis of 2-azecyclone An expression vector of a biosynthetic metabolic pathway gene and a synthesis method of 2-azone.
  • 2-Azicyclone is the main monomer for the synthesis of nylon-5 and nylon-6,5.
  • Nylon-5 and nylon-6,5 can not only be used as important engineering plastics to replace metals such as steel, iron, and copper, but also can be used to make tires, carpets, etc.
  • 2-azicyclone is mainly synthesized by chemical methods using petroleum as raw material. This synthesis process requires high equipment and is not friendly to the environment, and will produce more waste acid and waste gas. Its substitutes have important strategic value. Compared with traditional chemical synthesis, biosynthesis has the advantages of environmental friendliness, large space for cost reduction and sustainability. Therefore, the research on the biosynthesis of 2-azidine is of great significance in terms of sustainable development and ecological environment protection.
  • lysine is catalyzed by lysine monooxygenase (L-lysine monooxygenase, DavB) and 5-aminoglutaramide hydrolase (5-aminovaleramide amidohydrolase, DavA) to form 5-aminovaleric acid (5 -AVA), and then cyclization reaction can form 2-azepinecyclone.
  • 5-aminovaleric acid is an important precursor for the synthesis of 2-azidine and other C5 compounds, and its biosynthetic pathway has been extensively studied.
  • the existing technologies and methods are to construct the biosynthetic pathway of 2-azone in Escherichia coli, and use exogenous lysine as the raw material for the synthesis of 2-azone, and the use of lysine as a raw material increases the fermentation cost ;
  • the enzyme activity limits the biosynthesis of 2-azecyclone, resulting in low biosynthesis of 2-azecyclone.
  • the synthetic route of 2-azepinecyclone (valerolactam) is shown in the following formula.
  • 2-azidine has been successfully biosynthesized in Escherichia coli, but its yield is only 1.18g/L, and additional lysine is needed as a synthetic raw material.
  • the present invention aims to provide an expression vector of a biosynthetic metabolic pathway gene of 2-azone and a method for synthesizing 2-azone.
  • the present invention obtains a strain with high lysine production by modifying the genome of Corynebacterium glutamicum, and successfully heterologously expresses the synthetic metabolic pathway of 2-azecyclone in the Corynebacterium glutamicum with high production of lysine, By optimizing the expression of key enzymes in the 2-azecyclone synthesis pathway, the high yield of 2-azecyclone in Corynebacterium glutamicum was achieved.
  • the first aspect of the present invention provides an expression vector of a biosynthetic metabolic pathway gene of 2-azecyclone, the expression vector includes a first promoter and davA gene, davB gene, act gene or the orf26 gene or the caiC gene; or,
  • the expression vector includes a first promoter and davB gene, davA gene, act gene or orf26 gene or caiC gene inserted sequentially downstream of the first promoter;
  • the expression vector is based on a vector suitable for expression in Corynebacterium glutamicum;
  • the vector suitable for expression in Corynebacterium glutamicum is p208CES;
  • the vector suitable for expression in Corynebacterium glutamicum is a high-copy plasmid p208-HCP.
  • the second aspect of the present invention provides an expression vector of a biosynthetic metabolic pathway gene of 2-azecyclone, the expression vector includes a first promoter and davA gene, davB gene, Pb a promoter or a mutant thereof, an act gene or an orf26 gene or a caiC gene, a ChnR gene or a mutant thereof; or,
  • the expression vector includes a first promoter and a davB gene, a davA gene, a Pb promoter or a mutant thereof, an act gene or an orf26 gene or a caiC gene, a ChnR gene or a mutant thereof inserted sequentially downstream of the first promoter; or ,
  • the expression vector comprises a first promoter and a davA gene, a davB gene, a Pb promoter or a mutant thereof, a ChnR gene or a mutant thereof, an act gene or an orf26 gene or a caiC gene inserted sequentially downstream of the first promoter; or ,
  • the expression vector includes the first promoter and davB gene, davA gene, Pb promoter or its mutant, ChnR gene or its mutant, act gene or orf26 gene or caiC gene inserted sequentially downstream of the first promoter.
  • the Pb promoter mutant is a mutant that responds to 1mM 2-azone in the presence of the ChnR gene
  • the ChnR gene mutant is a mutant that responds better to 2-azone than ChnR in the presence of a Pb promoter or a mutant thereof.
  • the expression vector uses a vector suitable for expression in Corynebacterium glutamicum as a backbone.
  • the vector suitable for expression in Corynebacterium glutamicum is p208CES;
  • the vector suitable for expression in Corynebacterium glutamicum is a high-copy plasmid p208-HCP.
  • the Pb promoter has a nucleotide sequence as shown in SEQ ID NO.1;
  • the ChnR gene has a nucleotide sequence as shown in SEQ ID NO.3;
  • the Pb promoter mutant has a nucleotide sequence as shown in SEQ ID NO.2;
  • the ChnR gene mutant has a nucleotide sequence as shown in SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6.
  • the first promoter is a constitutive promoter
  • the first promoter is a strong promoter
  • the first promoter is H1, H2, H9 or H10;
  • the first promoter is H1.
  • the third aspect of the present invention provides a strain comprising the above expression vector.
  • the bacterial strain is obtained by transferring the expression vector into a high-lysine-producing Corynebacterium glutamicum.
  • the high-lysine-producing Corynebacterium glutamicum is obtained by performing a C932T point mutation on the aspartokinase gene of wild-type Corynebacterium glutamicum ATCC13032, or through other methods for lysine synthesis and metabolism pathway genes or Obtained after genetic modification related to other pathways.
  • the fourth aspect of the present invention provides a kind of synthetic method of 2-azone, comprising:
  • the seed liquid is inoculated into the fermentation medium, and the fermentation culture is carried out.
  • the formula of the seed culture medium is:
  • the conditions for culturing the bacterial strain in the seed medium are: temperature 30° C., rotation speed 200 rpm, time 17-18 hours. Further, the seed liquid is inoculated in 400mL fermentation medium with an inoculation amount of OD600 of 1.5; preferably, the formulation of the fermentation medium is;
  • the conditions of the fermentation culture are: temperature 30°C, maximum rotation speed 1200rpm, oxygen flow rate 30%, time 96h;
  • the pH is controlled to be 7.0 by adding 28% by volume of ammonia water, and 10% by volume of the defoamer 204 is used to eliminate foam.
  • fermentation culture is fed-batch fermentation
  • the fed-batch fermentation uses 50% by mass volume of glucose as an additional carbon source to control the residual sugar content at 5-10g/L, and controls the pH to 7.0 with 28% by volume of ammonia water. 10% of defoamer 204 inhibits foam.
  • the fifth aspect of the present invention provides a transcription factor biosensor of 2-azidine, said biosensor comprising the first promoter to promote the expression of the ChnR gene or its mutant, the Pb promoter or its mutant and the Pb promoter The reporter gene that the child or its mutants promote expression;
  • the first promoter is a constitutive promoter
  • the reporter gene is mCherry or sfGFP;
  • the reporter gene is sfGFP.
  • the Pb promoter has a nucleotide sequence as shown in SEQ ID NO.1;
  • the ChnR gene has a nucleotide sequence as shown in SEQ ID NO.3;
  • the Pb promoter mutant has a nucleotide sequence as shown in SEQ ID NO.2;
  • the ChnR gene mutant has a nucleotide sequence as shown in SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6.
  • the sixth aspect of the present invention provides that the transcription factor biosensor of 2-azacyclone is used in the biosynthesis of 2-azecyclone, 2-pyrrolidone or caprolactam and in the screening of highly active Act enzyme, CaiC enzyme or Orf26 enzyme Applications.
  • the expression vector of the biosynthetic metabolic pathway gene of 2-azicyclone provided by the present invention is to use the molecular probe system ChnR-Pb of 2-azone as act, caiC,
  • the promoter of the orf26 gene has a high response to low concentration of 2-azepinone.
  • operon davA-davB-cyclization gene (act/caiC/orf26) was promoted as the biosynthetic metabolic pathway of 2-azicyclone with a highly active promoter, and the high-copy plasmid p208-HCP (about 40- 50 copies) as the backbone carrier, which optimizes the biosynthetic pathway of 2-azecyclone.
  • the present invention synthesizes 2-azicyclone in Corynebacterium glutamicum for the first time, and utilizes the optimized 2-azehexone molecular probe system ChnR-Pb for the first time in the biosynthetic metabolic pathway of 2-azehexone
  • the expression of key genes realizes the high yield of 2-azone.
  • the expression vector of the biosynthetic metabolic pathway gene of 2-azepinone includes the H1 promoter and the davB-davA gene or davA-davB gene, pBb-E1 inserted sequentially downstream of the H1 promoter Promoter, act gene or orf26 gene or caiC gene, ChnR-B1 gene, the strain obtained by transferring the expression vector into high-yield lysine Corynebacterium glutamicum is used for fermentation and cultivation, and can realize 2-azone efficient biosynthesis.
  • the 2-azecyclone transcription factor biosensor provided by the present invention can also respond to 2-pyrrolidone or caprolactam, so the biosensor can also be applied to the expression of rate-limiting genes in the 2-pyrrolidone or caprolactam synthesis pathway.
  • the transcription factor biosensor for 2-azecyclone can obtain enzymes with higher activity through directed evolution of the key enzymes Act, CaiC and Orf26 in the 2-azecyclone synthetic metabolic pathway, thereby further improving the 2-nitrogen Biosynthesis of hexanone.
  • Fig. 1 is the lysine production of the Corynebacterium glutamicum mutant XT1 strain in the shake flask.
  • Fig. 2 is the activity analysis of different promoters in the XT1 strain of Corynebacterium glutamicum mutant.
  • Fig. 3 shows the response of the molecular probe pBblactam of 2-valerolactam and the mutant pBb-E1 to low concentration of 2-valerolactam.
  • Fig. 4 is the response of different fluorescent proteins to low concentration of 2-azecyclone in molecular probe pBb-E1.
  • Fig. 5 shows the mutants with better response to low concentration of 2-azecyclone screened from the ChnR gene random mutation library of molecular probe pBb-E1.
  • Fig. 6 is an analysis of metabolites of different 2-azecyclone synthetic metabolic pathways in Corynebacterium glutamicum XT1.
  • Fig. 7 is the analysis of metabolites of strains containing different 2-azecyclone biosynthetic pathways after 96 hours of culture in the fermenter.
  • Fig. 8 shows the response of the optimized 2-azidine transcription factor biosensor to 2-pyrrolidone and caprolactam. .
  • the key gene lysC (aspartokinase) in the lysine synthesis and metabolism pathway of wild-type Corynebacterium glutamicum ATCC13032 was point-mutated to release the feedback inhibition of the enzyme by lysine, thereby obtaining High lysine producing Corynebacterium glutamicum strain XT1.
  • the build method is as follows:
  • primer 1-1 and primer 1-2 to amplify fragment A containing the point mutation C932T from the wild-type Corynebacterium glutamicum genome, wherein the point mutation site is in primer 1-2 (marked with bold font), marked with Primers 1-3 and primers 1-4 amplified fragments of the second half of the lysC gene and its downstream genes from the genome to obtain Fragment B, in which primers 1-3 (marked in bold font) also contained the lysC point mutation site C932T , fragment A and B obtained by amplification are carried out fusion PCR amplification with primer 1-1 and primer 1-4, obtain fragment AB, then fragment AB is passed through Gibson and the Backbone of corynebacterium glutamicum genome transformation plasmid pK18 (with Primers 1-5 and primers 1-6 are amplified from the pK18mobsacB plasmid) to connect, then transfer to the point mutation plasmid pK18-lysC (C932T) of the ly
  • the fermentation medium (pH is 7.2) used in shake flask fermentation in the present embodiment is:
  • Corynebacterium glutamicum strain XT1 was 0.1 OD, which was inoculated into 25 ml of the above-mentioned medium, and 0.75 g of CaCO 3 was added at the same time to maintain the pH of the shake flask fermentation.
  • the fermentation conditions were 30° C., 200 rpm.
  • promoter activity of Corynebacterium glutamicum reported in the literature, strong promoters H1, H2, H9, H10 (promoter sequences are listed below) were selected, and mCherry was used as the reporter protein (derived from the plasmid pBblactam containing the mCherry gene) , pEC-XK99E is an expression vector, and the activity of the above-mentioned promoters in the XT1 strain is tested.
  • coli DH5 ⁇ and the pEC-H1-mCherry, pEC-H2-mCherry, pEC-H9-mCherry, pEC-H10-mCherry plasmids were respectively obtained by sequencing, and the obtained plasmids were Transformed into Corynebacterium glutamicum XT1 strain respectively.
  • LBHIS medium The formula of LBHIS medium is:
  • point mutations were performed on the expression vector p208 (p208CES) of Corynebacterium glutamicum to obtain a high-copy expression vector p208-HCP, and at the same time, the genes davA, davB, act, caiC, orf26 of the 2-azepinone synthesis pathway
  • the codon-optimized gene synthesis based on Corynebacterium glutamicum was carried out, and the biosynthetic pathway of 2-azicyclone was constructed on the p208-HCP vector with H1 as the promoter (the last step of cyclization was catalyzed by Act, CaiC, and Orf26, respectively). reaction).
  • the build method is as follows:
  • the 382 of the ORFA2 region of the p208 vector is mutated from g to t to obtain a high-copy plasmid p208-HCP (10-fold increase in copy number), using primer 3-1 (containing the mutation site, marked in bold) and primer 3-2 Amplify the p208 plasmid, then use Gibson ligation to purify the PCR fragment, transfer it into E. coli DH5 ⁇ strain, and obtain the high-copy plasmid p208-HCP correctly by sequencing.
  • the genes davA, davB, act, caiC, orf26 based on the codon-optimized 2-azecyclone synthesis pathway of Corynebacterium glutamicum were synthesized in Jinweizhi Co., Ltd., using primers 3-3 and 3 -4 Amplify the H1-davA fragment containing the H1 promoter sequence from the synthetic gene plasmid pUC-davA; amplify the davB1 fragment from the synthetic gene plasmid pUC-davB1 with primer 3-5 and primer 3-6, and use primer 3- 7 and primers 3-8 amplify the davB2 fragment from the synthetic gene plasmid pUC-davB2, and use primers 3-5 and primers 3-8 to fuse and amplify the davB1 and davB2 fragments to obtain the davB gene fragment; use primers 3-9 Amplify the act fragment from the synthetic gene plasmid pUC-act with
  • coli DH5 ⁇ strain and the 2-azecyclone synthetic metabolic pathway plasmids p208-HCP-H1-davA-davB-act and p208-HCP were respectively obtained by sequencing -H1-davA-davB-orf26, p208-HCP-H1-davA-davB-caiC.
  • the molecular probe pBblactam (ChnR-Pb) of 2-azecyclone was optimized, and the molecular probe mutant (ChnR-Pb-E1B1) with better response to low concentration of 2-azecyclone was obtained.
  • the build method is as follows:
  • the saturation mutation design was carried out on the binding region ttgtttggatc between the Pb promoter region and the ChnR protein in the 2-azidine molecular probe ChnR-Pb system.
  • the product was transformed into DH5 ⁇ strain by electroporation, and the plasmid was collected to extract the plasmid, then sequenced to evaluate the quality of the mutant library, and then transformed into Escherichia coli DH10B strain to obtain a saturated mutant library of the binding site of the Pb promoter region.
  • the mutant library and the control bacterial strain were all cultivated in LB containing 1mM 2-azhexone plus chloramphenicol resistance medium for 10h, and the mutant library and the control thalli were collected for flow cytometry sorting ( FACS); according to the mCherry fluorescence signal, set parameters in FACS to select 0.1% of the clones in the mutant library; collect and cultivate 0.1% of the clones, and randomly select the clones from the cultured plate to a 96-deep well plate (containing 1ml LB plus 1mM 2-azicyclone plus chloramphenicol) and pBblactam as a positive control, the selected clones were cultured at 37 degrees 800rpm, 10h after sampling in a microplate reader to detect fluorescence, according to the fluorescence signal to obtain the strongest fluorescence mutant E1 ( The sequence of the binding region is: TGTAGCCCACC), and the mutant was named pBb-E1, and its response to low concentration of 2-azepinone was
  • the molecular probe with sfGFP as the reporter protein has a better response effect; next, we constructed a random mutation library for the ChnR protein in pBb-E1, using a random mutation kit, with primers 4-5 and primers 4-6 Amplify the ChnR gene fragment in pBb-E1 to obtain a random mutation fragment of the ChnR gene, use primers 4-7 and primers 4-8 to amplify the backbone fragment of the plasmid from pBb-E1, and then pass the obtained ChnR The random mutation fragment of the gene is fused with the backbone fragment, and then transferred into the DH5 ⁇ strain by electric shock, 30 clones are randomly selected and sent for sequencing (for evaluating the quality of the random mutation library), the remaining clones are collected and the plasmid is extracted, and the plasmid is transferred into the DH10B strain by electric shock , a random mutation library of the ChnR gene was obtained.
  • the Pb promoter has the nucleotide sequence shown in SEQ ID NO.1
  • the Pb promoter mutant pBb-E1 has the nucleotide sequence shown in SEQ ID NO.2
  • the ChnR gene has the nucleotide sequence shown in SEQ ID NO.3 Nucleotide sequence shown.
  • the optimized molecular probe system ChnR-Pb-E1B1 was used to start the expression of the act/caiC/orf26 gene in the 2-azone biosynthetic pathway constructed in Example 3, and the optimized 2-azone was obtained Biosynthetic metabolic pathway plasmid; transfer it and the 2-azicyclone biosynthetic metabolic pathway plasmid constructed in Example 3 into the XT1 strain producing lysine respectively, and shake flask fermentation to verify the output of 2-azecyclone;
  • the build method is as follows:
  • the fermentation conditions were 30° C., 200 rpm. After 48 hours of fermentation, samples were taken to detect OD600, and the remaining samples were centrifuged to obtain the supernatant for LC-MS analysis of the yield of 2-azecyclone. The results are shown in Figure 6.
  • the high-yield XT1-Act (p208-HCP-H1-davA-davB-Pb-act-chnR), XT1-Orf26 (p208-HCP-H1-davA-davB-Pb-orf26-chnR) were verified by the shake flask fermentation of Example 5 ), XT1-CaiC (p208-HCP-H1-davA-davB-Pb-caiC-chnR) strains were used for fed-batch fermentation tests in fermentors.
  • the specific operation is: inoculate the bacterial strain into the seed culture medium to obtain the seed liquid, and the cultivation conditions are: temperature 30° C., rotation speed 200 rpm, and time 17-18 hours.
  • the seed solution was inoculated into 400 mL of fermentation medium with an inoculum size of OD600 of 1.5, and 50% by mass volume of glucose was used as feeding material to carry out fermentation culture.
  • the pH was controlled to be 7.0 by adding 28% by volume of ammonia water, and 10% by volume of the defoamer 204 was used to eliminate foam.
  • the fermentation culture temperature is 30° C.
  • the maximum rotation speed is 1200 rpm
  • the oxygen flow rate is 30%.
  • XT1-Act can obtain 8.8g/L of 2-azone
  • XT1-Orf26 can obtain 8.13g/L of 2-azecyclone
  • XT1-CaiC can obtain 12.6g/L of 2-azecyclone. The results are shown in Figure 7.
  • the formula for the seed medium is:
  • the formula of fermentation medium is;
  • This embodiment provides a transcription factor biosensor for 2-azidine, which includes the first promoter to promote the expression of the ChnR gene or its mutant, the Pb promoter or its mutant, and the Pb promoter or its mutant A reporter gene that initiates expression.
  • the Pb promoter has the nucleotide sequence shown in SEQ ID NO.1
  • the Pb promoter mutant pBb-E1 has the nucleotide sequence shown in SEQ ID NO.2
  • the ChnR gene has the nucleotide sequence shown in SEQ ID NO.3
  • the ChnR gene mutant has the nucleotide sequence shown in SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6, and the reporter gene is mCherry or sfGFP.
  • the first promoter is a constitutive promoter
  • the reporter gene is sfGFP
  • the Pb promoter mutant pBb-E1 is selected
  • the ChnR gene with the nucleotide sequence shown in SEQ ID NO.4 is selected for use Mutant B1.
  • the biosensor is responsive to 2-azecyclone, 2-pyrrolidone and caprolactam, and can be used to increase the expression of rate-limiting genes in the synthesis pathways of 2-azecyclone, 2-pyrrolidone and caprolactam.
  • the response of the transcription factor biosensor of 2-azecyclone to 2-pyrrolidone and caprolactam in this preferred scheme is shown in FIG. 8 .

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Abstract

提供了一种2-氮己环酮的生物合成代谢通路基因的表达载体和2-氮己环酮的合成方法。该表达载体包括第一启动子以及在第一启动子下游依次插入的davA-davB基因或davB-davA基因、Pb启动子或其突变体、act基因或orf26基因或caiC基因、ChnR基因或其突变体。进一步将表达载体转入高产赖氨酸的谷氨酸棒杆菌中，发酵培养以高效合成2-氮己环酮。

Description

一种2-氮己环酮的生物合成代谢通路基因的表达载体和2-氮己环酮的合成方法 技术领域
本发明属于微生物技术领域，涉及一种2-氮己环酮的生物合成代谢通路关键基因表达的优化和2-氮己环酮的高效生物合成方法，具体涉及一种2-氮己环酮的生物合成代谢通路基因的表达载体和2-氮己环酮的合成方法。
背景技术
2-氮己环酮是合成尼龙-5和尼龙-6,5的主要单体。尼龙-5以及尼龙-6,5除了可以作为重要的工程塑料用于替代钢、铁、铜等金属，也可以用于制作轮胎、地毯等。目前2-氮己环酮主要以石油为原料通过化学方法合成，该合成过程对设备要求高且对环境不友好，会产生较多的废酸、废气，此外石油作为不可再生资源，寻找或开发其替代品具有重要战略价值。相较于传统的化学合成，生物合成的方法具有环境友好、成本可降的空间大以及可持续的优势。因此对2-氮己环酮进行生物合成的研究，在可持续发展以及生态环境保护方面具有重要意义。
根据现有研究报道，赖氨酸经赖氨酸加氧酶(L-lysine monooxygenase，DavB)、5-氨基戊二酰胺水解酶(5-aminovaleramide amidohydrolase，DavA)催化形成5-氨基戊酸(5-AVA)，再经环化反应可形成2-氮己环酮。5-氨基戊酸作为2-氮己环酮和其他C5类化合物合成的重要前体，其生物合成途径已被进行了大量研究。在经过代谢工程改造的谷氨酸棒状杆菌中，利用来源于假单胞菌(Pseudomonas putida KT2440)的davB、davA基因合成5-氨基戊酸，其产量已经达到了39.93g/L；将改造后的大肠杆菌培养在含有120g/L的赖氨酸培养基中，同样以davB、davA基因构建5-氨基戊酸的合成通路，5-氨基戊酸的产量则达到了90.59g/L，然而现有报道2-氮己环酮的最高生物合成产量只有1.18g/L。如Chae等在大肠杆菌中导入假单胞菌来源的davB和davA基因，并以丙酸梭菌来源的丙二酰辅酶A转移酶(Act)催化2-氮己环酮合成中的最后一步反应，通过在发酵培养基中添加赖氨酸，以分批补料发酵方式最终获得了1.18g/L的2-氮己环酮。Zhang等以大肠杆菌来源的甜菜碱辅酶A连接酶(CaiC)和链霉菌来源的酰基辅酶A连接酶(ORF26)分别作为2-氮己环酮生物合成途径中环化反应的催化剂，与假单胞菌来源的davB和davA基因在大肠杆菌中构建2-氮己环酮的合成代谢通路，同样以赖氨酸为合成原料通过发酵获得了705mg/L的2-氮己环酮。
现有技术和方法均是在大肠杆菌中构建2-氮己环酮的生物合成通路，并以外源赖氨酸作为2-氮己环酮的合成原料，以赖氨酸作为原料增加了发酵成本；此外由于2-氮己环酮合成通 路中催化最后一步环化反应的酶活较低，酶活性限制了2-氮己环酮的生物合成，导致2-氮己环酮生物合成量低。
发明内容
2-氮己环酮(valerolactam)的合成路径如下式所示。目前2-氮己环酮成功的在大肠杆菌中实现了生物合成，然而其产量只有1.18g/L，且需要额外添加赖氨酸作为合成原料。为了解决现有技术中的不足，本发明旨在提供一种2-氮己环酮的生物合成代谢通路基因的表达载体和2-氮己环酮的合成方法。本发明通过对谷氨酸棒状杆菌基因组的改造获得赖氨酸高产的菌株，并成功在该高产赖氨酸的谷氨酸棒状杆菌中异源表达了2-氮己环酮的合成代谢通路，通过优化2-氮己环酮合成通路中关键酶的表达，实现了2-氮己环酮在谷氨酸棒状杆菌中的高产。
Figure PCTCN2021135394-appb-000001
本发明第一方面提供一种2-氮己环酮的生物合成代谢通路基因的表达载体，所述表达载体包括第一启动子以及在第一启动子下游依次插入的davA基因、davB基因、act基因或orf26基因或caiC基因；或，
所述表达载体包括第一启动子以及在第一启动子下游依次插入的davB基因、davA基因、act基因或orf26基因或caiC基因；
优选地，所述表达载体以适用于谷氨酸棒状杆菌表达的载体为骨架；
优选地，所述适用于谷氨酸棒状杆菌表达的载体为p208CES；
优选地，所述适用于谷氨酸棒状杆菌表达的载体为高拷贝质粒p208-HCP。
本发明第二方面提供一种2-氮己环酮的生物合成代谢通路基因的表达载体，所述表达载体包括第一启动子以及在第一启动子下游依次插入的davA基因、davB基因、Pb启动子或其突变体、act基因或orf26基因或caiC基因、ChnR基因或其突变体；或，
所述表达载体包括第一启动子以及在第一启动子下游依次插入的davB基因、davA基因、Pb启动子或其突变体、act基因或orf26基因或caiC基因、ChnR基因或其突变体；或，
所述表达载体包括第一启动子以及在第一启动子下游依次插入的davA基因、davB基因、Pb启动子或其突变体、ChnR基因或其突变体、act基因或orf26基因或caiC基因；或，
所述表达载体包括第一启动子以及在第一启动子下游依次插入的davB基因、davA基因、Pb启动子或其突变体、ChnR基因或其突变体、act基因或orf26基因或caiC基因。
进一步地，所述Pb启动子突变体为在ChnR基因存在条件下，对1mM 2-氮己环酮具有响应的突变体；
所述ChnR基因突变体为在有Pb启动子或其突变体存在下，对2-氮己环酮具有比ChnR更好响应的突变体。
进一步地，所述表达载体以适用于谷氨酸棒状杆菌表达的载体为骨架。
优选地，所述适用于谷氨酸棒状杆菌表达的载体为p208CES；
优选地，所述适用于谷氨酸棒状杆菌表达的载体为高拷贝质粒p208-HCP。
进一步地，所述Pb启动子具有如SEQ ID NO.1所示的核苷酸序列；
所述ChnR基因具有如SEQ ID NO.3所示的核苷酸序列；
优选地，所述Pb启动子突变体具有如SEQ ID NO.2所示的核苷酸序列；
优选地，所述ChnR基因突变体具有如SEQ ID NO.4、SEQ ID NO.5或SEQ ID NO.6所示的核苷酸序列。
进一步地，所述第一启动子为组成型启动子；
优选地，所述第一启动子为强启动子；
优选地，所述第一启动子为H1、H2、H9或H10；
优选地，所述第一启动子为H1。
本发明第三方面提供包含上述表达载体的菌株。
进一步地，所述菌株通过将所述表达载体转入高产赖氨酸的谷氨酸棒杆菌中获得。
进一步地，所述高产赖氨酸的谷氨酸棒杆菌通过对野生型谷氨酸棒杆菌ATCC13032的天冬氨酸激酶基因进行C932T点突变获得或通过其他方法对赖氨酸合成代谢通路基因或其他途径相关基因改造后获得。
本发明第四方面提供一种2-氮己环酮的合成方法，包括：
将转入所述表达载体的高产赖氨酸的谷氨酸棒杆菌菌株接种于种子培养基中，获得种子液；
将种子液接种到发酵培养基中，进行发酵培养。
进一步地，所述种子培养基的配方为：
Figure PCTCN2021135394-appb-000002
菌株在种子培养基中培养的条件为：温度30℃，转速200rpm，时间17-18h。进一步地，所述种子液以OD600为1.5的接种量接种于400mL发酵培养基中；优选地，所述发酵培养基的配方为；
Figure PCTCN2021135394-appb-000003
优选地，所述发酵培养的条件为：温度30℃，最大转速1200rpm，通氧量为30％，时间96h；
优选地，在发酵培养时，通过添加体积百分比28％的氨水控制pH为7.0，体积百分比10％的消泡剂204用于消除泡沫。
进一步地，所述发酵培养为分批补料发酵；
优选地，所述分批补料发酵以质量体积百分比50％葡萄糖作为补加碳源，使残糖含量控制在5-10g/L，以体积百分比28％的氨水控制pH为7.0，以体积百分比10％的消泡剂204抑制泡沫。
本发明第五方面提供一种2-氮己环酮的转录因子型生物传感器，所述生物传感器包括第一启动子启动表达的ChnR基因或其突变体，Pb启动子或其突变体和Pb启动子或其突变体启动表达的报告基因；
优选地，所述第一启动子为组成型启动子；
优选地，所述报告基因为mCherry或sfGFP；
优选地，所述报告基因为sfGFP。
优选地，所述Pb启动子具有如SEQ ID NO.1所示的核苷酸序列；
所述ChnR基因具有如SEQ ID NO.3所示的核苷酸序列；
优选地，所述Pb启动子突变体具有如SEQ ID NO.2所示的核苷酸序列；
优选地，所述ChnR基因突变体具有如SEQ ID NO.4、SEQ ID NO.5或SEQ ID NO.6所示的核苷酸序列。
本发明第六方面提供所述的2-氮己环酮的转录因子型生物传感器在2-氮己环酮、2-吡咯烷酮或己内酰胺生物合成以及在筛选高活性Act酶、CaiC酶或Orf26酶中的应用。
本发明的有益效果为：
1、本发明提供的2-氮己环酮的生物合成代谢通路基因的表达载体以2-氮己环酮的分子探针系统ChnR-Pb作为2-氮己环酮代谢通路中act、caiC、orf26基因的启动子，其对低浓度2-氮己环酮有较高响应。
进一步地，以高活性启动子启动操纵子davA-davB-cyclization gene(act/caiC/orf26)的表达作为2-氮己环酮的生物合成代谢通路，以高拷贝质粒p208-HCP(约40-50个拷贝)为骨架载体，优化了2-氮己环酮生物合成通路。
2、本发明首次在谷氨酸棒状杆菌中合成2-氮己环酮，首次利用优化的2-氮己环酮的分子探针系统ChnR-Pb启动2-氮己环酮生物合成代谢通路中关键基因的表达，实现2-氮己环酮 的高产。通过在高产赖氨酸的谷氨酸棒状杆菌中构建2-氮己环酮的合成通路，减少了以赖氨酸为合成原料所需的生产成本；利用2-氮己环酮的分子探针系统持续高表达催化环化反应所需的酶，解除了2-氮己环酮生物合成途径中的限速步骤，极大地提高了2-氮己环酮的生物合成产量。
在一个最优的实施方案中，2-氮己环酮的生物合成代谢通路基因的表达载体包括H1启动子以及在H1启动子下游依次插入的davB-davA基因或davA-davB基因、pBb-E1启动子、act基因或orf26基因或caiC基因、ChnR-B1基因，将该表达载体转入高产赖氨酸的谷氨酸棒杆菌中获得的菌株用于发酵培养，可以实现2-氮己环酮的高效生物合成。
3、本发明提供的2-氮己环酮的转录因子型生物传感器也可对2-吡咯烷酮或己内酰胺响应，因此该生物传感器也可应用于2-吡咯烷酮或己内酰胺合成通路中限速基因的表达。
该2-氮己环酮的转录因子型生物传感器可以通过定向进化2-氮己环酮合成代谢通路中的关键酶Act、CaiC、Orf26，获得具有更高活性的酶，从而进一步提高2-氮己环酮的生物合成量。
附图说明
图1为谷氨酸棒状杆菌突变体XT1菌株在摇瓶中的赖氨酸产量。
图2为不同启动子在谷氨酸棒状杆菌突变体XT1菌株中的活性分析。
图3为2-氮己环酮(valerolactam)的分子探针pBblactam以及突变体pBb-E1对低浓度2-氮己环酮的响应。
图4为不同荧光蛋白在分子探针pBb-E1中对低浓度2-氮己环酮的响应。
图5为从分子探针pBb-E1的ChnR基因随机突变库中筛选出的对低浓度2-氮己环酮有更好响应的突变体。
图6为不同的2-氮己环酮合成代谢通路在谷氨酸棒状杆菌XT1中的代谢产物分析。
图7为发酵罐培养96h后含有不同2-氮己环酮生物合成通路菌株的代谢产物分析。
图8为优化后的2-氮己环酮的转录因子型生物传感器对2-吡咯烷酮和己内酰胺的响应。。
具体实施方式
为了更清楚地理解本发明，现参照下列实施例及附图进一步描述本发明。实施例仅用于解释而不以任何方式限制本发明。实施例中，各原始试剂材料均可商购获得，未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件，或按照仪器制造商所建议的条件。
实施例1
对野生型谷氨酸棒杆菌(Corynebacterium glutamicum ATCC13032)的赖氨酸合成代谢通路中的关键基因lysC(天冬氨酸激酶)进行点突变，以解除赖氨酸对该酶的反馈抑制，从而获得高产赖氨酸的谷氨酸棒状杆菌菌株XT1。构建方法如下：
以引物1-1和引物1-2从野生型谷氨酸棒状杆菌基因组中扩增含有点突变C932T的片段A，其中点突变位点在引物1-2(以加粗字体标注)中，以引物1-3和引物1-4从基因组中扩增lysC基因后半段及其下游基因的部分片段获得片段B，其中引物1-3(加粗字体标注)中也含有lysC点突变位点C932T，将扩增获得的片段A和B以引物1-1和引物1-4进行融合PCR扩增，获得片段AB，然后将片段AB通过Gibson与谷氨酸棒状杆菌基因组改造质粒pK18的Backbone(以引物1-5和引物1-6从pK18mobsacB质粒中扩增)进行连接，然后转入大肠杆菌DH5α菌株中获得谷氨酸棒状杆菌的lysC基因的点突变质粒pK18-lysC(C932T)，将该质粒转入野生型谷氨酸棒状杆菌中，通过同源重组以及蔗糖诱导质粒基因消除，获得高产赖氨酸的谷氨酸棒状杆菌菌株XT1，摇瓶发酵验证赖氨酸的产量，结果如图1。
本实施例中摇瓶发酵用到的发酵培养基(pH为7.2)为：
Figure PCTCN2021135394-appb-000004
Figure PCTCN2021135394-appb-000005
谷氨酸棒状杆菌菌株XT1接种量为0.1OD，接种至25ml上述培养基中，同时加入0.75g CaCO 3，用于维持摇瓶发酵的pH。发酵条件为30℃，200rpm。
引物序列：
引物编号 Seq(5’-3’)  
1-1 atgcctgcaggtcgactATGGCCCTGGTCGTACAG SEQ ID NO.7
1-2 CGAGGGCAGGTGAAGATGATGTCGGTGGTGC SEQ ID NO.8
1-3 ATCTTCACCTGCCCTCGTTC SEQ ID NO.9
1-4 ctatgaccatgattacgCATCATGGACGAACTCAACG SEQ ID NO.10
1-5 catgcctgcaggtcgact SEQ ID NO.11
1-6 cgtaatcatggtcatagctg SEQ ID NO.12
实施例2
在XT1菌株中，测试文献(Promoter library-based module combination(PLMC)technology for optimization ofthreonine biosynthesis in Corynebacterium glutamicum.)报道的强启动子的活性，选取活性最强的启动子用于启动2-氮己环酮生物合成通路基因的表达。具体操作如下：
根据文献报道的谷氨酸棒状杆菌的组成型启动子活性，选取强启动子H1、H2、H9、H10(启动子序列如下表)，以mCherry为报告蛋白(来源于含有mCherry基因的质粒pBblactam)，pEC-XK99E为表达载体，测试上述启动子在XT1菌株中的活性。以引物2-1和引物2-2从pBblactam质粒扩增H1-mCherry片段，引物2-3和引物2-4从质粒pEC-XK99E扩增pEC-H1片段；以引物2-5和引物2-2扩增从pBblactam质粒H2-mCherry片段，引物2-6和引物2-4从质粒pEC-XK99E扩增pEC-H2片段；以引物2-7和引物2-2从pBblactam质粒扩增H9-mCherry片段，引物2-8和引物2-4从质粒pEC-XK99E扩增pEC-H9片段；以引物2-9和引物2-2从pBblactam质粒扩增H10-mCherry片段，引物2-10和引物2-4扩增从质粒pEC-XK99EpEC-H10片段；通过Gibson分别将上述H1-mCherry片段和pEC-H1片段，H2-mCherry片段和pEC-H2片段，H9-mCherry片段和pEC-H9片段，H10-mCherry片段和pEC-H10片段进行连接，并转入大肠杆菌DH5α，经测序分别获得pEC-H1-mCherry、pEC-H2-mCherry、pEC-H9-mCherry、pEC-H10-mCherry质粒，将获得的质粒分别转入谷氨酸棒状杆菌XT1菌株中。针对不同的启动子测试菌株，从平板上随机挑选三个克隆，在LBHIS培养基中过夜12h，然后以1：100的接种比例接种于含有1ml的LBHIS的96深孔板中，在96深孔板中测试培养不同时间上述启动子的活性，结果如图2所示， 根据结果显示，H1启动子在XT1菌株中具有最强活性，H1启动子将会用于2-氮己环酮生物合成通路的构建。
LBHIS培养基的配方为：
Figure PCTCN2021135394-appb-000006
启动子序列：
Figure PCTCN2021135394-appb-000007
引物序列：
Figure PCTCN2021135394-appb-000008
实施例3
根据文献报道，对谷氨酸棒状杆菌的表达载体p208(p208CES)进行点突变获得高拷贝表达载体p208-HCP，同时对2-氮己环酮合成通路的基因davA、davB以及act、caiC、orf26进行基于谷氨酸棒状杆菌的密码子优化基因合成，以H1为启动子，在p208-HCP载体上构建2-氮己环酮的生物合成通路(分别以Act、CaiC、Orf26催化最后一步环化反应)。构建方法如下：
根据文献报道，p208载体的ORFA2区域的382由g突变为t可获得高拷贝质粒p208-HCP(拷贝数增加10倍)，以引物3-1(含有突变位点，加粗字体标注)和引物3-2扩增p208质粒，然后以Gibson连接纯化PCR片段，转入大肠杆菌DH5α菌株中，经测序正确获得高拷贝质粒p208-HCP。
根据已有研究报道，在金唯智公司分别合成基于谷氨酸棒状杆菌密码子优化的2-氮己环酮合成通路的基因davA、davB以及act、caiC、orf26，以引物3-3和引物3-4从合成基因质粒pUC-davA中扩增含有H1启动子序列的H1-davA片段；以引物3-5和引物3-6从合成基因质粒pUC-davB1中扩增davB1片段，以引物3-7和引物3-8从合成基因质粒pUC-davB2中扩增davB2片段，并以引物3-5和引物3-8对davB1和davB2片段进行融合扩增，获得davB基因片段；以引物3-9和引物3-10从合成基因质粒pUC-act中扩增act片段；以引物3-11和引物3-12从合成基因质粒pUC-orf26中扩增orf26片段；以引物3-13和引物3-14从合成基因质粒pUC-caiC1中扩增caiC1片段，以引物3-15和引物3-16从合成基因质粒pUC-caiC2中扩增caiC2片段，并以引物3-13和引物3-16对caiC1和caiC2片段进行融合扩增，获得caiC基因片段；以引物3-17和引物3-18从质粒p208-HCP中扩增p208-HCP-H1骨架。回收上述扩增PCR片段，然后将H1-davA、davB、act、p208-HCP-H1进行Gibson连接，H1-davA、davB、orf26、p208-HCP-H1进行Gibson连接，H1-davA、davB、caiC、p208-HCP-H1进行Gibson连接，连接产物转入大肠杆菌DH5α菌株中，经测序正确分别获得2-氮己环酮合成代谢通路质粒p208-HCP-H1-davA-davB-act、p208-HCP-H1-davA-davB-orf26、p208-HCP-H1-davA-davB-caiC。
引物序列：
Figure PCTCN2021135394-appb-000009
Figure PCTCN2021135394-appb-000010
实施例4
对2-氮己环酮的分子探针pBblactam(ChnR-Pb)进行优化，获得对低浓度2-氮己环酮有更好响应的分子探针突变体(ChnR-Pb-E1B1)。构建方法如下：
根据文献报道(doi:10.1128/jb.182.17.4744-4751.2000)，对2-氮己环酮分子探针ChnR-Pb系统中Pb启动子区与ChnR蛋白的结合区域ttgtttggatc进行饱和突变设计，在引物4-1中设计上述区域对应的NNNNNNNNNNN饱和突变序列，以引物4-1和引物4-2从pBblactam中扩增含有结合位点饱和突变的片段，以引物4-3和引物4-4从pBblactam中扩增质粒骨架片段；将上述两个片段纯化回收后均进行限制性内切酶SacI和HindIII的酶切，回收酶切后的片段后进行T4DNA连接酶进行4℃连接16h，将连接后的产物电击转入DH5α菌株中，收集菌体提取质粒，然后测序评估突变库的质量，并转入大肠杆菌DH10B菌株中获得Pb启动子区结合位点的饱和突变库。将该突变库以及对照菌株均培养于含有1mM 2-氮己环酮的LB加氯霉素抗性培养基中10h，收集突变库和对照菌体用于进行流式细胞分选仪分选(FACS)；根据mCherry荧光信号，在FACS中设置参数选取突变库中0.1％的克隆；收集并培养0.1％的克隆，从培养的平板上随机挑选克隆至96深孔板中(含有1ml LB加1mM 2-氮己环酮加氯霉素)并以pBblactam为阳性对照，将挑选的克隆培养于37度800rpm、10h后取样在酶标仪中检测荧光，根据荧光信号获得荧光最强突变体E1(结合区域序列为：TGTAGCCCACC)，将该突变体命名为pBb-E1，并检测其对低浓度2-氮己环酮的响应效果，结果如图3所示。为了提高该分子探针对低浓度2-氮己环酮的灵敏度即有更明显的响应效果，我们首先将pBb-E1的报告蛋白mCherry更换为 sfGFP，经培养测试，结果如图4所示，以sfGFP为报告蛋白的分子探针有更好的响应效果；接下来我们对pBb-E1中的ChnR蛋白进行随机突变库的构建，采用随机突变试剂盒，以引物4-5和引物4-6对pBb-E1中的ChnR基因片段进行扩增，获得ChnR基因的随机突变片段，以引物4-7和引物4-8从pBb-E1中扩增质粒的骨架片段，然后通过Gibson将获得的ChnR基因随机突变片段和骨架片段进行融合，然后电击转入DH5α菌株中，随机挑选30个克隆送样测序(用于评估随机突变库质量)，收集剩余克隆并提取质粒，将质粒电击转入DH10B菌株中，获得ChnR基因的随机突变库。然后参考上述方法，对该随机突变库进行FACS分选，并以pBb-E1为阳性对照，获得具有更好响应突变体A4、E5、B1(突变位点信息如下表所示)，测试这三个突变体相对于pBb-E1对低浓度2-氮己环酮的响应效果，结果如图5所示，结果表明突变体B1具有更好的响应，因此后续2-氮己环酮的分子探针pBb-E1B1的ChnR-Pb系统会用于启动act、caiC、orf26基因的表达。
Pb启动子具有如SEQ ID NO.1所示的核苷酸序列，Pb启动子突变体pBb-E1具有如SEQ ID NO.2所示的核苷酸序列，ChnR基因具有如SEQ ID NO.3所示的核苷酸序列。
引物序列：
Figure PCTCN2021135394-appb-000011
ChnR基因突变体的具体信息：
Colony Mutation  
M-B1 63 agt-ggt,S-G,121 gtg-gcg,V-A,242 gct-gcc,A-A SEQ ID NO.4
M-E5 22 aat-act,N-T,63 agt-ggt,S-G. SEQ ID NO.5
M-A4 281 acc-aac,T-N SEQ ID NO.6
实施例5
以优化后的分子探针系统ChnR-Pb-E1B1启动实施例3构建好的2-氮己环酮生物合成通路中的act/caiC/orf26基因的表达，获得优化后的2-氮己环酮生物合成代谢通路质粒；将其与实施例3构建的2-氮己环酮生物合成代谢通路质粒分别转入产赖氨酸的XT1菌株中，摇瓶发酵验证2-氮己环酮的产量；构建方法如下：
以引物5-1和引物5-2从p208-HCP-H1-davA-davB-act中扩增p208-HCP-H1-davA-davB片段，以引物5-3和引物5-4从pBb-E1B1质粒中扩增Pb启动子区片段，以引物5-5和引物5-6从pBb-E1B1质粒中扩增chnR基因片段，以引物5-7和引物5-8从p208-HCP-H1-davA-davB-act中扩增act片段，以引物5-9和引物5-10从p208-HCP-H1-davA-davB-orf26中扩增orf26片段，以引物5-11和引物5-12从p208-HCP-H1-davA-davB-caiC扩增caiC片段。回收上述扩增PCR片段，然后将p208-HCP-H1-davA-davB、Pb、act、chnR进行Gibson连接，p208-HCP-H1-davA-davB、Pb、orf26、chnR进行Gibson连接，p208-HCP-H1-davA-davB、Pb、caiC、chnR进行Gibson连接，连接产物转入大肠杆菌DH5α菌株中，经测序正确分别获得2-氮己环酮合成代谢通路质粒p208-HCP-H1-davA-davB-Pb-act-chnR、p208-HCP-H1-davA-davB-Pb-orf26-chnR、p208-HCP-H1-davA-davB-Pb-caiC-chnR。将此步获得的3个质粒与实施例3得到的3个质粒分别电击转入产赖氨酸的谷氨酸棒状杆菌XT1菌株中，将获得的每个突变体菌株随机挑选三个克隆作为生物学重复于摇菌管中培养起来，并以OD600为0.1的接种量接种于含有25ml培养基的摇瓶中，所用培养基同实施例1，同时加入0.75g CaCO 3，用于维持摇瓶发酵的pH。发酵条件为30℃，200rpm。发酵48h后取样检测OD600，并将剩余样品离心取上清进行LC-MS分析2-氮己环酮的产量，结果如图6所示。
引物序列：
Figure PCTCN2021135394-appb-000012
Figure PCTCN2021135394-appb-000013
实施例6
将实施例5摇瓶发酵验证高产的XT1-Act(p208-HCP-H1-davA-davB-Pb-act-chnR)、XT1-Orf26(p208-HCP-H1-davA-davB-Pb-orf26-chnR)、XT1-CaiC(p208-HCP-H1-davA-davB-Pb-caiC-chnR)菌株进行发酵罐分批补料发酵测试。具体操作为：将菌株接种于种子培养基中，获得种子液，培养条件为：温度30℃，转速200rpm，时间17-18h。将种子液以OD600为1.5的接种量接种于400mL发酵培养基中，以质量体积百分比50％的葡萄糖作为流加物料，进行发酵培养。在发酵培养期间，通过添加体积百分比28％的氨水控制pH为7.0，体积百分比10％的消泡剂204用于消除泡沫。发酵培养温度为30℃，最大转速1200rpm，通氧量为30％。间隔12h取样保存用于残糖含量、OD600、发酵产物以及中间物的分析；根据发酵结果数据，经过96h的分批补料发酵，XT1-Act可获得8.8g/L的2-氮己环酮，XT1-Orf26可获得8.13g/L的2-氮己环酮，XT1-CaiC可获得12.6g/L的2-氮己环酮，结果如图7所示。
种子培养基的配方为：
Figure PCTCN2021135394-appb-000014
发酵培养基的配方为；
Figure PCTCN2021135394-appb-000015
Figure PCTCN2021135394-appb-000016
实施例7
本实施例提供一种2-氮己环酮的转录因子型生物传感器，其包括第一启动子启动表达的ChnR基因或其突变体，Pb启动子或其突变体和Pb启动子或其突变体启动表达的报告基因。Pb启动子具有如SEQ ID NO.1所示的核苷酸序列，Pb启动子突变体pBb-E1具有如SEQ ID NO.2所示的核苷酸序列，ChnR基因具有如SEQ ID NO.3所示的核苷酸序列，ChnR基因突变体具有如SEQ ID NO.4、SEQ ID NO.5或SEQ ID NO.6所示的核苷酸序列，报告基因为mCherry或sfGFP。
在一个优选的实施方案中，第一启动子为组成型启动子，报告基因为sfGFP，选用Pb启动子突变体pBb-E1，选用具有如SEQ ID NO.4所示核苷酸序列的ChnR基因突变体B1。该生物传感器对2-氮己环酮、2-吡咯烷酮和己内酰胺均有响应，可用于提高2-氮己环酮、2-吡咯烷酮和己内酰胺合成通路中限速基因的表达。该优选方案2-氮己环酮的转录因子型生物传感器对2-吡咯烷酮和己内酰胺的响应如图8所示。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (13)

  1. 一种2-氮己环酮的生物合成代谢通路基因的表达载体，其特征在于，所述表达载体包括第一启动子以及在第一启动子下游依次插入的davA基因、davB基因、act基因或orf26基因或caiC基因；或，
    所述表达载体包括第一启动子以及在第一启动子下游依次插入的davB基因、davA基因、act基因或orf26基因或caiC基因；
    优选地，所述表达载体以适用于谷氨酸棒状杆菌表达的载体为骨架；
    优选地，所述适用于谷氨酸棒状杆菌表达的载体为p208CES；
    优选地，所述适用于谷氨酸棒状杆菌表达的载体为高拷贝质粒p208-HCP。
  2. 一种2-氮己环酮的生物合成代谢通路基因的表达载体，其特征在于，所述表达载体包括第一启动子以及在第一启动子下游依次插入的davA基因、davB基因、Pb启动子或其突变体、act基因或orf26基因或caiC基因、ChnR基因或其突变体；或，
    所述表达载体包括第一启动子以及在第一启动子下游依次插入的davB基因、davA基因、Pb启动子或其突变体、act基因或orf26基因或caiC基因、ChnR基因或其突变体；或，
    所述表达载体包括第一启动子以及在第一启动子下游依次插入的davA基因、davB基因、Pb启动子或其突变体、ChnR基因或其突变体、act基因或orf26基因或caiC基因；或，
    所述表达载体包括第一启动子以及在第一启动子下游依次插入的davB基因、davA基因、Pb启动子或其突变体、ChnR基因或其突变体、act基因或orf26基因或caiC基因。
  3. 根据权利要求2所述的表达载体，其特征在于，所述Pb启动子突变体为在ChnR基因存在条件下，对1mM 2-氮己环酮具有响应的突变体；
    所述ChnR基因突变体为在有Pb启动子或其突变体存在下，对2-氮己环酮具有比ChnR更好响应的突变体。
  4. 根据权利要求2所述的表达载体，其特征在于，所述表达载体以适用于谷氨酸棒状杆菌表达的载体为骨架；
    优选地，所述适用于谷氨酸棒状杆菌表达的载体为p208CES；
    优选地，所述适用于谷氨酸棒状杆菌表达的载体为高拷贝质粒p208-HCP。
  5. 根据权利要求2所述的表达载体，其特征在于，所述Pb启动子具有如SEQ ID NO.1所示的核苷酸序列；
    所述ChnR基因具有如SEQ ID NO.3所示的核苷酸序列；
    优选地，所述Pb启动子突变体具有如SEQ ID NO.2所示的核苷酸序列；
    优选地，所述ChnR基因突变体具有如SEQ ID NO.4、SEQ ID NO.5或SEQ ID NO.6所示的核苷酸序列。
  6. 根据权利要求1或2所述的表达载体，其特征在于，所述第一启动子为组成型启动子；
    优选地，所述第一启动子为强启动子；
    优选地，所述第一启动子为H1、H2、H9或H10；
    优选地，所述第一启动子为H1。
  7. 包含权利要求1-6任一项所述表达载体的菌株。
  8. 根据权利要求7所述的菌株，其特征在于，所述菌株通过将权利要求1-6任一项所述表达载体转入高产赖氨酸的谷氨酸棒杆菌中获得。
  9. 根据权利要求8所述的菌株，其特征在于，所述高产赖氨酸的谷氨酸棒杆菌通过对野生型谷氨酸棒杆菌ATCC13032的天冬氨酸激酶基因进行C932T点突变获得或通过其他方法对赖氨酸合成代谢通路基因或其他途径相关基因改造后获得。
  10. 一种2-氮己环酮的合成方法，其特征在于，包括：
    将权利要求8所述菌株接种于种子培养基中，获得种子液；
    将种子液接种到发酵培养基中，进行发酵培养。
  11. 一种2-氮己环酮的转录因子型生物传感器，其特征在于，所述生物传感器包括第一启动子启动表达的ChnR基因或其突变体，Pb启动子或其突变体和Pb启动子或其突变体启动表达的报告基因；
    优选地，所述第一启动子为组成型启动子；
    优选地，所述报告基因为mCherry或sfGFP；
    优选地，所述报告基因为sfGFP。
  12. 根据权利要求11所述的转录因子型生物传感器，其特征在于，所述Pb启动子具有如SEQ ID NO.1所示的核苷酸序列；
    所述ChnR基因具有如SEQ ID NO.3所示的核苷酸序列；
    优选地，所述Pb启动子突变体具有如SEQ ID NO.2所示的核苷酸序列；
    优选地，所述ChnR基因突变体具有如SEQ ID NO.4、SEQ ID NO.5或SEQ ID NO.6所示的核苷酸序列。
  13. 权利要求11所述的2-氮己环酮的转录因子型生物传感器在2-氮己环酮、2-吡咯烷酮或己内酰胺生物合成以及在筛选高活性Act酶、CaiC酶或Orf26酶中的应用。
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