WO2018090288A1 - 一种高效合成α-氨基丁酸的单细胞工厂及其构建与应用 - Google Patents

一种高效合成α-氨基丁酸的单细胞工厂及其构建与应用 Download PDF

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WO2018090288A1
WO2018090288A1 PCT/CN2016/106241 CN2016106241W WO2018090288A1 WO 2018090288 A1 WO2018090288 A1 WO 2018090288A1 CN 2016106241 W CN2016106241 W CN 2016106241W WO 2018090288 A1 WO2018090288 A1 WO 2018090288A1
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dehydrogenase
recombinant
single cell
gene
cell factory
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饶志明
周俊平
杨套伟
张显
徐美娟
张蔡喆
戚云龙
郑俊贤
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江南大学
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    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
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    • C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)
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Definitions

  • the invention relates to a single cell factory for efficiently synthesizing ⁇ -aminobutyric acid and its construction and application, and belongs to the technical field of microorganisms.
  • Non-natural alpha-amino acids are a large class of amino acids that differ from 22 natural alpha-amino acids that can be synthesized by organisms. They have important biological and physiological effects and are widely used in peptides, chiral drugs, and alkaloids. Synthesis. --aminobutyric acid is an unnatural amino acid that inhibits the transmission of human neural information, and has the function of enhancing glucose phosphatase activity and promoting brain cell metabolism. Alpha-aminobutyric acid is also an important chemical raw material and pharmaceutical intermediate. It has been widely used in the synthesis of drugs, such as the anti-tuberculosis drug ethambutol hydrochloride and the antiepileptic drug levetiracetam. The market is huge.
  • the synthesis method of ⁇ -aminobutyric acid mainly includes three methods of chemical synthesis, enzymatic resolution and enzymatic conversion.
  • the chemical method includes desulfurization reaction, ammoniation hydrolysis reaction, butanone acid reduction method, etc.
  • the chemical synthesis is simple, the reaction conditions are harsh and easy to generate by-products, and sometimes it is necessary to utilize a large amount of organic solvents harmful to the environment, such as Jeffery EA.
  • the ⁇ -aminobutyric acid was prepared by electrochemical method, and the yield was only 48%, and the by-product glutamic acid was present.
  • the preparation of ⁇ -aminobutyric acid by microbial method has the advantages of high specificity, mild conditions and environmental friendliness.
  • the construction of recombinant microbial synthetic non-natural amino acid metabolic pathways can be completed.
  • the microbial preparation of ⁇ -aminobutyric acid is mainly carried out by extracellular enzymatic conversion, including enzymatic resolution of racemic ⁇ -aminobutyric acid and use of 2-butyric acid as a raw material by dehydrogenase or transaminase. Catalytic preparation.
  • the inventors constructed a one-step method using the bulk chemical L-threonine as an inexpensive substrate, through the enzyme system of L-threonine deaminase, L-amino acid dehydrogenase and coenzyme regeneration system. Preparation of ⁇ -aminobutyric acid.
  • the amount of L-threonine deaminase found during the enzymatic conversion process needs to be precisely controlled, otherwise it will cause the accumulation of the intermediate ketobutyric acid, thereby inhibiting the conversion of ketobutyrate to ⁇ -aminobutyric acid, resulting in enzyme conversion. Production of alpha-aminobutyric acid is interrupted.
  • the present invention provides an RBs sequence optimization for controlling the expression level of L-threonine deaminase, and simultaneously ligating it with a L-amino acid dehydrogenase and a dehydrogenase providing a cofactor NADH cycle to a plasmid. And expressed in E. coli, constructed into a recombinant E. coli single cell factory, using this single cell factory for whole cell transformation to efficiently prepare ⁇ -ammonia The method of butylbutyrate.
  • the invention constructs a single cell factory capable of efficiently synthesizing ⁇ -aminobutyric acid, mainly by controlling the expression level of L-threonine deaminase (ltd) in the recombinant bacteria by using rbs to control the intermediate product keto acid.
  • a first object of the present invention is to provide a recombinant single cell factory for efficiently synthesizing ⁇ -aminobutyric acid, which is obtained by transforming a recombinant co-expression vector into a host strain;
  • the expression vector is a L-threonine deaminase gene, an L-amino acid dehydrogenase gene, and a dehydrogenase gene providing a cofactor NADH cycle in a plasmid vector; wherein the expression of the L-amino acid dehydrogenase gene is Benchmark, controlling the expression level of the dehydrogenase for the cofactor NADH cycle makes the production rate of the cofactor NADH at a relatively high level, and controls the expression level of L-threonine deaminase at a relatively appropriate level.
  • the recombinant single cell plant can obtain a superior equilibrium rate from L-threonine to the intermediate ketobutyrate and from ketobutyrate to ⁇ -aminobutyric acid without causing accumulation of the intermediate ketobutyric acid. Therefore, it does not cause inhibition of the reaction.
  • the recombinant single cell factory does not require exogenous addition of cofactors, which reduces the path of substrate entry and exit or diffusion and increases the rate of transformation compared to other methods.
  • control is by, but not limited to, a promoter and rbs sequence optimization, and may also be an enhancer, a terminator, a silencer optimization, or the like.
  • control is performed by promoter and/or rbs sequence optimization.
  • the method for constructing the recombinant single cell factory comprises:
  • the supply rate of the cofactor NADH was regulated, mainly by optimizing the promoter and rbs sequence to control the expression of the dehydrogenase providing the cofactor NADH cycle to control the regeneration rate of the cofactor NADH;
  • the L-threonine deaminase gene is preceded by a promoter carried by the plasmid vector itself, for L-
  • the expression level of the threonine deaminase gene and the plasmid vector is lower than that of the rbs sequence of the plasmid vector itself; the L-threonine deaminase gene and the L-amino acid dehydrogenase gene are carried by the plasmid vector itself.
  • the rbs linkage; the dehydrogenase gene providing the cofactor NADH cycle is preceded by a promoter designed for the dehydrogenase gene and the plasmid vector providing the cofactor NADH cycle, and the rbs sequence having an expression intensity higher than or equal to the plasmid vector itself rbs .
  • the host strain may be Escherichia coli or other hosts such as Bacillus subtilis, Corynebacterium, yeast, and the like.
  • the host strain is E. coli BL21.
  • the plasmid vector can be any commercially available plasmid vector or any of the previously reported engineered plasmid vectors.
  • the L-threonine deaminase is selected from, but not limited to, E. coli-derived L-threonine deaminase (the nucleotide sequence is Gene ID: 948287 on NCBI).
  • the L-amino acid dehydrogenase is selected from, but not limited to, Bacillus-derived L-leucine dehydrogenase (Gene ID: 1206507), Bacillus-derived L-C Dehydrogenase (Gene ID: 936557 on NCBI), L-proline dehydrogenase from Streptomyces (Gene ID: 1099526), Rhodococcus-derived L-phenylalanine dehydrogenase (NCBI) Gene ID: 4219741).
  • the dehydrogenase providing a cofactor NADH cycle is selected from, but not limited to, formate dehydrogenase of Candida boidin (GenBank: KM454879.1 on NCBI), glucose of Bacillus subtilis Dehydrogenase (GeneID: 938261 on NCBI), glucose dehydrogenase from Pseudomonas putida (Gene ID: 1045820).
  • the rbs sequences can be rbs sequences of different intensities.
  • the promoter or rbs sequence preceding the L-threonine deaminase gene can be optimized for different expression systems.
  • the promoter or rbs sequence preceding the dehydrogenase gene providing a cofactor NADH cycle may also be optimized for different expression systems.
  • the expression system includes, but is not limited to, an E. coli expression system, and may also be a Bacillus subtilis expression system, a Corynebacterium expression system, a yeast expression system, and the like.
  • the host is Escherichia coli; the promoter prior to the L-threonine deaminase gene is a T7 promoter; the rbs sequence directly linked to the L-threonine deaminase gene is Any one of the sequences of SEQ ID NO: 1 to SEQ ID NO: 6; the L-threonine deaminase gene and the L-amino acid dehydrogenase gene have the sequence shown by SEQ ID NO: Sequence RBS connection.
  • the promoter prior to the dehydrogenase gene providing a cofactor NADH cycle is a tac promoter
  • the rbs sequence is any one of SEQ ID NO: 33 to SEQ ID NO: 39.
  • the rbs sequence is SEQ ID NO: 37 the sequence of.
  • the recombinant co-expression vector is constructed based on the plasmid vector pET-28a; the L-threonine deaminase gene and the L-amino acid dehydrogenase gene are carried by pET-28a itself The rbs are directly connected.
  • a second object of the present invention is to provide a method for the fermentation synthesis of ⁇ -aminobutyric acid by using the recombinant single cell factory of the present invention.
  • the method comprises: activating a recombinant single cell plant and transferring it to a fermentation medium, inducing expression or directly expressing the recombinant protein by IPTG, collecting the cells by centrifugation, and then using the whole cell transformation of the cells to produce Alpha-aminobutyric acid.
  • the fermentation medium has nutrients required for the growth of microorganisms, that is, a carbon source, a nitrogen source, an inorganic salt, a growth factor, etc.;
  • the carbon source includes glucose, glycerin, etc.;
  • the nitrogen source mainly has a yeast extract, peptone Etc., as well as phosphate (phosphorus source) and sulfate (sulfur source), etc.; in addition, a suitable amount of metal ions can be added to the medium.
  • the fermentation medium is TB medium, TY medium, TYG medium or GP medium.
  • the whole cell transformation is after the obtained cells are washed, and then resuspended in 50 mM PB buffer at pH 7.5, and then the substrate L-threonine and formic acid or a group are added at 30 ° C.
  • the acid salt or glucose is controlled to have a pH of about 7.5 with 20% formic acid or 1 M hydrochloric acid and 5 M aqueous ammonia.
  • the present invention also claims the use of the recombinant single cell factory for the synthesis of alpha-aminobutyric acid, ketobutyric acid or related accessory products thereof.
  • ⁇ -Aminobutyric acid is an important chemical raw material and pharmaceutical intermediate, and is widely used in the synthesis of drugs such as the antituberculosis drug ethambutol hydrochloride and the antiepileptic drug levetiracetam.
  • the present invention constructs a single cell factory for recombinant L-threonine deaminase, L-amino acid dehydrogenase and a dehydrogenase providing a cofactor NADH cycle for the first time, and optimizes the control of L-threonine deaminase by using rbs sequence.
  • the amount of expression can effectively control the accumulation of ketobutyric acid as an intermediate in the conversion process, because a certain amount of ketobutyric acid inhibits the transformation process, and the dehydrogenation of the cofactor NADH is controlled by the promoter and rbs sequence optimization.
  • the expression rate of the enzyme was optimized, and the regeneration rate of the cofactor NADH was optimized.
  • the high-efficiency amino acid dehydrogenase was ligated into the plasmid and expressed in the E. coli expression system to construct a recombinant single cell factory. The plant performs whole cell transformation to efficiently prepare ⁇ -aminobutyric acid.
  • the whole cell transformation of L-threonine into ⁇ -aminobutyric acid is carried out by the single cell factory, the conversion process is simple, the culture cost of the recombinant bacteria is low, and no cofactor is added during the conversion process, and the conversion batch is stable and uninterrupted.
  • the phenomenon reduces the cost while improving the conversion efficiency and has important industrial application value.
  • Example 1 Optimization of rbs sequence of L-threonine deaminase and construction of recombinant L-threonine deaminase Escherichia coli
  • the rbs sequence of different expression intensity was designed based on the expression sequence of E. coli L-threonine deaminase gene and the T7 promoter for its expression in E. coli, and then sent to Shanghai Biotech for gene synthesis.
  • the PCR primers include primers rbs1, rbs2, rbs3, rbs4, rbs5, rbs6, and L-su with rbs sequences containing different expression intensities (shown in bold underlined, sequences as shown in SEQ ID NO: 1 to SEQ ID NO: 6).
  • the terminal primer of the tyrosine deaminase gene, LtdR (sequences are SEQ ID NO: 7 to SEQ ID NO: 13).
  • Primers with rbs sequences with different expression intensities and terminal primers (R) are used as primer pairs, and PCR amplification is performed using chromosomal DNA of Escherichia coli as a template to obtain L-threonine capable of expressing different expression levels.
  • the nucleotide sequence of the deaminase For example, by using rbs1,ltdR as a primer to obtain a nucleotide fragment, the nucleotide fragment contains the L-threonine deaminase gene and is directly linked to the L-threonine deaminase gene. There is a sequence of rbs.
  • nucleotide fragments of the L-threonine deaminase capable of expressing different expression levels obtained in the previous step were respectively ligated to the plasmid vector pET-28a (double digestion with Xba I and BamH I, respectively).
  • the recombinant plasmid was transformed into competent E. coli BL21, and the correct transformant was screened to obtain recombinant L-threonine deaminase Escherichia coli.
  • the enzyme activity of L-threonine deaminase was tested, and the cultured bacterial solution was taken, centrifuged at 8000 r/min for 10 min at 4 ° C, and the cells were collected and washed twice with 100 mL of 50 mM PB buffer of pH 7.0 to reconstitute the large intestine.
  • the bacilli were resuspended in 10 mL of 50 mM PB buffer.
  • the suspended cells were placed in an ultrasonic cell disrupter to break the cells, and the cells were broken for 1 s, stopped for 3 s, and the power operation time of 300 W was 10 min.
  • the crushed liquid was centrifuged at 4 ° C in a centrifuge at 10000 r / min for 30 min to remove the precipitate, and the supernatant was assayed for enzyme activity.
  • [5] L-threonine deaminase enzyme activity assay method A 40 mM threonine substrate solution was prepared using 0.1 M of pH 7.5 PB buffer. 0.96 mL of substrate buffer was added to the cuvette, and 40 ⁇ L of the enzyme solution was added and mixed immediately. The change of the concentration of ketobutyric acid was determined by calculating the change of the absorbance of the enzyme reaction solution under ultraviolet light at 230 nm, and then the change of the concentration of ketobutyrate was obtained by comparison with the standard curve of ketobutyrate. The enzyme activity is defined as the amount of enzyme required for 1 ⁇ mol of ⁇ -ketobutyrate produced per minute.
  • Example 2 Construction of Recombinant Plasmid and Recombinant Bacteria Co-expressing L-Threonine Deaminase and L-Amino Acid Dehydrogenase
  • Genomic DNA of Bacillus cereus, Rhodococcus, Bacillus subtilis, and Streptomyces coelicolor was used as a template.
  • PBcldhF 5'-CGGGATCCAAGGAGATATACATGACATTAGAAATCTTCG-3' (BamH I)
  • PBcldhR 5'-CGAGCTCTTAGCGACGGCTAATAATAT C-3' (Sac I)
  • PRjpdhF 5'-CGGGATCCAAGGAGATATACATGACTCTCACCGCGGAAC-3' (BamH I)
  • PBsadhF 5'-CGGGATCCAAGGAGATATACATGATCATAGGGGTTCCT-3' (BamH I)
  • PBsadhR 5'-CGAGCTCTTAAGCACCCGCCACAGATG-3' (Sac I)
  • PScvdhF 5'-CGGGATCCAAGGAGATATACATGGTGACCGACGTAAACGG-3' (BamH I)
  • PScvdhR 5'-CGAGCTCTCACGGCCGGGGACGGGCCT-3' (Sac I)
  • PBcldhF represents the upstream of the ldh gene used to amplify Bacillus cereus Primer.
  • nucleotide fragment and plasmid were double digested with BamH I and Sac I, respectively, and then ligated a recombinant plasmid pET-28a-rbs1-ltd+Bcldh, pET-28a-rbs2-ltd+Rjpdh, pET-28a-rbs3- capable of co-expressing L-threonine deaminase and L-amino acid dehydrogenase Ltd+Bsadh, pET-28a-rbs4-ltd+Scvdh, pET-28a-rbs5-ltd+Bcldh, pET-28a-rbs6-l
  • Example 3 Construction of recombinant Escherichia coli providing dehydrogenase of cofactor NADH cycle and promoter of formate dehydrogenase and construction of recombinant Escherichia coli optimized for rbs sequence
  • PCR was performed with the corresponding primers and genomic template, and the gene fragment of the dehydrogenase providing the cofactor NADH cycle derived from the corresponding strain was obtained and ligated to the pET-28a plasmid (the nucleotide fragment and the plasmid were respectively BamH I and Sac).
  • PBsglcdhF PBsglcdhR
  • Bacillus subtilis chromosomal DNA as a template to obtain a glucose dehydrogenase gene fragment derived from Bacillus subtilis, and ligating it to a pET-28a plasmid (nucleotide fragment and plasmid)
  • Recombinant plasmid pET-28a-rbs2-Bsglcdh expressing a glucose dehydrogenase derived from Bacillus subtilis was obtained by double digestion with BamH I and Sac I, respectively, and the recombinant plasmid was transformed into competent E. coli BL21.
  • the correct transformant is screened to obtain a recombinant strain derived from Bacillus subtilis-derived glucose dehydrogenase which provides a cofactor NADH cycle.
  • primer sequences eg SEQ ID NO: 23 to SEQ ID NO: 32.
  • PBsglcdhF 5'-CGGGATCCATGTATCCGGATTTAAAAGG-3' (BamH I)
  • PBsglcdhR 5'-CCCAAGCTTTTAACCGCGGCCTGCCTGG-3' (Hind III)
  • P28aPromoterF 5'-ACATGCATGCCGATCCCGCGAAATTAATAC-3' (Sph I)
  • PBsglcdhRBglII 5'-GAAGATCTTTAACCGCGGCCTGCCTGG-3' (Bgl II)
  • PfdhRBglII 5'-GAAGATCTTTATTTTTTGTCGTGTTTACC-3' (Bgl II)
  • the tac promoter was selected, and the rbs sequences containing different intensities were designed according to the tac promoter and formic acid dehydrogenase gene sequences on the pXMJ-19 plasmid (expressed underlined, such as SEQ ID NO: 33 to SEQ ID).
  • NO: 39 PCR primers r1FDH, r2FDH, r3FDH, r4FDH, r5FDH, r6FDH, r7FDH, and the terminal primer pFDHRBamHI of the formate dehydrogenase gene.
  • primer sequences eg SEQ ID NO: 40 to SEQ ID NO: 49.
  • pTacFSphI 5'-CATGCATGCTGACAATTAATCATCGGCT-3' (Sph I)
  • prrnBRBglII 5'-GAAGATCTAGAGTTTGTAGAAACGC-3' (Bgl II)
  • primers containing rbs sequences of different intensities and pFDHRBamHI were used to construct primer pairs, and PCR was carried out to obtain a plurality of gene fragments containing rbs sequence and formate dehydrogenase, and pXMJ-19 plasmid ligation (nucleotide fragment and plasmid were digested with Hind III and BamH I, respectively, and ligated) to obtain recombinant plasmid pXMJ-19-r1fdh, pXMJ-19-r2fdh, pXMJ expressing formate dehydrogenase.
  • the recombinant plasmid was transformed into competent E. coli BL21, and the correct transformant was screened to obtain expression.
  • r1FDH pFDHRBamHI
  • Candida boidin genome as a template to obtain a formate dehydrogenase gene fragment derived from Candida boidin, which is ligated to the pXMJ-19 plasmid (nuclei)
  • the cleavage fragment and the plasmid were digested with Hind III and BamH I, respectively, and ligated to obtain the recombinant plasmid pXMJ-19-r1fdh expressing formate dehydrogenase, and the recombinant plasmid was transformed into competent E. coli BL21, and the screening was correct.
  • a transformant that is, a recombinant strain expressing a formate dehydrogenase that provides a cofactor NADH cycle.
  • the cultured bacteria solution was taken, centrifuged at 8000 r/min for 10 min at 4 ° C, and the cells were collected and washed twice with 100 mL of 50 mM PB buffer, pH 7.0, and the recombinant E. coli was resuspended. In 10 mL of 50 mM PB buffer.
  • the suspended cells were placed in an ultrasonic cell disrupter to break the cells, and the cells were broken for 1 s, stopped for 3 s, and the power operation time of 300 W was 10 min.
  • the crushed liquid was centrifuged at 4 ° C in a centrifuge at 10000 r / min for 30 min to remove the precipitate, and the supernatant was assayed for enzyme activity.
  • [4] Formate dehydrogenase enzyme activity assay method A 100 mM sodium formate substrate solution was prepared using 0.1 M of a pH 7.5 PB buffer. 0.96 mL of substrate buffer was added to the cuvette, and 40 ⁇ L of the enzyme solution was added and mixed immediately. The change of the concentration of NADH is determined by calculating the change of the absorbance of the enzyme reaction solution under ultraviolet light at 340 nm, and then the change of the concentration of NADH is obtained by comparison with the standard curve of NADH, and can also be calculated by the formula according to the molar extinction coefficient of NADH. Enzyme activity. The enzyme activity is defined as the amount of enzyme required for 1 ⁇ mol of NADH produced per minute.
  • the next step was expressed by using the pXMJ-19-r5fdh plasmid as a gene source.
  • Example 4 Construction of a recombinant E. coli single cell plant for efficient synthesis of ⁇ -aminobutyric acid
  • PCR was carried out using pET-28a-Bsglcdh as a template, P28aPromoterF and PBsglcdhRBglII as primers.
  • the pET-28a self-banding rbs and the glucose-derived glucose dehydrogenase using pET-28a-Ppglcdh as a template, P28aPromoterF and PPpglcdhRBglII as primers
  • PCR amplification was carried out to obtain a T7 promoter carrying the pET-28a plasmid vector itself, a pET-28a self-banding rbs, and a nucleotide fragment of a glucose dehydrogenase derived from Pseudomonas putida; using the pXMJ-19-r5fdh plasmi
  • E. coli competent state The monoclonal Escherichia coli was activated in 10 ml of LB medium, and then cultured at 37 ° C with shaking to OD 600 0.35 to prepare the competent state; the cultured bacterial solution was placed in ice water, and the bacterial liquid was rapidly cooled by shaking gently.
  • Transformation of plasmids Take the prepared competent cells [1], add the plasmid to be transformed, gently pipette and place in ice for 45 min; place the centrifuge tube in a 42 ° C water bath for 90 seconds, then take it out and put it into the ice. 5 min; add 800 ⁇ L of LB medium, gently mix, incubate at 37 ° C for 1 - 1.5 h; centrifuge the cells for 2 min, discard most of the supernatant, and then re-absorb and suspend, take 200 ⁇ L on the target resistant plate, placed in 37 Culture in a °C incubator; after the transformant grows, the plasmid is verified.
  • Example 6 Fermentation medium for recombinant Escherichia coli
  • LB medium g/L: Tryptone 10, yeast extract 5, NaCl 10.
  • TB medium g/L: glycerol 4, tryptone 12, yeast extract 24, K 2 HPO 4 12.5, KH 2 PO 4 2.3, MgSO 4 0.2, pH 7.0-7.2.
  • TY medium glucose 10, tryptone 10, yeast extract 5, NaCl 3, K 2 HPO 4 6, KH 2 PO 4 3, sodium citrate 1, MgSO 4 0.2, pH 7.0 -7.2.
  • TYG medium g/L: glycerol 10, tryptone 10, yeast extract 5, K 2 HPO 4 6, KH 2 PO 4 3, sodium citrate 1, MgSO 4 0.2, pH 7.0-7.2.
  • GP medium glucose 30, tryptone 10, yeast extract 5, K 2 HPO 4 6, KH 2 PO 4 3, sodium citrate 1, MgSO 4 0.2, pH 7.0-7.2.
  • Example 7 Whole cell transformation of recombinant E. coli single cell factory expressing formate dehydrogenase to produce ⁇ -aminobutyric acid
  • the recombinant Escherichia coli single cell factory obtained in the step [3] of Example 4 is pET-28a-rbs1-ltd+Bcldh+r5fdh/BL21, pET-28a-rbs2-ltd+Rjpdh+r5fdh/BL21, pET-28a -rbs3-ltd+Bsadh+r5fdh/BL21, pET-28a-rbs4-ltd+Scvdh+r5fdh/BL21, pET-28a-rbs5-ltd+Bcldh+r5fdh/BL21, pET-28a-rbs6-ltd+Bcldh+r5fdh /BL21 was activated by LB medium, cultured at 37 ° C, 160 r / min overnight, and then transferred to 100 mL of LB medium.
  • the inoculation amount was 1%, the culture temperature was 37 ° C, and the shaking speed was 160 r/min.
  • IPTG was added to a final concentration of 1 mmol/L, and the expression was induced by shaking at 16 ° C for 24 h.
  • Perform whole-cell transformation experiments take the cultured bacteria in different culture medium, centrifuge at 10 °C, 8000r/min for 10min, collect the cells, wash them twice with 100mL pH 7.0 50mM PB buffer, and resuspend the recombinant E. coli in 100mL. pH 7.5 in 50 mM PB buffer.
  • Example 8 Recombinant bacteria pET-28a-rbs4-ltd+Scvdh+r5fdh/BL21 were whole cell transformed to produce ⁇ -aminobutyric acid after being cultured in different fermentation media.
  • the recombinant strain pET-28a-rbs4-ltd+Scvdh+r5fdh/BL21 was activated by LB medium, and cultured overnight at 37° C. and 160 r/min, and then transferred to 2 L of TB group, TY group, TYG group and GP group, respectively.
  • the inoculation amount was 8%
  • the culture temperature was 37 ° C
  • the rotation speed was 300 r/min
  • the aeration amount was 1.0 vvm.
  • IPTG was added to a final concentration of 0.5 mM, and the temperature was decreased to 28 ° C.
  • the cells were collected by centrifugation at 8000 r/min for 10 min at 4 ° C, and the recombinant Escherichia coli was washed with 50 mM PB buffer at pH 7.5.
  • 50 mM PB buffer pH 7.5
  • 1.8 M L-threonine and 1.8 M ammonium formate were added to the system, and the transformation was carried out at 30 ° C, 300 r / min, and 20% formic acid or 5M aqueous ammonia was adjusted to pH 7.5.
  • Example 9 Recombinant E. coli single cell factory expressing glucose dehydrogenase whole cell transformation to produce ⁇ -aminobutyric acid
  • the recombinant Escherichia coli single cell factory obtained in the step [3] of Example 4 is pET-28a-rbs3-ltd+Bcldh+Bsglcdh/BL21, pET-28a-rbs4-ltd+Bcldh+Bsglcdh/BL21, pET-28a -rbs5-ltd+Rjpdh+Ppglcdh/BL21 and pET-28a-rbs6-ltd+Bsadh+Ppglcdh/BL21 recombinant bacteria were activated by LB medium, cultured at 37°C, 160r/min overnight and transferred to 2L LB.
  • the inoculation amount was 8%, the culture temperature was 37 ° C, the rotation speed was 300 r/min, and the aeration amount was 1.0 vvm.
  • IPTG was added to a final concentration of 0.5 mM, and the temperature was decreased to 28 ° C.
  • the cells were collected by centrifugation at 8000 r/min for 10 min at 4 ° C, and the recombinant Escherichia coli was washed with 50 mM PB buffer at pH 7.5.
  • pET-28a-rbs3-ltd+Bcldh+Bsglcdh/BL21 pET-28a-rbs4-ltd+Bcldh+Bsglcdh/BL21
  • pET-28a-rbs5-ltd+Rjpdh+Ppglcdh/BL21 recombinant cells in whole cell transformation No accumulation of ketobutyrate was detected, and 73.6 g/L of intermediate ketobutyrate was detected in the whole cell transformation of recombinant pET-28a-rbs6-ltd+Bsadh+Ppglcdh/BL21, indicating that glucose was removed.
  • rbs4 and rbs5 sequences to induce L-threonine deaminase expression is beneficial to the preparation of ⁇ -aminobutyric acid, in which rbs5 sequence is L-threonine deaminase.
  • the ribosome binding site sequence is optimal.
  • Example 10 Recombinant strain pET-28a-rbs5-ltd+Rjpdh+Ppglcdh/BL21 is subjected to whole cell transformation to produce ⁇ -aminobutyric acid after being cultured in different fermentation media.
  • the recombinant strain pET-28a-rbs5-ltd+Rjpdh+Ppglcdh/BL21 was activated by LB medium, cultured at 37°C, 160r/min overnight, and then transferred to 2L TB medium, TY medium, TYG medium and GP. In the medium.
  • the inoculation amount was 8%, the culture temperature was 37 ° C, the rotation speed was 300 r/min, and the aeration amount was 1.0 vvm. After incubation for 2-3 h, IPTG was added to a final concentration of 0.5 mM, and the temperature was decreased to 28 ° C.
  • the cells were collected by centrifugation at 8000 r/min for 10 min at 4 ° C, and the recombinant Escherichia coli was washed with 50 mM PB buffer at pH 7.5.
  • the present invention can achieve better results by the following means: the L-threonine deaminase gene has a promoter carried by the plasmid vector itself, and the expression of the L-threonine deaminase gene and the plasmid vector is designed.
  • the rbs sequence whose intensity is lower than the rbs of the plasmid vector itself; the L-threonine deaminase gene and the L-amino acid dehydrogenase gene are linked by the rbs carried by the plasmid vector itself; and the dehydrogenase gene of the cofactor NADH cycle is provided before
  • the recombinant single cell factory of the invention can obtain a better equilibrium rate from L-threonine to the intermediate ketobutyric acid and from ketobutyric acid to ⁇ -aminobutyric acid, without causing accumulation of the intermediate ketobutyric acid. Therefore, it does not cause inhibition of the reaction.
  • the recombinant single cell factory does not require exogenous addition of cofactors, which reduces the path of substrate entry and exit or diffusion and increases the rate of transformation compared to other methods.

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Abstract

提供了一种合成α-氨基丁酸的工程菌，其制备方法是将L-苏氨酸脱氨酶、L-氨基酸脱氢酶及提供辅因子NADH循环的脱氢酶基因串联后构建共表达载体，然后转入宿主菌。该载体中包含了分别设计的启动子和核糖体结合位点序列，用以控制三种酶实现协同表达，提高α-氨基丁酸的最终产量。

Description

一种高效合成α-氨基丁酸的单细胞工厂及其构建与应用 技术领域
本发明涉及一种高效合成α-氨基丁酸的单细胞工厂及其构建与应用，属于微生物技术领域。
背景技术
非天然α-氨基酸是区别于能够由生物体自身合成的22种天然α-氨基酸的一大类氨基酸，它们具有重要的生物活性和生理作用，广泛用于多肽、手性药物以及生物碱等化合物的合成。α-氨基丁酸是抑制人体神经信息传递的非天然氨基酸，具有加强葡萄糖磷酸酯酶的活性，促进脑细胞代谢的作用。α-氨基丁酸也是一种重要的化工原料和医药中间体，已广泛用于药物的合成，如抗结核药物盐酸乙胺丁醇和抗癫痫药物左乙拉西坦的合成，市场巨大。
α-氨基丁酸的合成方法主要包括化学合成法、酶拆分法及酶转化法三种。其中化学法包括脱硫反应、氨化水解反应、丁酮酸还原法等，化学合成虽然操作简单，但往往反应条件苛刻且易生成副产物，有时需要利用对环境有害的大量有机溶剂，如Jeffery E.A.等利用电化学法制备得α-氨基丁酸，产率仅有48％，同时存在副产物谷氨酸。相比之下，微生物法制备α-氨基丁酸具有特异性较强，条件温和，对环境友好等优点。并且随着基因工程技术的发展，构建重组微生物合成非天然氨基酸代谢途径已经可以完成。目前，α-氨基丁酸的微生物法制备主要通过细胞外酶转化法，包括对消旋α-氨基丁酸进行酶法拆分制备及以2-丁酮酸为原料通过脱氢酶或转氨酶来催化制备。
在之前的研究中，发明人构建了以大宗化学品L-苏氨酸为廉价底物，通过L-苏氨酸脱氨酶、L-氨基酸脱氢酶及辅酶再生系统的酶体系进行一步法制备α-氨基丁酸。以酶转化过程中发现L-苏氨酸脱氨酶的量需要精确控制，不然会造成中间产物酮丁酸的积累，从而抑制了酮丁酸到α-氨基丁酸的转化，造成了酶转化生产α-氨基丁酸的中断。同时，利用酶转化体系去进行α-氨基丁酸的生产需要对产酶的三种重组菌进行细胞破碎，工艺繁琐成本高昂，同时在转化的过程中因酶的失活而影响了转化的稳定性，另外因辅因子的损耗需要不断地外源添加，进一步增加了α-氨基丁酸的生产成本。因此，有必要寻找一种高效稳定但成本低廉的制备α-氨基丁酸的方法。
发明内容
为了解决上述问题，本发明提供了利用rbs序列优化控制L-苏氨酸脱氨酶的表达量，同时将其与L-氨基酸脱氢酶及提供辅因子NADH循环的脱氢酶串联到质粒上并在大肠杆菌中的表达，构建成重组大肠杆菌单细胞工厂，利用该单细胞工厂进行全细胞转化高效制备α-氨 基丁酸的方法。
本发明构建了能高效合成α-氨基丁酸的单细胞工厂，主要通过利用rbs优化控制L-苏氨酸脱氨酶(ltd)在重组菌中的表达量，以达到控制中间产物酮酸的积累量，同时利用启动子以及rbs序列进行优化来控制提供辅因子NADH循环的脱氢酶表达量从而控制辅因子NADH的生成速率，最后将不同rbs强度的L-苏氨酸脱氨酶与L-氨基酸脱氢酶基因及经过启动子以及rbs序列优化后的提供辅因子NADH循环的脱氢酶构建在大肠杆菌表达系统中的重组共表达载体，并将其转化到大肠杆菌E.coli BL21中，成功构建了不同的基因工程菌单细胞工厂。在不添加任何外源辅因子的条件下，利用这些单细胞工厂进行全细胞转化法对廉价底物L-苏氨酸进行转化高效制备α-氨基丁酸，为α-氨基丁酸的工业化生产提供了一种实际有效策略。
本发明的第一个目的是提供一种高效合成α-氨基丁酸的重组菌单细胞工厂，所述重组菌单细胞工厂是将重组共表达载体转化到宿主菌中得到的；所述重组共表达载体是在质粒载体上串联L-苏氨酸脱氨酶基因、L-氨基酸脱氢酶基因、提供辅因子NADH循环的脱氢酶基因；其中，以L-氨基酸脱氢酶基因的表达为基准，控制供辅因子NADH循环的脱氢酶的表达量使辅因子NADH的生成速率处于相对较高的水平，控制L-苏氨酸脱氨酶的表达量处于相对较合适的水平。
所述的重组菌单细胞工厂能得到较优的从L-苏氨酸到中间产物酮丁酸及从酮丁酸到α-氨基丁酸的平衡速率，不会造成中间产物酮丁酸的积累因而不会造成对反应的抑制。同时所述的重组菌单细胞工厂不需要外源添加辅因子，相比于其他方法减少了底物进出细胞或扩散的路径从而增加了转化速率。
在一种实施方式中，所述控制，是通过但不限于，启动子以及rbs序列优化，也可以是增强子、终止子、沉默子优化等。
在一种实施方式中，所述控制是通过启动子和/或rbs序列优化来进行。
所述重组菌单细胞工厂的构建方法，包括：
(1)根据启动子及L-苏氨酸脱氨酶的基因序列，设计不同强度的rbs序列用来控制L-苏氨酸脱氨酶的表达量从而控制L-苏氨酸到酮丁酸的转化速率；
(2)对辅因子NADH的供应速率进行了调控，主要是对启动子以及rbs序列进行优化来控制提供辅因子NADH循环的脱氢酶表达量从而控制辅因子NADH的再生速率；
(3)将rbs优化后的L-苏氨酸脱氨酶基因、L-氨基酸脱氢酶基因、启动子以及rbs优化后的提供辅因子NADH循环的脱氢酶基因按顺序连接，构建重组共表达载体，并将重组共表达载体转化宿主菌中，构建基因工程菌单细胞工厂。
在一种实施方式中，所述L-苏氨酸脱氨酶基因前有质粒载体自身携带的启动子、针对L- 苏氨酸脱氨酶基因和质粒载体设计的表达强度低于质粒载体本身rbs的rbs序列；所述L-苏氨酸脱氨酶基因与L-氨基酸脱氢酶基因之间通过质粒载体自身携带的rbs连接；所述提供辅因子NADH循环的脱氢酶基因前有针对提供辅因子NADH循环的脱氢酶基因和质粒载体设计的启动子、表达强度高于或等于质粒载体本身rbs的rbs序列。
在一种实施方式中，所述宿主菌可以是大肠杆菌，也可以是其他宿主，比如枯草芽孢杆菌、棒杆菌、酵母等。
在一种实施方式中，所述宿主菌为E.coli BL21。
在一种实施方式中，所述质粒载体可以是任意一种可商业化购买的质粒载体，或者是任意一种已有报道的经改造的质粒载体。
在一种实施方式中，所述L-苏氨酸脱氨酶选自但不限于大肠杆菌来源的L-苏氨酸脱氨酶(核苷酸序列是NCBI上Gene ID:948287)。
在一种实施方式中，所述L-氨基酸脱氢酶选自：但不限于，芽孢杆菌来源的L-亮氨酸脱氢酶(NCBI上Gene ID:1206507)、芽孢杆菌来源的L-丙氨酸脱氢酶(NCBI上Gene ID:936557)、链霉菌来源的L-缬氨酸脱氢酶(NCBI上Gene ID:1099526)、红球菌来源的L-苯丙氨酸脱氢酶(NCBI上Gene ID:4219741)。
在一种实施方式中，所述提供辅因子NADH循环的脱氢酶选自：但不限于博伊丁假丝酵母的甲酸脱氢酶(NCBI上GenBank:KM454879.1)、枯草芽孢杆菌的葡萄糖脱氢酶(NCBI上GeneID:938261)、恶臭假单胞杆菌的葡萄糖脱氢酶(NCBI上Gene ID:1045820)。
在一种实施方式中，所述rbs序列，可以是不同强度的rbs序列。
在一种实施方式中，所述L-苏氨酸脱氨酶基因前的启动子或rbs序列，可以是针对不同表达系统进行优化得到的。
在一种实施方式中，所述提供辅因子NADH循环的脱氢酶基因前的启动子或rbs序列，也可以是针对不同表达系统进行优化得到的。
在一种实施方式中，所述表达系统包括但不限于大肠杆菌表达系统，也可以是枯草芽孢杆菌表达系统、棒杆菌表达系统、酵母表达体系等。
在一种实施方式中，所述宿主为大肠杆菌；所述L-苏氨酸脱氨酶基因前的启动子为T7启动子；L-苏氨酸脱氨酶基因前直接连接的rbs序列为序列SEQ ID NO:1～SEQ ID NO:6所示序列中的任意一种；所述L-苏氨酸脱氨酶基因和L-氨基酸脱氢酶基因通过序列为SEQ ID NO:22所示序列的RBS连接。
在一种实施方式中，所述提供辅因子NADH循环的脱氢酶基因前的启动子为tac启动子，rbs序列为SEQ ID NO:33～SEQ ID NO:39中的任意一种。可选地，rbs序列为SEQ ID NO:37 的序列。
在一种实施方式中，所述重组共表达载体是在质粒载体pET-28a的基础上构建得到的；L-苏氨酸脱氨酶基因和L-氨基酸脱氢酶基因通过pET-28a自身携带的rbs直接连接。
本发明的第二个目的是提供一种发酵合成α-氨基丁酸的方法，是利用本发明的重组菌单细胞工厂。
在一种实施方式中，所述方法，是将重组菌单细胞工厂活化后转接于发酵培养基中，IPTG诱导表达或直接表达重组蛋白，离心收集菌体，然后利用菌体全细胞转化生产α-氨基丁酸。
在一种实施方式中，发酵培养基中具备微生物生长所需的营养成分，即碳源、氮源、无机盐、生长因子等；碳源包括葡萄糖、甘油等；氮源主要有酵母膏、蛋白胨等，以及磷酸盐(磷源)和硫酸盐(硫源)等；另外，培养基中还可以适量加入金属离子。
在一种实施方式中，所述发酵培养基为TB培养基、TY培养基、TYG培养基或者GP培养基。
在一种实施方式中，所述全细胞转化是将获得的菌体洗涤后，再加入pH 7.5的50mM PB缓冲液重悬，然后于30℃下加入底物L-苏氨酸及甲酸或甲酸盐或葡萄糖，以20％甲酸或1M盐酸及5M氨水控制pH在7.5左右。
本发明还要求保护所述的重组菌单细胞工厂应用于α-氨基丁酸、酮丁酸或其相关附属产物的合成。
本发明的有益效果：
α-氨基丁酸一种重要的化工原料和医药中间体，被广泛用于药物的合成如抗结核药物盐酸乙胺丁醇和抗癫痫药物左乙拉西坦的合成。本发明首次构建了重组L-苏氨酸脱氨酶、L-氨基酸脱氢酶及提供辅因子NADH循环的脱氢酶的单细胞工厂，利用rbs序列优化控制L-苏氨酸脱氨酶的表达量，可以有效控制作为转化过程中中间产物酮丁酸的积累量，因为一定量的酮丁酸会抑制转化过程的进行，同时利用启动子及rbs序列优化控制了提供辅因子NADH的脱氢酶表达量，优化了辅因子NADH的再生速率，最后将其与高效的氨基酸脱氢酶串联到质粒上并分别在大肠杆菌表达体系中的表达，构建了重组菌单细胞工厂，利用该单细胞工厂进行全细胞转化高效制备α-氨基丁酸。利用该单细胞工厂对L-苏氨酸进行全细胞转化为α-氨基丁酸，转化过程操作简单、重组菌培养成本低廉，且转化过程中不需要添加任何辅因子，转化批次稳定无中断现象，在提高转化效率的同时降低了成本，具有重要的工业应用价值。
具体实施方式
下面结合实施例对本发明做详细的说明，以下实施例不对本发明产生限制。
实施例1：L-苏氨酸脱氨酶的rbs序列优化及重组L-苏氨酸脱氨酶大肠杆菌的构建
[1]将来源于大肠杆菌L-苏氨酸脱氨酶基因ltd序列结合T7启动子针对其在大肠杆菌中的表达量设计了不同表达强度的rbs序列，然后送至上海生工生物进行基因合成。PCR引物包括含有不同表达强度的rbs序列(用下划线加粗表示，序列如SEQ ID NO:1～SEQ ID NO:6所示)的引物rbs1、rbs2、rbs3、rbs4、rbs5、rbs6及L-苏氨酸脱氨酶基因的末端引物ltdR(序列如SEQ ID NO:7～SEQ ID NO:13)。
Figure PCTCN2016106241-appb-000001
ltdR：5’-CGGGATCCTTAACCCGCCAAAAAGAACCTG-3’(BamH I)
[2]以含有不同表达强度的rbs序列的引物、末端引物ltdR组成引物对，利用大肠杆菌的染色体DNA作为模板，进行PCR扩增，即可得到能够表达出不同表达量的L-苏氨酸脱氨酶的核苷酸序列。比如，以rbs1、ltdR为引物进行扩增，即可得到一段核苷酸片段，这段核苷酸片段含有L-苏氨酸脱氨酶基因且L-苏氨酸脱氨酶基因前直接连接有一段rbs序列。
[3]将上一步得到的能够表达出不同表达量的L-苏氨酸脱氨酶的核苷酸片段分别连接到质粒载体pET-28a上(分别用Xba I和BamH I进行双酶切，然后连接)，得到6个重组质粒pET-28a-rbs1-ltd、pET-28a-rbs2-ltd、pET-28a-rbs3-ltd、pET-28a-rbs4-ltd、pET-28a-rbs5-ltd、pET-28a-rbs6-ltd，将重组质粒转化到感受态E.coli BL21，筛选正确的转化子，即得到重组L-苏氨酸脱氨酶大肠杆菌。
[4]将[3]中所构建好的重组L-苏氨酸脱氨酶大肠杆菌利用LB培养基活化，37℃、160r/min培养过夜后分别转接于100ml的LB基中。接种量1％，培养温度37℃，摇床转速160r/min。培养至OD600约0.6～0.8时加入终浓度为1mM的IPTG，置于16℃摇床24h诱导表达。对L-苏氨酸脱氨酶的酶活力进行测试，取培养好的菌液，4℃，8000r/min离心10min 收集菌体，用100mL pH 7.0的50mM PB缓冲液洗涤二次，将重组大肠杆菌重悬于10mL的50mM PB缓冲液中。将悬浮好的细胞放入超声波细胞破碎仪中进行细胞破碎，破1s，停3s，300W的功率工作时间10min。取破碎液于离心机内4℃，10000r/min离心30min去除沉淀，测定上清液酶活。
[5]L-苏氨酸脱氨酶酶活测定方法：利用0.1M的pH 7.5的PB缓冲液来配制40mM的苏氨酸底物溶液。将0.96mL的底物缓冲液加入到比色皿中，再加入40μL的酶液并立即混匀。通过计算酶反应液在230nm紫外光下吸光值的变化去测定酮丁酸浓度的变化，然后与酮丁酸的标准曲线对照得出酮丁酸的浓度变化值。酶活的定义为：每分钟产生的1μmol的α-酮丁酸所需的酶量。
[6]结果显示重组L-苏氨酸脱氨酶大肠杆菌pET-28a-rbs1-ltd/BL21、pET-28a-rbs2-ltd/BL21、pET-28a-rbs3-ltd/BL21、pET-28a-rbs4-ltd/BL21、pET-28a-rbs5-ltd/BL21及pET-28a-rbs6-ltd/BL21在LB培养基中所诱导出的酶活分别为0.13U/mL、0.34U/mL、0.72U/mL、2.56U/mL、4.89U/mL、11.6U/mL。
实施例2：共表达L-苏氨酸脱氨酶和L-氨基酸脱氢酶的重组质粒和重组菌的构建
[1]以蜡样芽孢杆菌、红球菌、枯草芽孢杆菌及天蓝色链霉菌的基因组DNA作为模板。
[2]根据L-氨基酸脱氢酶基因序列和pET-28a质粒上的酶切位点设计L-氨基酸脱氢酶基因引物，包括蜡样芽孢杆菌的L-亮氨酸脱氢酶基因Bcldh(引物为PBcldhF、PBcldhR)、红球菌的L-苯丙氨酸脱氢酶基因Rjpdh(引物为PRjpdhF、PRjpdhR)、枯草芽孢杆菌的L-丙氨酸脱氢酶基因Bsadh(引物为PBsadhF、PBsadhR)、天蓝色链霉菌的缬氨酸脱氢酶基因Scvdh(引物为PScvdhF、PScvdhR)。引物序列如下(如SEQ ID NO:14～SEQ ID NO:21)：
PBcldhF：5’-CGGGATCCAAGGAGATATACATGACATTAGAAATCTTCG-3’(BamH I)
PBcldhR：5’-CGAGCTCTTAGCGACGGCTAATAATAT C-3’(Sac I)
PRjpdhF：5’-CGGGATCCAAGGAGATATACATGACTCTCACCGCGGAAC-3’(BamH I)
PRjpdhR：5’-CGAGCTCCTACCTGGCTGCAGCGATG-3’(Sac I)
PBsadhF：5’-CGGGATCCAAGGAGATATACATGATCATAGGGGTTCCT-3’(BamH I)
PBsadhR：5’-CGAGCTCTTAAGCACCCGCCACAGATG-3’(Sac I)
PScvdhF：5’-CGGGATCCAAGGAGATATACATGGTGACCGACGTAAACGG-3’(BamH I)
PScvdhR：5’-CGAGCTCTCACGGCCGGGGACGGGCCT-3’(Sac I)
其中下划线为pET-28a质粒载体上本身携带的rbs序列；引物命名方式：P+菌株种属首字母+基因名称+引物方向，即PBcldhF代表用于扩增来源于蜡样芽孢杆菌的ldh基因的上游引物。
[3]分别以蜡样芽孢杆菌、红球菌、枯草芽孢杆菌及天蓝色链霉菌的基因组DNA作为模板，利用上述对应的引物进行PCR扩增，得到含有L-氨基酸脱氢酶基因的核苷酸片段且该基因前直接连接有pET-28a质粒载体上本身携带的rbs序列AAGGAG(序列如SEQ ID NO:22所示)。
[4]将上一步得到的多个含有L-氨基酸脱氢酶基因的核苷酸片段与实施例1构建的重组质粒pET-28a-rbs1-ltd、pET-28a-rbs2-ltd、pET-28a-rbs3-ltd、pET-28a-rbs4-ltd、pET-28a-rbs5-ltd、pET-28a-rbs6-ltd连接(核苷酸片段与质粒分别用BamH I和Sac I进行双酶切，然后连接)，得到能够共表达L-苏氨酸脱氨酶和L-氨基酸脱氢酶的重组质粒pET-28a-rbs1-ltd+Bcldh、pET-28a-rbs2-ltd+Rjpdh、pET-28a-rbs3-ltd+Bsadh、pET-28a-rbs4-ltd+Scvdh、pET-28a-rbs5-ltd+Bcldh、pET-28a-rbs6-ltd+Bcldh等，然后将重组质粒转化到感受态E.coli BL21，筛选正确的转化子，即得到共表达L-苏氨酸脱氨酶和L-氨基酸脱氢酶的重组菌。
实施例3：提供辅因子NADH循环的脱氢酶重组大肠杆菌的构建及甲酸脱氢酶的启动子及rbs序列优化的重组大肠杆菌的构建
[1]根据不同来源的提供辅因子NADH循环的脱氢酶的基因序列及串联到pET-28a质粒上的酶切位点设计引物，其中包括博伊丁假丝酵母的甲酸脱氢酶fdh(引物为PfdhF、PfdhR)、枯草芽孢杆菌的葡萄糖脱氢酶Bsglcdh(引物为PBsglcdhF、PBsglcdhR)、恶臭假单胞杆菌的葡萄糖脱氢酶Ppglc(引物为PPpglcdhF、PPpglcdhR)。分别以相应的引物、基因组模板进行PCR，得到相应菌株来源的提供辅因子NADH循环的脱氢酶的基因片段，将其与pET-28a质粒连接(核苷酸片段与质粒分别用BamH I和Sac I进行双酶切，然后连接)，得到表达提供辅因子NADH循环的脱氢酶的重组质粒pET-28a-fdh、pET-28a-Bsglcdh、pET-28a-Ppglcdh，将重组质粒转化到感受态E.coli BL21，筛选正确的转化子，即得到表达提供辅因子NADH循环的脱氢酶的重组菌。
比如，以PBsglcdhF、PBsglcdhR为引物对、枯草芽孢杆菌染色体DNA作为模板进行扩增，得到来源于枯草芽孢杆菌的葡萄糖脱氢酶基因片段，将其与pET-28a质粒连接(核苷酸片段与质粒分别用BamH I和Sac I进行双酶切，然后连接)，得到表达来源于枯草芽孢杆菌的葡萄糖脱氢酶的重组质粒pET-28a-rbs2-Bsglcdh，将重组质粒转化到感受态E.coli BL21，筛选正确的转化子，即得到表达提供辅因子NADH循环的来源于枯草芽孢杆菌的葡萄糖脱氢酶的重组菌。
其中引物序列(如SEQ ID NO:23～SEQ ID NO:32)如下：
PBsglcdhF：5’-CGGGATCCATGTATCCGGATTTAAAAGG-3’(BamH I)
PBsglcdhR：5’-CCCAAGCTTTTAACCGCGGCCTGCCTGG-3’(Hind III)
P28aPromoterF：5’-ACATGCATGCCGATCCCGCGAAATTAATAC-3’(Sph I)
PBsglcdhRBglII：5’-GAAGATCTTTAACCGCGGCCTGCCTGG-3’(Bgl II)
PPpglcdhF：5’-CGGGATCCATGAGCACTGAAGGTGCGAACC-3’(BamH I)
PPpglcdhR：5’-CCCAAGCTTTTACTCGGCTAATTTGTAAG-3’(Hind III)
PPpglcdhRBglII：5’-GAAGATCTTTACTCGGCTAATTTGTAAG-3’(Bgl II)
PfdhF：5’-ACCGGGATCCATGAAAATCGTTCTGGTTCTG-3’(BamH I)
PfdhR：5’-CGCGTCGACTTATTTTTTGTCGTGTTTACC-3’(Sal I)
PfdhRBglII：5’-GAAGATCTTTATTTTTTGTCGTGTTTACC-3’(Bgl II)
[2]以甲酸脱氢酶为例，还可以进行以下优化：
同时选取了tac启动子，并根据pXMJ-19质粒上的tac启动子及甲酸脱氢酶的基因序列，设计含有不同强度的rbs序列(用下划线加粗表示，如SEQ ID NO:33～SEQ ID NO:39所示)的PCR引物r1FDH、r2FDH、r3FDH、r4FDH、r5FDH、r6FDH、r7FDH，及甲酸脱氢酶基因的末端引物pFDHRBamHI。
其中引物序列(如SEQ ID NO:40～SEQ ID NO:49)如下：
Figure PCTCN2016106241-appb-000002
pFDHRBamHI:5’-CGGGATCCTTATTTCTTATCGTGTTTAC-3’(BamHI)
pTacFSphI:5’-CATGCATGCTGACAATTAATCATCGGCT-3’(Sph I)
prrnBRBglII:5’-GAAGATCTAGAGTTTGTAGAAACGC-3’(Bgl II)
利用博伊丁假丝酵母的染色体DNA作为模板，分别以含有不同强度的rbs序列的引物和pFDHRBamHI组成引物对，进行PCR，得到多条含有rbs序列和甲酸脱氢酶的基因片段，将其与pXMJ-19质粒连接(核苷酸片段与质粒分别用Hind III和BamH I进行双酶切，然后连接)，得到表达甲酸脱氢酶的重组质粒pXMJ-19-r1fdh、pXMJ-19-r2fdh、pXMJ-19-r3fdh、pXMJ-19-r4fdh、pXMJ-19-r5fdh、pXMJ-19-r6fdh、pXMJ-19-r7fdh，将重组质粒转化到感受态E.coli BL21，筛选正确的转化子，即得到表达提供辅因子NADH循环的甲酸脱氢酶的重组菌。
比如，以r1FDH、pFDHRBamHI为引物对、博伊丁假丝酵母基因组作为模板进行扩增，得到来源于博伊丁假丝酵母的甲酸脱氢酶基因片段，将其与pXMJ-19质粒连接(核苷酸片段与质粒分别用Hind III和BamH I进行双酶切，然后连接)，得到表达甲酸脱氢酶的重组质粒pXMJ-19-r1fdh，将重组质粒转化到感受态E.coli BL21，筛选正确的转化子，即得到表达提供辅因子NADH循环的甲酸脱氢酶的重组菌。
[3]将[1]和[2]中所构建好的表达有甲酸脱氢酶的重组菌利用LB培养基活化，37℃、160r/min培养过夜后分别转接于100ml的LB基中。接种量1％，培养温度37℃，摇床转速160r/min。培养至OD600约0.6～0.8时加入终浓度为1mmol/L的IPTG，置于16℃摇床24h诱导表达。对甲酸脱氢酶的酶活力进行测试实验，取培养好的菌液，4℃，8000r/min离心10min收集菌体，用100mL pH 7.0的50mM PB缓冲液洗涤二次，将重组大肠杆菌重悬于10mL的50mM PB缓冲液中。将悬浮好的细胞放入超声波细胞破碎仪中进行细胞破碎，破1s，停3s，300W的功率工作时间10min。取破碎液于离心机内4℃，10000r/min离心30min去除沉淀，测定上清液酶活。
[4]甲酸脱氢酶酶活测定方法：利用0.1M的pH 7.5的PB缓冲液来配制100mM的甲酸钠底物溶液。将0.96mL的底物缓冲液加入到比色皿中，再加入40μL的酶液并立即混匀。通过计算酶反应液在340nm紫外光下吸光值的变化去测定生成NADH浓度的变化，然后与NADH的标准曲线对照得出NADH的浓度变化值，也可以根据NADH的摩尔消光系数利用公式计算得出酶活。酶活的定义为：每分钟产生的1μmol的NADH所需的酶量。
[5]结果显示重组菌pET-28a-fdh/BL21、pXMJ-19-r1fdh/BL21、pXMJ-19-r2fdh/BL21、pXMJ-19-r3fdh/BL21、pXMJ-19-r4fdh/BL21、pXMJ-19-r5fdh/BL21、pXMJ-19-r6fdh/BL21以及pXMJ-19-r7fdh/BL21在LB培养基中所诱导出的酶活分别为0.34U/mL、0.12U/mL、0.15U/mL、0.14U/mL、0.27U/mL、0.56U/mL、0.37U/mL、0.41U/mL。因此，以pXMJ-19-r5fdh质粒为基因来源进行下一步串联表达。
实施例4：高效合成α-氨基丁酸的重组大肠杆菌单细胞工厂的构建
[1]以pET-28a-Bsglcdh为模板、P28aPromoterF和PBsglcdhRBglII为引物，进行PCR扩 增，得到携带pET-28a质粒载体本身的T7启动子、pET-28a自带rbs以及枯草来源的葡萄糖脱氢酶的核苷酸片段；以pET-28a-Ppglcdh为模板、P28aPromoterF和PPpglcdhRBglII为引物，进行PCR扩增，得到携带pET-28a质粒载体本身的T7启动子、pET-28a自带rbs以及恶臭假单胞杆菌来源的葡萄糖脱氢酶的核苷酸片段；以pXMJ-19-r5fdh质粒作为模板、pTacFSphI和prrnBRBglII为引物进行扩增，得到携带pXMJ-19质粒载体本身的tac启动子、pXMJ-19自带rbs以及博伊丁假丝酵母来源的甲酸脱氢酶的核苷酸片段。
[2]上一步得到的3个核苷酸片段连接到pMD18-T上，构建得到重组质粒pMD18-T-promoter+Bsglcdh、pMD18-T-promoter+Ppglcdh、pMD18-T-tac-promoter+r5fdh，导入感受态E.coli JM109，验证转化子。
[3]从[2]中转化子中提取相应的质粒，与实施例2中已经串联有不同rbs序列的L-苏氨酸脱氨酶和L-氨基酸脱氢酶的表达载体pET-28a-rbs1-ltd+Bcldh、pET-28a-rbs2-ltd+Rjpdh、pET-28a-rbs3-ltd+Bsadh、pET-28a-rbs4-ltd+Scvdh、pET-28a-rbs5-ltd+Bcldh、pET-28a-rbs6-ltd+Bcldh分别用Sph I和Bgl II进行双酶切，然后连接。将连接好的重组质粒pET-28a-rbs1-ltd+Bcldh+r5fdh、pET-28a-rbs2-ltd+Rjpdh+r5fdh、pET-28a-rbs3-ltd+Bsadh+r5fdh、pET-28a-rbs4-ltd+Scvdh+r5fdh、pET-28a-rbs5-ltd+Bcldh+r5fdh、pET-28a-rbs6-ltd+Bcldh+r5fdh、pET-28a-rbs3-ltd+Bcldh+Bsglcdh、pET-28a-rbs4-ltd+Bcldh+Bsglcdh、pET-28a-rbs5-ltd+Rjpdh+Ppglcdh及pET-28a-rbs6-ltd+Bsadh+Ppglcdh转化到感受态E.coliBL21，酶切验证正确的菌株即为合成α-氨基丁酸重组大肠杆菌单细胞工厂。
实施例5：大肠杆菌感受态的制备及质粒的转化
[1]大肠杆菌感受态的制备。将单克隆大肠杆菌于10ml LB培养基中活化，之后转接于37℃振荡培养至OD6000.35即可制备感受态；将培养好的菌液置于冰水中，轻轻摇晃使菌液迅速冷却约10min；准备灭好菌的1.5ml离心管若干个，分装菌液于管中，每管装菌量1.2ml，将离心管放置于冰中；菌液离心8000r/min 10-20s，冰水中静置2min，弃上清，加入预冷好的0.1M CaCl2400μL，轻轻吹吸悬浮液，放入冰中15min(该步骤重复2-3次)；最后，每管菌液离心弃上清后加入预冷好的0.1M CaCl280μL，轻轻吹吸悬浮菌液放入冰中。
[2]质粒的转化。取[1]制备好的感受态细胞，加入需要转化的质粒，轻轻反复吹吸，并在冰中放置45min；将离心管放入42℃水浴锅准确放置90s，然后取出迅速放入冰中5min；加入LB培养基800μL，轻轻混合，37℃摇床培养1-1.5h；菌体离心2min，弃大部分上清，再重新吹吸悬浮，取200μL于目标抗性平板，置于37℃培养箱中培养；待转化子长出之后提质粒验证。
实施例6：大肠杆菌重组菌的发酵培养基
[1]LB培养基(g/L)：胰蛋白胨10，酵母提取物5，NaCl 10。
[2]TB培养基(g/L)：甘油4，胰蛋白胨12，酵母提取物24，K2HPO4 12.5，KH2PO4 2.3，MgSO4 0.2，pH 7.0-7.2。
[3]TY培养基(g/L)：葡萄糖10，胰蛋白胨10，酵母提取物5，NaCl 3，K2HPO4 6，KH2PO4 3，柠檬酸钠1，MgSO4 0.2，pH 7.0-7.2。
[4]TYG培养基(g/L)：甘油10，胰蛋白胨10，酵母提取物5，K2HPO4 6，KH2PO4 3，柠檬酸钠1，MgSO4 0.2，pH 7.0-7.2。
[5]GP培养基(g/L)：葡萄糖30，胰蛋白胨10，酵母提取物5，K2HPO4 6，KH2PO4 3，柠檬酸钠1，MgSO4 0.2，pH 7.0-7.2。
实施例7：表达甲酸脱氢酶的重组大肠杆菌单细胞工厂全细胞转化产α-氨基丁酸
[1]将实施例4步骤[3]得到的重组大肠杆菌单细胞工厂pET-28a-rbs1-ltd+Bcldh+r5fdh/BL21、pET-28a-rbs2-ltd+Rjpdh+r5fdh/BL21、pET-28a-rbs3-ltd+Bsadh+r5fdh/BL21、pET-28a-rbs4-ltd+Scvdh+r5fdh/BL21、pET-28a-rbs5-ltd+Bcldh+r5fdh/BL21、pET-28a-rbs6-ltd+Bcldh+r5fdh/BL21分别利用LB培养基活化，37℃、160r/min培养过夜后分别转接于100mL的LB培养基中。接种量1％，培养温度37℃，摇床转速160r/min。培养至OD600约0.6～0.8时加入终浓度为1mmol/L的IPTG，置于16℃摇床24h诱导表达。进行全细胞转化实验，取不同培养基中培养好的菌液，4℃，8000r/min离心10min收集菌体，用100mLpH 7.0的50mM PB缓冲液洗涤二次，分别将重组大肠杆菌重悬于100mL的pH7.5的50mM PB缓冲液中。向该体系中投入0.8M L-苏氨酸和0.8M甲酸铵及0.1％(v/v)tween-80，置30℃摇床中继续培养，培养过程中每隔0.5h加入20％甲酸或5M氨水以保持反应液pH为7.5。分不同时间取样，离心并用0.22μm滤膜过滤后经HPLC分析。
[2]氨基酸的HPLC分析条件：在EP管中依次加入转化液样品200μL，衍生剂400μL(取10mg邻苯二甲醛+0.5ml无水乙醇，再加入2ml pH 9.5的0.l M硼砂缓冲液及50μL 2-巯基乙醇)，混匀后等待2分钟加入400μL 0.1M KH2PO4缓冲液，严格控制时间和试剂添加量，然后进样。色谱柱：dimosoil C18(5μl,250mm×4.6mm)，流动相：0.05M醋酸钠缓冲液:甲醇-63:35，检测器：UVDetector，检测波长：338nm，柱温：40℃，进样量：20μL，流速：1.0ml/min。
[3]有机酸的HPLC分析条件：色谱柱：Aminex HPX-87(300mm×7.8mm)，流动相：5mM H2SO4，检测器：UV Detector，检测波长：210nm，柱温：30℃，进样量：10μL，流速：0.6ml/min。
[4]氨基酸测定结果显示pET-28a-rbs1-ltd+Bcldh+r5fdh/BL21、pET-28a-rbs2-ltd+Rjpdh+r5fdh/BL21、pET-28a-rbs3-ltd+Bsadh+r5fdh/BL21、pET-28a-rbs4-ltd+Scvdh+r5fdh/BL21、pET-28a-rbs5-ltd+Bcldh+r5fdh/BL21、pET-28a-rbs6-ltd+Bcldh+r5fdh/BL21全细胞转化制备α-氨基丁酸的产量分别为22.1g/L、48.5g/L、66.2g/L、81.8g/L、40.6g/L、39.2g/L。其中pET-28a-rbs1-ltd+Bcldh+r5fdh/BL21、pET-28a-rbs2-ltd+Rjpdh+r5fdh/BL21、pET-28a-rbs3-ltd+Bsadh+r5fdh/BL21重组菌全细胞转化液中L-苏氨酸均有残留，且并未检测到中间产物酮丁酸的积累，说明rbs1/rbs2/rbs3所诱导出的L-苏氨酸脱氨酶酶活较低，不能够满足α-氨基丁酸的高效生产。而pET-28a-rbs5-ltd+Bcldh+r5fdh/BL21、pET-28a-rbs6-ltd+Bcldh+r5fdh/BL21重组菌全细胞转化液中分别检测出40.7g/L及41.4g/L的中间产物酮丁酸，因前期实验已经显示来源于蜡样的亮氨酸脱氢酶转化速率较快完全满足产物的快速积累，说明可能是L-苏氨酸脱氨酶的表达量较高导致中间产物的积累进而抑制了转化的进行。
[5]将重组菌pET-28a-rbs3-ltd+Bsadh+r5fdh/BL21、pET-28a-rbs4-ltd+Scvdh+r5fdh/BL21、pET-28a-rbs5-ltd+Bcldh+r5fdh/BL21利用LB培养基活化，37℃、160r/min培养过夜后分别转接于2L的LB基中。接种量8％，培养温度37℃，转速300r/min，通气量1.0vvm。培养2-3h后加入终浓度为0.5mM的IPTG，诱导温度降低为28℃，诱导16h后，4℃，8000r/min离心10min收集菌体，用pH 7.5的50mM PB缓冲液将重组大肠杆菌洗涤二次，重悬于培养时同等体积的pH 7.5的50mM PB缓冲液中，向该体系中投入1M L-苏氨酸及1M甲酸铵，于30℃、300r/min进行转化，并以20％甲酸或者5M氨水调节使pH6.0。待转化20h后取样，离心并用0.22μm滤膜过滤后经HPLC分析，得到α-氨基丁酸的产率分别为86.2g/L、99.6g/L及43.1g/L。
实施例8：重组菌pET-28a-rbs4-ltd+Scvdh+r5fdh/BL21在不同发酵培养基培养后进行全细胞转化产α-氨基丁酸
将重组菌pET-28a-rbs4-ltd+Scvdh+r5fdh/BL21利用LB培养基活化，37℃、160r/min培养过夜后分别转接于2L的TB基、TY基、TYG基及GP基中。接种量8％，培养温度37℃，转速300r/min，通气量1.0vvm。培养2-3h后加入终浓度为0.5mM的IPTG，诱导温度降低为28℃，诱导16h后，4℃，8000r/min离心10min收集菌体，用pH 7.5的50mM PB缓冲液将重组大肠杆菌洗涤二次，重悬于培养时同等体积的pH 7.5的50mM PB缓冲液中，向该体系中投入1.8M L-苏氨酸和1.8M甲酸铵，于30℃、300r/min进行转化，并以20％甲酸或者5M氨水调节使pH7.5。待转化10h及20h后分别取样，离心并用0.22μm滤膜过滤后经HPLC分析。发现L-苏氨酸到α-氨基丁酸在TB基、TY基、TYG基及GP基发酵后的全细胞 转化20h后转化率均可以达到98％以上，但是在10h时的转化率差别较大，分别为68％，46％，49％，64％，因酵母提取物的成本相比于葡萄糖来说较高，因此确定最佳的发酵培养基为GP培养基，其最终α-氨基丁酸的产量达到181g/L，时空产率为9.05g/L·h。
实施例9：表达葡萄糖脱氢酶的重组大肠杆菌单细胞工厂全细胞转化产α-氨基丁酸
[1]将实施例4步骤[3]得到的重组大肠杆菌单细胞工厂pET-28a-rbs3-ltd+Bcldh+Bsglcdh/BL21、pET-28a-rbs4-ltd+Bcldh+Bsglcdh/BL21、pET-28a-rbs5-ltd+Rjpdh+Ppglcdh/BL21及pET-28a-rbs6-ltd+Bsadh+Ppglcdh/BL21重组菌重组菌利用LB培养基活化，37℃、160r/min培养过夜后分别转接于2L的LB基中。接种量8％，培养温度37℃，转速300r/min，通气量1.0vvm。培养2-3h后加入终浓度为0.5mM的IPTG，诱导温度降低为28℃，诱导16h后，4℃，8000r/min离心10min收集菌体，用pH 7.5的50mM PB缓冲液将重组大肠杆菌洗涤二次，重悬于培养时同等体积的pH 7.5的50mM PB缓冲液中，向该体系中投入1M L-苏氨酸及1M葡萄糖，于30℃、300r/min进行转化，并以1M盐酸或者5M氨水调节使pH7.5。待转化18h后取样，离心并用0.22μm滤膜过滤后经HPLC分析。
[2]氨基酸测定结果显示pET-28a-rbs3-ltd+Bcldh+Bsglcdh/BL21、pET-28a-rbs4-ltd+Bcldh+Bsglcdh/BL21、pET-28a-rbs5-ltd+Rjpdh+Ppglcdh/BL21及pET-28a-rbs6-ltd+Bsadh+Ppglcdh/BL21全细胞转化制备α-氨基丁酸的产量分别为33.5g/L、89.1g/L、100.2g/L、27.8g/L。其中pET-28a-rbs3-ltd+Bcldh+Bsglcdh/BL21、pET-28a-rbs4-ltd+Bcldh+Bsglcdh/BL21、pET-28a-rbs5-ltd+Rjpdh+Ppglcdh/BL21重组菌全细胞转化液中并未检测到中间产物酮丁酸的积累，而重组菌pET-28a-rbs6-ltd+Bsadh+Ppglcdh/BL21全细胞转化液中检测出73.6g/L的中间产物酮丁酸，说明在以葡萄糖脱氢酶为辅酶NADH再生用酶时利用rbs4及rbs5序列去诱导L-苏氨酸脱氨酶表达时有利于α-氨基丁酸的制备，其中以rbs5序列为L-苏氨酸脱氨酶的核糖体结合位点序列最佳。
实施例10：重组菌pET-28a-rbs5-ltd+Rjpdh+Ppglcdh/BL21在不同发酵培养基培养后进行全细胞转化产α-氨基丁酸
将重组菌pET-28a-rbs5-ltd+Rjpdh+Ppglcdh/BL21利用LB培养基活化，37℃、160r/min培养过夜后分别转接于2L的TB培养基、TY培养基、TYG培养基及GP培养基中。接种量8％，培养温度37℃，转速300r/min，通气量1.0vvm。培养2-3h后加入终浓度为0.5mM的IPTG，诱导温度降低为28℃，诱导16h后，4℃，8000r/min离心10min收集菌体，用pH 7.5的50mM PB缓冲液将重组大肠杆菌洗涤二次，重悬于培养时同等体积的pH 7.5的50mM PB 缓冲液中，向该体系中投入2M L-苏氨酸和2M葡萄糖，于30℃、300r/min进行转化，并以1M盐酸或者5M氨水调节使pH7.5。待转化10h及20h后分别取样，离心并用0.22μm滤膜过滤后经HPLC分析。发现重组菌利用L-苏氨酸转化成α-氨基丁酸在TB培养基、TY培养基、TYG培养基及培养GP基发酵后的全细胞转化20h后转化率也均可以达到98％以上，但是在10h时的转化率差别较大，分别为47％，62％，66％，53％，因此确定最佳的发酵培养基为TYG培养基，其最终葡萄糖酸的产量达390.8g/L，α-氨基丁酸的产量达到204g/L，α-氨基丁酸的时空产率为10.2g/L·h。
综上，本发明采用如下手段可以取得较好的效果：L-苏氨酸脱氨酶基因前有质粒载体自身携带的启动子、针对L-苏氨酸脱氨酶基因和质粒载体设计的表达强度低于质粒载体本身rbs的rbs序列；L-苏氨酸脱氨酶基因与L-氨基酸脱氢酶基因之间通过质粒载体自身携带的rbs连接；提供辅因子NADH循环的脱氢酶基因前有针对提供辅因子NADH循环的脱氢酶基因和质粒载体设计的启动子、表达强度高于或等于质粒载体本身rbs的rbs序列。
本发明的重组菌单细胞工厂能得到较优的从L-苏氨酸到中间产物酮丁酸及从酮丁酸到α-氨基丁酸的平衡速率，不会造成中间产物酮丁酸的积累因而不会造成对反应的抑制。同时所述的重组菌单细胞工厂不需要外源添加辅因子，相比于其他方法减少了底物进出细胞或扩散的路径从而增加了转化速率。
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
Figure PCTCN2016106241-appb-000003
Figure PCTCN2016106241-appb-000004
Figure PCTCN2016106241-appb-000005
Figure PCTCN2016106241-appb-000006
Figure PCTCN2016106241-appb-000007
Figure PCTCN2016106241-appb-000008
Figure PCTCN2016106241-appb-000009
Figure PCTCN2016106241-appb-000010
Figure PCTCN2016106241-appb-000011
Figure PCTCN2016106241-appb-000012

Claims (14)

  1. 一种高效合成α-氨基丁酸的重组菌单细胞工厂，其特征在于，所述重组菌单细胞工厂是将重组共表达载体转化到宿主菌中得到的；所述重组共表达载体是在质粒载体上串联L-苏氨酸脱氨酶基因、L-氨基酸脱氢酶基因、提供辅因子NADH循环的脱氢酶基因；其中，以L-氨基酸脱氢酶基因的表达为基准，控制供辅因子NADH循环的脱氢酶的表达量使辅因子NADH的生成速率处于相对较高的水平，控制L-苏氨酸脱氨酶的表达量处于相对较合适的水平。
  2. 根据权利要求1所述的重组菌单细胞工厂，其特征在于，所述的重组菌单细胞工厂能得到较优的从L-苏氨酸到中间产物酮丁酸及从酮丁酸到α-氨基丁酸的平衡速率，不会造成中间产物酮丁酸的积累因而不会造成对反应的抑制；同时所述的重组菌单细胞工厂不需要外源添加辅因子，相比于其他方法减少了底物进出细胞或扩散的路径从而增加了转化速率。
  3. 根据权利要求1所述的重组菌单细胞工厂，其特征在于，所述控制，是通过但不限于，启动子和/或rbs序列优化，也可以是增强子、终止子、沉默子优化等。
  4. 根据权利要求1所述的重组菌单细胞工厂，其特征在于，所述重组菌单细胞工厂的构建方法，包括：
    (1)根据启动子及L-苏氨酸脱氨酶的基因序列，设计不同强度的rbs序列用来控制L-苏氨酸脱氨酶的表达量从而控制L-苏氨酸到酮丁酸的转化速率；
    (2)对辅因子NADH的供应速率进行了调控，主要是对启动子以及rbs序列进行优化来控制提供辅因子NADH循环的脱氢酶表达量从而控制辅因子NADH的再生速率；
    (3)将rbs优化后的L-苏氨酸脱氨酶基因、L-氨基酸脱氢酶基因、启动子以及rbs优化后的提供辅因子NADH循环的脱氢酶基因按顺序连接，构建重组共表达载体，并将重组共表达载体转化宿主菌中，构建基因工程菌单细胞工厂。
  5. 根据权利要求1所述的重组菌单细胞工厂，其特征在于，所述L-苏氨酸脱氨酶基因前有质粒载体自身携带的启动子、针对L-苏氨酸脱氨酶基因和质粒载体设计的表达强度低于质粒载体本身rbs的rbs序列；所述L-苏氨酸脱氨酶基因与L-氨基酸脱氢酶基因之间通过质粒载体自身携带的rbs连接；所述提供辅因子NADH循环的脱氢酶基因前有针对提供辅因子NADH循环的脱氢酶基因和质粒载体设计的启动子、表达强度高于或等于质粒载体本身rbs的rbs序列。
  6. 根据权利要求1所述的重组菌单细胞工厂，其特征在于，所述宿主菌可以是大肠杆菌，，也可以是其他宿主，比如枯草芽孢杆菌、棒杆菌、酵母等。
  7. 根据权利要求1所述的重组菌单细胞工厂，其特征在于，所述质粒载体可以是任意一种可商业化购买的质粒载体，或者是任意一种已有报道的经改造的质粒载体。
  8. 根据权利要求1所述的重组菌单细胞工厂，其特征在于，所述L-苏氨酸脱氨酶选自但不限于大肠杆菌来源的L-苏氨酸脱氨酶。
  9. 根据权利要求1所述的重组菌单细胞工厂，其特征在于，所述L-氨基酸脱氢酶选自：但不限于，芽孢杆菌来源的L-亮氨酸脱氢酶、芽孢杆菌来源的L-丙氨酸脱氢酶、链霉菌来源的L-缬氨酸脱氢酶、红球菌来源的L-苯丙氨酸脱氢酶。
  10. 根据权利要求1所述的重组菌单细胞工厂，其特征在于，所述提供辅因子NADH循环的脱氢酶选自：但不限于博伊丁假丝酵母的甲酸脱氢酶、枯草芽孢杆菌的葡萄糖脱氢酶、恶臭假单胞杆菌的葡萄糖脱氢酶。
  11. 根据权利要求1所述的重组菌单细胞工厂，其特征在于，所述宿主为大肠杆菌；所述L-苏氨酸脱氨酶基因前的启动子为T7启动子；L-苏氨酸脱氨酶基因前直接连接的rbs序列为序列SEQ ID NO:1～SEQ ID NO:6所示序列中的任意一种；所述L-苏氨酸脱氨酶基因和L-氨基酸脱氢酶基因通过序列为SEQ ID NO:22所示序列的RBS连接。
  12. 根据权利要求11所述的重组菌单细胞工厂，其特征在于，所述提供辅因子NADH循环的脱氢酶基因前的启动子为tac启动子，rbs序列为SEQ ID NO:33～SEQ ID NO:39中的任意一种；可选地，rbs序列为SEQ ID NO:37的序列。
  13. 一种发酵合成α-氨基丁酸的方法，是利用权利要求1～12任一所述的重组菌单细胞工厂。
  14. 权利要求1～12任一所述的重组菌单细胞工厂应用于α-氨基丁酸、酮丁酸或其相关附属产物的合成。
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