WO2021143468A1 - 一种温度开关***及其在提高氨基酸产量中的应用 - Google Patents

一种温度开关***及其在提高氨基酸产量中的应用 Download PDF

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WO2021143468A1
WO2021143468A1 PCT/CN2020/138263 CN2020138263W WO2021143468A1 WO 2021143468 A1 WO2021143468 A1 WO 2021143468A1 CN 2020138263 W CN2020138263 W CN 2020138263W WO 2021143468 A1 WO2021143468 A1 WO 2021143468A1
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vector
gene
temperature switch
threonine
fermentation
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French (fr)
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王小元
方宇
王建莉
张淑嫣
胡晓清
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江南大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y604/00—Ligases forming carbon-carbon bonds (6.4)
    • C12Y604/01—Ligases forming carbon-carbon bonds (6.4.1)
    • C12Y604/01001—Pyruvate carboxylase (6.4.1.1)

Definitions

  • the invention relates to a temperature switch system and its application in increasing the output of amino acids, in particular to a method for regulating the intracellular metabolic flow distribution to increase the output of threonine by using the temperature switch system, and belongs to the technical field of genetic engineering and microbial fermentation.
  • biosensors can be used to dynamically adjust metabolic pathways, and through the method of genetic circuits to divert metabolic flow, the yield and productivity of engineered strains can be effectively improved.
  • these biosensors are designed to produce fatty acid ethyl esters, glucaric acid, 2-fucose lactose, ⁇ -aminobutyric acid and other substances.
  • most biosensors require a huge workload of genetic circuit element debugging to adapt to target strains under specific fermentation conditions.
  • inducers are rarely used in industrial fermentation production.
  • thermosensitive biosensor The activity of the thermosensitive biosensor is controlled by temperature, which can reduce the metabolic burden of the host during the cell growth stage. It has been applied to the expression of recombinant proteins, the synthesis of D-lactic acid, and the synthesis of itaconic acid. Although the temperature-dependent control module has great industrial application prospects, due to its uncertainty, few related products have been reported. For example, when a thermosensitive biosensor regulates temperature changes, it may cause unknown effects on global cell metabolism and leaky expression of thermosensitive promoters.
  • Threonine as a typical oxaloacetic acid derivative, is an essential amino acid that cannot be synthesized by humans and animals, and it can be industrially produced by microbial fermentation. So far, the threonine yield of engineered microorganisms has been greatly improved, but the complex metabolic flow distribution between the synthetic pathway and the TCA cycle has become a bottleneck for the further improvement of threonine conversion rate. The conversion rate is only 87.8%.
  • the threonine synthesis pathway competes with the central metabolic pathway for common precursor metabolites, but it also requires the TCA cycle to provide a variety of cofactors (NADPH and ATP), because the TCA cycle is the main energy supply pathway for cells to grow under aerobic conditions.
  • the engineered strains need to continuously supply the precursor metabolite oxaloacetate, which is the main metabolic branch between glycolysis, L-aspartic acid and the TCA cycle.
  • the accumulation of oxaloacetate in Escherichia coli is mainly through overexpression of phosphoenolpyruvate carboxylase (PPC) or heterologous expression of phosphoenolpyruvate carboxykinase (PCK) to catalyze phosphoenolpyruvate carboxykinase (PCK).
  • PPC phosphoenolpyruvate carboxylase
  • PCK heterologous expression of phosphoenolpyruvate carboxykinase
  • the acid generates oxaloacetic acid.
  • the intracellular pyruvate synthesis pathway mainly includes the coupling pathway of pyruvate kinase and the PTS system and the ED pathway. Abundant pyruvate is easily wasted, producing excess energy substances and unwanted by-products (acetate, formate, Lactate and ethanol). These organic acid by-products can cause disorders of glycolysis pathways and central carbon metabolism, thereby hindering cell growth and leading to unfavorable product synthesis.
  • Pyruvate carboxylase is another heterologous pathway from pyruvate to oxaloacetate, and it has been applied to produce a variety of derivatives of TCA cycle intermediate metabolites in E. coli.
  • PYC Pyruvate carboxylase
  • high-level expression of PYC enables as many carbon sources as possible to synthesize the target product.
  • low-level expression of PYC is difficult to accumulate more oxaloacetate, resulting in a lower conversion rate of oxaloacetate derivatives.
  • the present invention constructs a method for dynamically regulating the metabolic flow of the central metabolic pathway, by rebalancing the microbial cell carbon distribution, to increase the conversion rate of downstream products of oxaloacetate and make it industrially applicable.
  • the first object of the present invention is to provide a temperature switch carrier, which is obtained by connecting a temperature switch circuit to the carrier;
  • the temperature switch circuit includes a temperature sensitive circuit cI ts -p R -p L and a rigorous circuit tetR- P LtetO-1 ;
  • the temperature-sensitive loop cI ts -p R -p L is composed of a temperature-sensitive repressor gene cI ts and a tandem promoter p R -p L , and the nucleotide sequence is as shown in GenBank: AB248919.1;
  • the rigorous loop tetR-P LtetO-1 consists of a repressor gene tetR and a promoter P LtetO-1 , and the nucleotide sequence of the repressor gene tetR is shown in SEQ ID NO.1.
  • the temperature switch vector replaces the pMB1 in the plasmid pFW001 with the medium copy number replicon p15A, and replaces the PJ23101 promoter in the plasmid pFW001 with the temperature switch circuit.
  • the temperature switch circuit combines the temperature-sensitive suppressor gene cI ts , the promoter p R -p L , RBS, the suppressor gene tetR, the multiple cloning site sequence MCS1, the terminator T7, the promoter P LtetO-1 , and the multiple clone
  • the site sequence MCS2 and the terminator T1 are serially connected in sequence.
  • the temperature switch vector when the RBS nucleotide sequence is as shown in SEQ ID NO. 7, pFT22 is obtained; the temperature switch vector includes pFT22 or a recombinant vector constructed on the basis of pFT22.
  • pFT24 when the RBS nucleotide sequence is as shown in SEQ ID NO. 8, pFT24; pFT24 or a recombinant vector constructed on the basis of pFT24 is obtained.
  • the temperature switch vector includes pFT24r, pFT24p, pFT24pm, pFT24rp, pFT24t1, pFT24t2, pFT24t3, pFT24t4, pFT24rpt3, pFT24rpa1, pFT24rpa2, pFT24rpa3, or pFT24rpa4.
  • the nucleotide sequence of the RBS of the vector pFT24r, pFT24p, pFT24pm, pFT24rp, pFT24t1, pFT24t2, pFT24t3, pFT24t4, pFT24rpt3, pFT24rpa1, pFT24rpa2, pFT24rpa3, or pFT24rpa4 is as SEQ ID NO. 8 shown.
  • the vector pFT24r has the rhtC gene inserted into the MCS1 of the vector of claim 2;
  • the vector pFT24r has a pyc gene inserted at MCS1 of the vector of claim 2;
  • the vector pFT24pm has a pycmt gene inserted into the MCS1 of the vector of claim 2;
  • the vector pFT24rp has rhtC and pycmt genes inserted in sequence at MCS1 of the vector of claim 2;
  • the vector pFT24t1 has a pta gene inserted at MCS2 of the vector of claim 2;
  • the vector pFT24t2 is a pta gene linked to the RBS sequence shown in SEQ ID NO. 9 inserted into the MCS2 of the vector of claim 2;
  • the vector pFT24t3 is inserted into the MCS2 of the vector of claim 2 with a pta gene labeled with a standard SsrA degradation peptide chain;
  • the vector pFT24t4 is inserted into the MCS2 of the vector of claim 2 with an RBS labeled with a standard SsrA degradation peptide chain and a nucleotide sequence as shown in SEQ ID NO.9. Sequence-linked pta gene;
  • the vector pFT24rpt3 has rhtC and pycmt genes inserted in sequence at MCS1 of the vector described in claim 2, and MCS2 is inserted with a standard SsrA degradable peptide chain labeled with the original on the genome.
  • the vector pFT24rpa1 has rhtC and pycmt genes inserted in sequence at MCS1 of the vector described in claim 2, and MCS2 is inserted with a standard SsrA degradable peptide chain labeled with the original on the genome.
  • the vector pFT24rpa2 has rhtC and pycmt genes inserted in sequence at MCS1 of the vector of claim 2, and MCS2 is inserted with nucleotides labeled with a standard SsrA degradable peptide chain.
  • the rhtC and pycmt genes are inserted in the MCS1 of the vector of claim 2, and the alaC gene labeled with a standard SsrA degradation peptide chain is inserted into the MCS2;
  • the vector pFT24rpa4 has the rhtC and pycmt genes inserted in the MCS1 of the vector described in claim 2, and the RBS labeled with a standard SsrA degradation peptide chain and the nucleotide sequence shown in SEQ ID NO.9 are inserted into MCS2. Sequence-linked alaC gene;
  • amino acid sequence of the standard SsrA degradation peptide chain is shown in SEQ ID NO. 10, and the nucleotide sequence is shown in SEQ ID NO. 11.
  • the temperature control range of the temperature switch carrier is between 30°C and 42°C.
  • the second object of the present invention is to provide a method for regulating the relative levels of pyruvate and oxaloacetate in cells, the method controls the expression of pyruvate carboxylase through the above-mentioned temperature switch carrier, and turns off pyruvate during the cell growth stage of fermentation.
  • the expression of carboxylase ensures the rapid accumulation of strain biomass; when the fermentation biomass has accumulated enough, the expression of pyruvate carboxylase is turned on to provide sufficient oxaloacetate for the target product that needs to be synthesized.
  • the method combines the chemical properties of oxaloacetic acid: temperature can accelerate the spontaneous decarboxylation of oxaloacetic acid (see Figure 7).
  • the strain to synthesize threonine from glucose requires more oxaloacetate to provide it with precursor intermediate metabolites, and at the same time requires enough pyruvate to be oxidized to produce sufficient reducing power (NADPH, NADH and ATP).
  • the temperature switch vector controls the expression of pyruvate carboxylase and the chemical properties of oxaloacetate to dynamically adjust the metabolic flow distribution of the central metabolic pathway (see Figure 1).
  • the third object of the present invention is to provide a threonine-producing strain which expresses the above-mentioned temperature switch vector.
  • the above-mentioned temperature switch vector is transferred into threonine production platform strains, and the threonine production platform strains include E. coli TWF001, TWF101, TWF102, TWF103, TWF104, TWF105, TWF106 , TWF107, TWF108, TWF110, TWF111, TWF112 or TWF113.
  • the TWF001 overexpressed the gene encoding pyridine nucleotide transhydrogenase pntAB and related genes related to threonine production ppc, aspC, lysC, asd, thrA G433R BC and rhtA, the construction method is disclosed in the literature: Zhao, H. ,Fang,Y.,Wang,X.,Zhao,L.,Wang,J.,Li,Y.,2018.Increasing L-threonine production in Escherichia coli by engineering the glyoxylate shunt and the L-threonine biosynthesis pathway.
  • TWF101, TWF102, TWF103, TWF104, TWF105, TWF106, TWF107, TWF108, TWF110, TWF111, TWF112, and TWF113 are based on TWF001, further independently or in combination to knock out unimportant genes related to organic acid synthesis poxB, pflB , ldhA, adhE, pta, the gene encoding the threonine transporter tdcC, the alanine synthesis pathway related genes avtA, alaA, alaC (see Figure 2), the specific construction methods are shown in Table 1.
  • the strain uses a temperature switch vector to control the rhtC gene encoding the threonine extracellular transporter, the pyc gene encoding pyruvate carboxylase, and the pyc-based codon
  • the optimized pycmt gene encoding pyruvate carboxylase was expressed independently or in combination, and high threonine-producing strains TWF106/pFT24r, TWF106/pFT24p, TWF106/pFT24p and TWF106/pFT24rp were obtained, and their threonine yields were 17.24g/ L, 20.15g/L, 20.60g/L and 23.29g/L, the corresponding molar sugar acid conversion rates are 72.44%, 81.50%, 98.43% and 111.78%.
  • the strain further uses a temperature switch vector to close the alanine synthesis pathway to obtain a strain TWF113/pFT24rpa1 with a higher threonine conversion rate.
  • the construction method of the TWF113/pFT24rpa1 is based on TWF106, knocking out the three genes avtA, alaA and alaC at the same time to obtain TWF113; on the basis of the temperature switch vector pFT24rpa1, using the promoter P LtetO-1 to control the expression of alaA, The expression vector pFT24rpa1 was obtained; the pFT24rpa1 was transferred into TWF113 to obtain TWF113/pFT24rpa1, the threonine output reached 25.85g/L, and the molar sugar acid conversion rate was 124.03%.
  • the fourth object of the present invention is to provide a method for producing threonine, which uses the above-mentioned threonine-producing strain as a fermentation strain to fermentatively produce threonine.
  • the seed culture of the fermentation strain with an initial OD 600 of 0.2-0.3 is inoculated into the fermentation medium, and the fermentation is cultured until all the glucose is consumed.
  • seed cultures of fermentation strains with an initial OD 600 of 0.2-0.3 are inoculated into the fermentation medium, fermented at 36-38°C for 5-8 hours, and continued to grow at 41-43°C to the fermentation broth. All the glucose has been consumed.
  • the seed medium is STF medium, and the formula is: 10g/L sucrose, 20g/L peptone, 5g/L yeast extract, 15g/L (NH 4 ) 2 SO 4 , 1g /L MgSO 4 , pH 7.3; the fermentation medium formula is: 35g/L glucose, 25g/L (NH 4 ) 2 SO 4 , 7.46g/L KH 2 PO 4 , 2g/L yeast extract, 2g/L L citric acid, 2g/L MgSO 4 ⁇ 7H 2 O, 5 mg/L FeSO 4 ⁇ 7H 2 O, 5 mg/L MnSO 4 ⁇ 4H 2 O, pH 7.1.
  • the fifth object of the present invention is to provide the application of the above-mentioned temperature switch carrier in protein production.
  • the sixth object of the present invention is to provide the application of the above-mentioned temperature switch carrier in the production of aspartic acid family amino acids and their derivatives.
  • the aspartic acid family amino acids and their derivatives include aspartic acid, homoserine, threonine, lysine, methionine, isoleucine and their derivatives. derivative.
  • the present invention constructs a temperature switch system that regulates intracellular metabolic flux distribution to promote threonine production.
  • the switch system includes a temperature switch vector with a medium-copy replicon p15A and a triclosan-resistant screening gene fabV.
  • two metabolic pathway-independent control loop modules (temperature control loop cI ts -p R -p L and rigorous loop tetR-P LtetO-1 ), the system is gradually optimized and upgraded, and finally used to improve threonine Acid production.
  • the switch temperature control range is between 30 and 42°C, and its narrowest effective temperature control range is between 37 and 40°C.
  • the temperature switch system of the present invention is a metabolic pathway-independent regulatory system, which is not affected by the intracellular metabolic environment and can freely control the overexpression of target genes and the closed expression of other genes in the metabolic pathway.
  • the temperature switch system of the present invention is applied to production and fermentation without adding inducers and antibiotics, only a small amount of triclosan needs to be added to maintain plasmid stability. And the switch system is very suitable to be combined with factory-scale fermentation equipment to realize industrialized mass production.
  • the temperature switch system of the present invention is used to overexpress pyruvate carboxylase, combined with the chemical characteristics of oxaloacetic acid decarboxylation by heating, and can intelligently adjust the ratio of pyruvate and oxaloacetic acid in the central metabolic pathway, thereby achieving extreme The purpose of greatly increasing the output of downstream products of oxaloacetate, while ensuring the supply of sufficient cofactors (NADPH and ATP).
  • the temperature switch system of the present invention is applied to threonine production platform strains, which greatly improves the conversion rate of threonine, and obtains two threonine production strains TWF106/pFT24rp and TWF113/pFT24rpa1.
  • the threonine yields were 23.29g/L and 25.85g/L, and the corresponding threonine molar conversion rates were 111.78% and 124.03%, respectively.
  • Figure 1 Schematic diagram of the temperature switch system dynamically regulating the intracellular levels of oxaloacetate; the thick line indicates that genes in the metabolic pathway are overexpressed.
  • Figure 2 Gene knockout of the alternative pathway involved in the construction of platform strains.
  • FIG. 3 Schematic diagram of temperature switch carrier components and loop control.
  • Figure 4 Schematic diagram of the optimization of the temperature switch carrier transformation.
  • Figure 5 Detecting the feasibility of the temperature switch system to control gene expression from the level of translation and transcription.
  • Figure 6 Detecting the sensitivity of the temperature switch system to control gene expression from the level of translation and transcription.
  • Figure 8 Seed medium optimization of shake flask fermentation and comparison of threonine fermentation by threonine-producing strain TWF001 using LB and STF as seed medium.
  • Figure 9 The comparison diagram of threonine shake flask fermentation under the conditions of 37°C constant temperature fermentation and variable temperature fermentation of the chassis strain with the knockout of the alternative pathway gene.
  • FIG. 10 The temperature switch system regulates the expression of threonine extracellular transport protein RhtC and pyruvate carboxylase PYC to produce threonine shake flask fermentation results.
  • Figure 11 Schematic diagram of temperature switch system controlling pta gene shut down.
  • Figure 12 Threonine-producing strains that control the pta gene to turn off the results of shaking flask fermentation for threonine production.
  • Figure 13 A schematic diagram of the temperature switch system controlling the shutdown of the L-alanine synthesis pathway.
  • Figure 14 The results of shake flask fermentation of threonine production by threonine-producing bacteria that control the closure of the L-alanine synthesis pathway.
  • FIG. 15 The temperature switch system regulates the threonine-producing bacteria to gradually increase the threonine conversion rate.
  • the system temperature switch mechanism CI ts thermosensitive repressor binding at rt p R -p L promoter and inhibit transcription, and high-temperature conditions that cause inactivation of repressor CI ts and p R -p L promoter activation.
  • TetR repressor inhibit P LtetO-1 transcriptional promoter, which is controlled tetR genes corresponding p R -p L promoter.
  • ⁇ -galactosidase LacZ and green fluorescent protein GFP are used as reporter proteins, and they are expressed under the p R -p L promoter and the P LtetO-1 promoter, respectively.
  • the SsrA degradation tail was added to the C-terminus of GFP to eliminate GFP residual protein to achieve the effect of complete gene shutdown.
  • the temperature switch vector can control some genes that are necessary for cell growth or that affect cell growth, and the expression is turned on and off in different fermentation stages.
  • Acetic acid synthesis pathway and alanine synthesis pathway are necessary for bacterial growth and biomass accumulation, but these two alternative pathways will compete with the target product synthesis pathway for carbon sources, which will reduce the conversion rate of the target product, so it is necessary to grow the cell Phase expression and cut off during the fermentation phase.
  • the overexpression of pyruvate carboxylase will compete for the carbon source entering the TCA cycle and hinder the normal growth of strains.
  • the expression of pyruvate carboxylase can accumulate more precursor intermediate metabolites-oxaloacetate, which promotes threonization.
  • the production of acid can increase the conversion rate of sugar and acid of the target product, so it is necessary to turn off its expression during the cell growth stage and turn on its expression during the fermentation stage.
  • the above gene expression control can all be achieved by changing the temperature using the temperature switch vector.
  • the CRISPR-Cas9 knockout system was used to efficiently edit the E. coli genome.
  • the editing plasmid pCas is first electrotransformed into threonine-producing E. coli.
  • the plasmid contains the constitutively expressed cas9 gene and the arabinose-induced expression of the gene encoding lambda Red recombinase to obtain a pCas-containing strain.
  • the strain containing pCas was cultured overnight (8-14h) to ensure an initial OD 600 of 0.04, inoculated into LB medium, and added with 50 mg/L kanamycin and 10 mM arabinose, and then grown at 30°C and 200 rpm until OD 600 reached 0.6.
  • PTargetF original vector (the pMBl replicon, spectinomycin resistance) as a template
  • N 20 embedded sequence of the target gene primers to amplify the whole plasmid.
  • the PCR product was digested with DpnI to eliminate the template plasmid, transformed into E. coli DH5 ⁇ , and a circular plasmid was generated by the overlapping sequence at the end.
  • the pTargetF-poxB and knockout template fragments were electroporated into strain TWF001.
  • the gene encoding lambda Red recombinase on the pCas plasmid of this strain was induced to express.
  • the transformants were picked and cultured overnight. 1 mL of cell culture was concentrated by centrifugation and spread on LB agar plates containing kanamycin (50 mg/L) and spectinomycin (50 mg/L) for selection.
  • Real-time quantitative PCR detects the relative transcription levels of the two reporter genes in the strain, see reference for details: Livak, KJ, Schmittgen, TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C (T)) Method.
  • UV-1800 spectrophotometer to measure the absorbance of OD 600 to monitor the bacterial cell concentration of fermentation samples at different time points in real time. After centrifugation at 13000 ⁇ g for 10 minutes, the culture supernatant was taken to analyze the content of extracellular metabolites, including residual glucose, amino acids and organic acids. A biosensor analyzer (SBA-40C, Institute of Biology, Shandong Academy of Sciences) was used to measure the residual glucose concentration in the culture broth. Amino acids were quantified by high performance liquid chromatography on Agilent 1200 or 1260 series instruments using Thermo ODS-2HYPERSIL C18 column (250mm ⁇ 4.0mm).
  • the sample was derivatized with a commercial o-phthalaldehyde reagent solution (Agilent Technologies), and the injection volume was 1 ⁇ L.
  • Amino acid analysis method program see reference: Koros,A.,Varga,Z.,Molnar-Perl,I.. Simultaneous analysis of amino acids and amines as their o-phthalaldehyde-ethanethiol-9-fluorenylmethyl chloroformate derivatives in cheese by high- performance liquid chromatography.
  • the threonine production was determined by comparison with amino acid standards (Sigma).
  • Aminex HPX-87H chromatographic column 300mm ⁇ 7.8mm; Bio-Rad Laboratories) is used for the determination of organic acids, and the column temperature is 55°C. 5mM sulfuric acid was used as the mobile phase, and the flow rate was 0.6mL/min.
  • STF seed medium 10g/L sucrose, 20g/L peptone, 5g/L yeast extract, 15g/L (NH 4 ) 2 SO 4 , 1g/L MgSO 4 , pH 7.3.
  • Fermentation medium 35g/L glucose, 25g/L(NH 4 ) 2 SO 4 , 7.46g/L KH 2 PO 4 , 2g/L yeast extract, 2g/L citric acid, 2g/L MgSO 4 ⁇ 7H 2 O, 5mg/L FeSO 4 ⁇ 7H 2 O, 5mg/L MnSO 4 ⁇ 4H 2 O, pH 7.1.
  • the method for constructing the threonine production platform strain and temperature switch vector involved in the present invention is shown in Table 1, and the source or sequence of the gene or protein is shown in Table 2.
  • ⁇ means knockout
  • (M), (L), (H) means different primers used when amplifying the target gene, such as P RL ::tetR, MCS1, P LtetO1 ::pta(M) means amplification
  • P RL ::tetR, MCS1, P LtetO1 ::pta(M) means amplification
  • the primer pair used is pta-Mrbs-F and pta-R
  • P RL ::tetR,MCS1,P LtetO1 ::pta(LAA)(H) means that when amplifying pta, the primer pair used is pta- Hrbs-F and pta-LAA-R, see Table 3 for details.
  • the medium copy number replicon p15A was used to replace pMB1
  • the temperature switch circuit was used to replace the PJ23101 promoter to obtain a temperature switch vector.
  • the plasmid pFW001 is derived from a published paper in 2018: Zhao,H.,Fang,Y.,Wang,X.,Zhao,L.,Wang,J.,Li,Y.,2018. Increasing L-threonine production in Escherichia coli by engineering the glyoxylate shunt and the L-threonine biosynthesis pathway.
  • the temperature switch circuit includes the temperature-sensitive circuit cI ts -p R -p L , the TetR suppressor gene tetR and the strictly regulated promoter P LtetO-1 . These genes are obtained by chemical synthesis. In order to obtain independent genetic regulation, double multiple cloning sites (MCS1 and MCS2) and different transcription terminators (T 7 from bacteriophage T7 RNA polymerase and T 1 from E. coli rrnB gene) were inserted into the temperature-sensitive circuit cI ts, respectively -p R -p L and rigorous loop tetR-P LtetO-1 in two independent modules.
  • MCS1 and MCS2 double multiple cloning sites
  • T 7 from bacteriophage T7 RNA polymerase
  • T 1 from E. coli rrnB gene
  • the temperature switch circuit obtained is to combine the temperature-sensitive suppressor gene cI ts , the promoter p R -p L , the suppressor gene tetR, the multiple cloning site sequence MCS1, the terminator T7, the promoter P LtetO-1 , and the multiple cloning site. Sequence MCS2 and terminator T1 are connected in series in sequence.
  • the temperature switch loop and the p15A replicon derived from pSU2718 were integrated into the backbone plasmid pFW001 to obtain a circular temperature switch vector.
  • the original temperature switch vectors were further optimized and modified to produce a series of temperature switch vectors to test whether these vectors can be used in low temperature conditions (30°C).
  • pFT22 and pFT24 have better performance.
  • the temperature switch circuit temperature-sensitive suppressor gene cI ts , promoter p R -p L , RBS, suppressor gene tetR, multiple cloning site sequence MCS1, terminator T7, promoter P LtetO-1 , multiple cloning site
  • the nucleotide sequence of RBS (high intensity) in sequence MCS2 and terminator T1 is TTAAAGAGGAGAAAGGTACC
  • the resulting temperature switch vector is pFT22
  • the nucleotide sequence of RBS (low intensity) in the temperature switch loop is In the case of AAACGAAGCATTGGGATCTT
  • the temperature switch vector obtained is pFT24 (see Figure 4).
  • the lacZ fragment was amplified from the E. coli genome using primers with high-strength RBS, and inserted into the SpeI site of MCS1 of pFT22 and pFT24 using the ClonExpress II one-step cloning kit (Vazyme, Jiangsu, China), respectively, to obtain pFT22-lacZ and pFT24-lacZ vectors.
  • the green fluorescent protein gene gfp was integrated into the EcoRI restriction site of MCS2 of the pFT22-lacZ and pFT24-lacZ vectors to obtain pFT22-lacZ-gfp and pFT24-lacZ-gfp.
  • the present invention also constructed another recombinant expression vector, labeled GFP with a standard SsrA degradation peptide chain (AADENYALAA, "LAA"), and produced pFT22-lacZ-gfp (LAA) and pFT24-lacZ-gfp (LAA).
  • LAA standard SsrA degradation peptide chain
  • the pFT22-lacZ-gfp, pFT24-lacZ-gfp, pFT22-lacZ-gfp (LAA) and pFT24-lacZ-gfp (LAA) were transformed into the lacZI cluster knockout strain TWF101 of E. coli TWF001 to remove lacZ from the genome Background expression.
  • RNA extraction kit BioFlux, Beijing, China
  • Nanodrop 2000 Thermo Fisher Scientific, Wilmington, MA, USA
  • HiScript II Q RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Jiangsu, China) to remove the remaining DNA in the total RNA, and reverse transcription to synthesize cDNA to ensure that each reaction contains the same amount of RNA for standardization.
  • TWF001 pyridine nucleotide transhydrogenase
  • ppc pyridine nucleotide transhydrogenase
  • ppc pyridine nucleotide transhydrogenase
  • ppc pyridine nucleotide transhydrogenase
  • ppc pyridine nucleotide transhydrogenase
  • ppc pyridine nucleotide transhydrogenase
  • ppc pyridine nucleotide transhydrogenase
  • ppc pyridine nucleotide transhydrogenase
  • ppc pyridine nucleotide transhydrogenase
  • ppc pyridine nucleotide transhydrogenase
  • ppc pyridine nucleotide transhydrogenase
  • ppc pyridine nucleotide transhydrogenase
  • ppc pyridine nucleotide transhydrogenase
  • the platform strains TWF102, TWF103, TWF104, TWF105, TWF106, TWF107, and TWF108 were generated, as shown in Table 1. These platform strains were respectively subjected to shake flask fermentation to produce threonine at 37°C constant temperature and variable temperature (from 37°C to 42°C) to evaluate the effect of temperature changes on the strain's threonine production ability.
  • the shake flask fermentation data shows that different platform strains fermented at 37°C constant temperature and variable temperature (from 37°C to 42°C), and the cell growth, glucose consumption, threonine production, and organic acid within 15 hours of fermentation The trend of production.
  • the variable temperature fermentation conditions have no obvious negative impact on the threonine production performance of the threonine-producing bacteria.
  • the strain TWF106/pFT24 has certain advantages by comparing the threonine production of various strains under variable temperature fermentation conditions.
  • the strain TWF107/pFT24 was knocked out of pta, and the growth of this strain was significantly inhibited in the early stage of fermentation, and its threonine production capacity was significantly reduced.
  • the temperature switch system is used to close some carbon source competition pathways that affect the growth of strains, including the acetic acid synthesis pathway and the L-alanine synthesis pathway.
  • the pta gene encoding phosphate acetyltransferase in the genome of threonine-producing bacteria was knocked out.
  • the expression vectors pFT24t1, pFT24t2, pFT24t3, pFT24t4, and pFT24rpt3 are obtained, so that the P LtetO-1 promoter controls the normal expression of the pta gene in the cell growth stage, and shuts down its expression during the fermentation production stage. Thereby saving carbon source (as shown in Figure 11).
  • the expression vectors pFT24t1, pFT24t2, pFT24t3, pFT24t4, and pFT24rpt3 were transferred into the pta-knockout strain TWF108, but their threonine production capacity was not improved compared to TWF106/pFT24.
  • the L-alanine synthesis pathway converts pyruvate to L-alanine through the L-alanine synthetic transaminase, which includes three main proteins: AvtA, AlaA, and AlaC.
  • a multi-knockout strain TWF113 (TWF106 ⁇ avtA ⁇ alaA ⁇ alaC) was constructed to further design a temperature switch system for shutting down L-alanine synthesis.
  • alaA and alaC genes containing different RBS sequences (see Table 3) were inserted into the P LtetO-1 promoter, pFT24rpa1, pFT24rpa2, pFT24rpa3 and pFT24rpa4 were obtained.
  • the temperature switch vector pFT24 controls its gene expression during the growth period of the strain. To restore the normal growth and biomass accumulation of the strain (as shown in Figure 13). These pathways are closed during the fermentation period to reduce the shunting of excessive carbon sources to other pathways, so as to concentrate more carbon sources to synthesize the target products.
  • the heterologous gene pyc was amplified from Lactococcus lactis using primers containing high-strength RBS sequences.
  • the rhtC gene containing the high-strength RBS sequence was amplified from the E. coli TWF001 genome, and the primers used are shown in Table 3.
  • the two genes rhtC and pycmt are co-expressed under the control of the cI ts -p R -p L loop to obtain the temperature switch vector pFT24rp.
  • temperature switch vectors were electrotransformed into engineering strain TWF106 to produce a series of recombinant strains.
  • TWF106 temperature switch carrier
  • pta, gltA, alaA, and alaC were amplified from the E. coli genome with primers attached with a "LAA" degrading tail, and cloned into the EcoRI restriction sites of the vector pFT24 or pFT24rp, respectively.
  • the restriction site is located at the P LtetO-1 promoter Child downstream.
  • Complementary plasmids containing target genes of different strengths of RBS are introduced into the target strains to restore cell growth.
  • STF seed medium is based on LB medium and is obtained after optimization and improvement. After adjusting the sucrose and peptone content in the LB medium, it was used to cultivate the TWF001 strain, and to detect the growth status and threonine production of the strain in the seed medium of different components (as shown in Figure 8). The results showed that the STF seed medium was used as the seed medium, and the 37°C constant temperature shake flask fermentation was used. After 18 hours of fermentation, the threonine output of TWF001 was 15.37g/L, which was 50.54% higher than that of LB as the seed medium.
  • the temperature switch vector was further used to close the alanine synthesis pathway, and the recombinant strain TWF113/pFT24rpa1 (as shown in Figure 14) was obtained, specifically: on the basis of TWF106, avtA was knocked out at the same time , AlaA and alaC three genes, the strain TWF113 was obtained; on the basis of the temperature expression vector pFT24rp, the promoter P LtetO-1 was used to control the expression of alaA, and the expression vector pFT24rpa1 was obtained.
  • the recombinant strain TWF113/pFT24rpa1 was fermented in a variable temperature shake flask, and the threonine output reached 25.85 g/L, and its molar sugar acid conversion rate was 124.03% (as shown in Figure 15). All strains were tested for the formation of organic acids during the shaking flask fermentation process, and it was found that the pyruvate and acetic acid produced by these strains in the early stage of fermentation can be re-absorbed and utilized after the temperature switch system is turned on.

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Abstract

提供了一种包含温度开关回路的载体及其在提高微生物细胞内苏氨酸产量中的应用，该载体通过控制丙酮酸羧化酶异源表达，并结合草酰乙酸温敏易脱羧的化学特性，在丙酮酸和草酰乙酸之间重新平衡代谢流量，动态调节中心代谢途径保证还原型辅因子的供应来促进L-苏氨酸生产。

Description

一种温度开关***及其在提高氨基酸产量中的应用 技术领域
本发明涉及一种温度开关***及其在提高氨基酸产量中的应用，具体涉及一种利用温度开关***调控胞内代谢流分布提高苏氨酸产量的方法，属于基因工程和微生物发酵技术领域。
背景技术
代谢工程与代谢调控相结合已被应用来提高天然化学品的产量，特别是大宗氨基酸产品。然而，长期以来，细胞生长和理想产物之间不平衡的细胞代谢流分布限制了产物的产率和生产力的进一步提高。传统的技术(如使旁路途径的相关基因失活和过表达异源途径的相关基因)不能处理更复杂的碳分布的挑战(如需要多个辅因子参与产物合成)。过强的异源途径会和细胞供能代谢途径竞争，将更多的碳源转变为中间代谢物，导致生长停滞，生产产量与产率下降。近年来，调控基因表达水平来优化代谢流分布的方法已经取得了一些进展，如构建工程启动子、调控核糖体结合序列的强度、表达小调控RNA等。虽然这些技术能有效和快速调节基因表达水平来提高工程菌株生产性能，但是这些调节方式都属于静态控制方式，很难响应动态的代谢环境，导致菌株在次优状态下合成目标产物。
目前，可以通过合成生物传感器动态调节代谢途径，并通过基因回路改道代谢流的方法，有效提高工程菌株的产率和生产力。这些生物传感器合成后经过设计被用来生产脂肪酸乙酯、葡萄糖二酸、2-岩藻糖乳糖、γ-氨基丁酸等物质。然而大多数生物传感器需要巨大工作量的基因回路元件调试，来适配特定发酵条件下的目标菌株。此外，由于罐体发酵过程中存在细菌感染的高风险和高昂的下游纯化成本，工业发酵生产中很少采用诱导剂。
热敏生物传感器的活性受温度控制，可以减轻细胞生长阶段宿主的代谢负担，已应用于重组蛋白的表达、D-乳酸的合成和衣康酸的合成。尽管温度依赖调控模块有很大的工业应用前景，但由于其不确定性，很少的相关产品被报告。例如，当热敏的生物传感器调节温度变化，可能会引起全局细胞代谢的未知影响和热敏启动子的渗漏表达。
苏氨酸作为一种典型的草酰乙酸衍生物，不能被人、动物合成的必需氨基酸，其已可以通过微生物发酵工业化生产。到目前为止，工程微生物的苏氨酸产率已经有了很大的提高，但是其合成途径与TCA循环之间复杂的代谢流分布成为苏氨酸转化率进一步提高的瓶颈，目前报道的最高摩尔转化率仅为87.8％。苏氨酸合成途径与中心代谢途径竞争共同前体代谢物，但也需要TCA循环提供多种辅因子(NADPH和ATP)，因为TCA循环是细胞在有氧条件下生长的主要供能途径。
为了提高苏氨酸生产能力，工程菌株需要连续供应前体代谢产物草酰乙酸，而草酰乙酸是糖酵解、L-天冬氨酸和TCA循环之间的主要代谢分支。传统方法中，在大肠杆菌中积累草酰乙酸主要是通过过表达磷酸烯醇式丙酮酸羧化酶(PPC)或异源表达磷酸烯醇式丙酮酸羧激酶(PCK)，催化磷酸烯醇丙酮酸生成草酰乙酸。然而，相对较丰富的丙酮酸通常会在TCA循环中脱羧并彻底氧化为二氧化碳，导致大量还原因子(NADH，NADPH和ATP)积累。细胞内丙酮酸合成途径主要包括丙酮酸激酶和PTS***偶联途径以及ED途径，丰富的丙酮酸很容易被浪费，产生多余的能量物质和不需要的副产物(醋酸盐、甲酸盐、乳酸盐和乙醇)。这些有机酸副产物会导致糖酵解途径和中心碳代谢的紊乱，从而阻碍细胞生长和导致不利的产物合成。丙酮酸羧化酶(PYC)是从丙酮酸到草酰乙酸的另一种异源途径，已被应用于在大肠杆菌中生产多种TCA循环中间代谢物的衍生产品。在以前的研究中，无需考虑辅助因子供应的情况下，高水平表达PYC使尽可能多的碳源用来合成目标产物。可是，低水平表达PYC很难积累更多的草酰乙酸，导致草酰乙酸衍生物转化率较低。
发明内容
为了解决上述问题，本发明构建了一种动态调控中心代谢途径代谢流的方法，通过再平衡微生物细胞碳分布，来提高草酰乙酸下游产物转化率并使其具有工业实用性。
本发明的第一个目的是提供一种温度开关载体，是将温度开关回路连接到载体上得到的；所述温度开关回路含温敏回路cI ts-p R-p L和严谨型回路tetR-P LtetO-1；所述温敏回路cI ts-p R-p L由温敏抑制子基因cI ts和串联启动子p R-p L组成，核苷酸序列如GenBank:AB248919.1所示；所述严谨型回路tetR-P LtetO-1由抑制子基因tetR和启动子P LtetO-1组成，所述抑制子基因tetR核苷酸序列如SEQ ID NO.1所示。
在本发明的一种实施方式中，所述温度开关载体是将质粒pFW001中的pMB1替换为中等拷贝数复制子p15A，并将质粒pFW001中的PJ23101启动子替换为温度开关回路。所述温度开关回路是将温敏抑制子基因cI ts、启动子p R-p L、RBS、抑制子基因tetR、多克隆位点序列MCS1、终止子T7、启动子P LtetO-1、多克隆位点序列MCS2、终止子T1依次串联。
在本发明的一种实施方式中，当RBS核苷酸序列如SEQ ID NO.7所示时，得到pFT22；所述温度开关载体包括pFT22或在pFT22的基础上构建的重组载体。
在本发明的一种实施方式中，当RBS核苷酸序列如SEQ ID NO.8所示时，得到pFT24；pFT24或在pFT24的基础上构建的重组载体。
在本发明的一种实施方式中，所述温度开关载体包括pFT24r、pFT24p、pFT24pm、pFT24rp、pFT24t1、pFT24t2、pFT24t3、pFT24t4、pFT24rpt3、pFT24rpa1、pFT24rpa2、pFT24rpa3 或pFT24rpa4。
在本发明的一种实施方式中，所述载体pFT24r、pFT24p、pFT24pm、pFT24rp、pFT24t1、pFT24t2、pFT24t3、pFT24t4、pFT24rpt3、pFT24rpa1、pFT24rpa2、pFT24rpa3或pFT24rpa4的RBS的核苷酸序列如SEQ ID NO.8所示。
在本发明的一种实施方式中，所述载体pFT24r是在权利要求2所述载体的MCS1处***了rhtC基因；
在本发明的一种实施方式中，所述载体pFT24r是在权利要求2所述载体的MCS1处***了pyc基因；
在本发明的一种实施方式中，所述载体pFT24pm是在权利要求2所述载体的MCS1处***了pycmt基因；
在本发明的一种实施方式中，所述载体pFT24rp是在权利要求2所述载体的MCS1处依次***了rhtC和pycmt基因；
在本发明的一种实施方式中，所述载体pFT24t1是在权利要求2所述载体的MCS2处***了pta基因；
在本发明的一种实施方式中，所述载体pFT24t2是在权利要求2所述载体的MCS2处***了与核苷酸序列如SEQ ID NO.9所示的RBS序列连接的pta基因；
在本发明的一种实施方式中，所述载体pFT24t3是在权利要求2所述载体的MCS2处***了用标准SsrA降解肽链标记的pta基因；
在本发明的一种实施方式中，所述载体pFT24t4是在权利要求2所述载体的MCS2处***了用标准SsrA降解肽链标记的与核苷酸序列如SEQ ID NO.9所示的RBS序列连接的pta基因；
在本发明的一种实施方式中，所述载体pFT24rpt3是在权利要求2所述载体的MCS1处依次***了rhtC和pycmt基因，在MCS2中***了用标准SsrA降解肽链标记的含有基因组上原始的RBS和pta基因；
在本发明的一种实施方式中，所述载体pFT24rpa1是在权利要求2所述载体的MCS1处依次***了rhtC和pycmt基因，在MCS2中***了用标准SsrA降解肽链标记的含有基因组上原始的RBS和alaA基因；
在本发明的一种实施方式中，所述载体pFT24rpa2是在权利要求2所述载体的MCS1处依次***了rhtC和pycmt基因，在MCS2中***了用标准SsrA降解肽链标记的与核苷酸序列如SEQ ID NO.9所示的RBS序列连接的alaA基因；
所述载体pFT24rpa3是在权利要求2所述载体的MCS1处依次***了rhtC和pycmt基因，MCS2中***了用标准SsrA降解肽链标记的alaC基因；
所述载体pFT24rpa4是在权利要求2所述载体的MCS1处依次***了rhtC和pycmt基因，MCS2中***了用标准SsrA降解肽链标记的与核苷酸序列如SEQ ID NO.9所示的RBS序列连接的alaC基因；
所述标准SsrA降解肽链的氨基酸序列如SEQ ID NO.10所示，核苷酸序列如SEQ ID NO.11所示。
在本发明的一种实施方式中，所述温度开关载体的温控范围在30～42℃之间。
本发明的第二个目的是提供一种调节细胞内丙酮酸和草酰乙酸相对水平的方法，所述方法通过上述温度开关载体控制丙酮酸羧化酶表达，在发酵的细胞生长阶段关闭丙酮酸羧化酶的表达，来保证菌株生物量快速积累；当发酵生物量积累足够后，开启丙酮酸羧化酶的表达，为需要合成的目标产物提供充足的草酰乙酸。
在本发明的一种实施方式中，所述方法结合了草酰乙酸的化学特性：温度可以加速草酰乙酸自发脱羧(见图7)。菌株从葡萄糖合成苏氨酸需要更多草酰乙酸为其提供前体中间代谢物，同时需要足够的丙酮酸被氧化产生充足的还原力(NADPH、NADH和ATP)。通过温度开关载体控制丙酮酸羧化酶的表达和草酰乙酸这一化学特性，来动态调节中心代谢途径代谢流分布(见图1)。
本发明的第三个目的是提供一种生产苏氨酸的菌株，其表达上述的温度开关载体。
在本发明的一种实施方式中，是将上述温度开关载体转入苏氨酸生产平台菌株中，所述苏氨酸生产平台菌株包括大肠杆菌TWF001、TWF101、TWF102、TWF103、TWF104、TWF105、TWF106、TWF107、TWF108、TWF110、TWF111、TWF112或TWF113。所述TWF001过表达了编码吡啶核苷酸转氢酶的基因pntAB和涉及苏氨酸生产的相关基因ppc、aspC、lysC、asd、thrA G433RBC和rhtA，构建方法公开于文献：Zhao,H.,Fang,Y.,Wang,X.,Zhao,L.,Wang,J.,Li,Y.,2018.Increasing L-threonine production in Escherichia coli by engineering the glyoxylate shunt and the L-threonine biosynthesis pathway中。所述TWF101、TWF102、TWF103、TWF104、TWF105、TWF106、TWF107、TWF108、TWF110、TWF111、TWF112、TWF113是在TWF001的基础上，进一步独立或组合敲除了与有机酸合成有关的不重要基因poxB,pflB,ldhA,adhE,pta、编码苏氨酸转运蛋白的基因tdcC、丙氨酸合成途径相关基因avtA,alaA,alaC(见图2)，具体构建方法见表1。
在本发明的一种实施方式中，所述菌株在TWF106的基础上，利用温度开关载体控制编 码苏氨酸胞外转运蛋白的rhtC基因、编码丙酮酸羧化酶的pyc基因和基于pyc密码子优化的编码丙酮酸羧化酶的pycmt基因独立表达或组合表达，获得高产苏氨酸的菌株TWF106/pFT24r、TWF106/pFT24p、TWF106/pFT24p和TWF106/pFT24rp，其苏氨酸产量分别是17.24g/L、20.15g/L、20.60g/L和23.29g/L，对应的摩尔糖酸转化率是72.44％、81.50％、98.43％和111.78％。
在本发明的一种实施方式中，所述菌株在TWF106/pFT24rp基础上，进一步使用温度开关载体关闭丙氨酸合成途径，获得苏氨酸转化率更高的菌株TWF113/pFT24rpa1。所述TWF113/pFT24rpa1的构建方法是在TWF106的基础上，同时敲除avtA、alaA和alaC三个基因，获得TWF113；在温度开关载体pFT24rpa1的基础上，用启动子P LtetO-1控制alaA表达，获得表达载体pFT24rpa1；将pFT24rpa1转入TWF113中，即得TWF113/pFT24rpa1，其苏氨酸产量达25.85g/L，其摩尔糖酸转化率为124.03％。
本发明的第四个目的是提供一种生产苏氨酸的方法，是以上述生产苏氨酸的菌株为发酵菌株，发酵生产苏氨酸。
在本发明的一种实施方式中，将初始OD 600为0.2～0.3的发酵菌株种子培养物接种到发酵培养基中，发酵培养至葡萄糖全部被消耗完。
在本发明的一种实施方式中，将初始OD 600为0.2～0.3的发酵菌株种子培养物接种到发酵培养基中，36～38℃发酵培养5～8h，41～43℃继续培养至发酵液中葡萄糖全部被消耗完。
在本发明的一种实施方式中，种子培养基为STF培养基，配方为：10g/L蔗糖、20g/L蛋白胨、5g/L酵母提取物、15g/L(NH 4) 2SO 4、1g/L MgSO 4，pH 7.3；所述发酵培养基配方为：35g/L葡萄糖、25g/L(NH 4) 2SO 4、7.46g/L KH 2PO 4、2g/L酵母提取物、2g/L柠檬酸、2g/L MgSO 4·7H 2O、5mg/L FeSO 4·7H 2O、5mg/L MnSO 4·4H 2O，pH 7.1。
本发明的第五个目的是提供上述温度开关载体在生产蛋白中的应用。
本发明的第六个目的是提供上述温度开关载体在生产天冬氨酸家族氨基酸及其衍生物的应用。
在本发明的一种实施方式中，所述天冬氨酸家族氨基酸及其衍生物包括天冬氨酸、高丝氨酸、苏氨酸、赖氨酸、甲硫氨酸、异亮氨酸及其衍生物。
本发明的优点和效果：
(1)本发明构建了一种温度开关***调控胞内代谢流分布来促进苏氨酸生产，该开关***包含一个温度开关载体，其具有中等拷贝的复制子p15A、耐三氯生筛选基因fabV和两个代谢途径非依赖的调控回路模块(温控回路cI ts-p R-p L和严谨性回路tetR-P LtetO-1)，该***经 过逐步的优化升级，最终被用来提高苏氨酸产量。温度开关载体经过升级改良后，开关温控范围在30～42℃之间，其有效最窄温控范围在37～40℃之间。
(2)本发明中温度开关***是代谢途径非依赖型调控***，不受胞内代谢环境的影响，可以随意控制目标基因的过表达和代谢途径中其他基因的关闭表达。通过弱化抑制子TetR的RBS强度，有效缓解启动子p R-p L的表达渗漏，减弱常温条件下严谨型回路tetR-P LtetO-1的表达抑制，保证目标基因在开关启动前拥有足够的表达水平。
(3)将本发明中的温度开关***应用于工程菌株进行分阶段发酵，可将整个发酵过程分成细胞生长和发酵生产两个阶段，使工程菌株同时具有快速的生物量积累和高效的发酵生产双重优势，来进一步提高菌株的生产性能，缩短发酵周期。
(4)将本发明中的温度开关***应用于生产发酵，无需添加诱导剂和抗生素，只需要添加微量的三氯生来维持质粒稳定。且该开关***非常适合与工厂规模的发酵设备结合，实现工业化大量生产。
(5)将本发明中的温度开关***用来过表达丙酮酸羧化酶，结合草酰乙酸受热脱羧的化学特性，可智能调节中心代谢途径中丙酮酸和草酰乙酸的比例，从而达到极大提高草酰乙酸下游产物产量的目的，同时保证足够的辅因子(NADPH和ATP)的供应。
(6)将本发明中的温度开关***应用到苏氨酸生产平台菌株中，大幅提高了苏氨酸的转化率，得到两株苏氨酸生产菌株TWF106/pFT24rp和TWF113/pFT24rpa1，经过变温摇瓶发酵，其苏氨酸产量分别是23.29g/L和25.85g/L，对应的苏氨酸摩尔转化率分别为111.78％和124.03％。
附图说明
图1：温度开关***动态调控草酰乙酸胞内水平示意图；粗线表示代谢途径中的基因被过表达。
图2：平台菌株构建所涉及旁路途径的基因敲除。
图3：温度开关载体构成元件及环路调控示意图。
图4：温度开关载体改造优化示意图。
图5：从翻译水平和转录水平检测温度开关***控制基因表达可行性。
图6：从翻译水平和转录水平检测温度开关***控制基因表达灵敏性。
图7：草酰乙酸自发脱羧受到温度和时间的影响。
图8：摇瓶发酵种子培养基优化及苏氨酸生产菌TWF001以LB和STF作为种子培养基进行苏氨酸发酵对比图。
图9：旁路途径基因敲除的底盘菌株在37℃恒温发酵和变温发酵条件下苏氨酸摇瓶发酵对照图。
图10：温度开关***调控苏氨酸胞外运输蛋白RhtC和丙酮酸羧化酶PYC表达生产苏氨酸摇瓶发酵结果。
图11：温度开关***控制pta基因关闭示意图。
图12：控制pta基因关闭的苏氨酸生产菌产苏氨酸摇瓶发酵结果。
图13：温度开关***控制L-丙氨酸合成途径关闭示意图。
图14：控制L-丙氨酸合成途径关闭的苏氨酸生产菌产苏氨酸摇瓶发酵结果。
图15：温度开关***调控苏氨酸生产菌逐步提高苏氨酸转化率示意图。
具体实施方式
1、温度开关***的工作机制：热敏CI ts阻遏物在室温下结合p R-p L启动子并抑制转录，而高温条件引起CI ts阻遏物失活和p R-p L启动子激活。TetR抑制子可抑制P LtetO-1启动子的转录，其对应的基因tetR被p R-p L启动子控制。检测温度开关***的效果时，以β-半乳糖苷酶LacZ和绿色荧光蛋白GFP作为报告蛋白，分别置于p R-p L启动子和P LtetO-1启动子下表达。将SsrA降解尾巴添加至GFP的C端以消除GFP残余蛋白，以达到基因完全关闭的效果。低温条件下，热敏CI ts阻遏物结合p R-p L启动子并抑制下游基因tetR和lacZ转录，则P LtetO-1启动子和gfp可正常转录；高温条件下，CI ts阻遏蛋白失活，不能与p R-p L启动子结合，下游基因tetR和lacZ正常转录，抑制P LtetO-1启动子和gfp的转录(见图3)。
当温度开关***应用于苏氨酸生产菌株中时，温度开关载体可以控制一些细胞生长必需或影响细胞生长的基因，在不同的发酵阶段开启表达和关闭表达。乙酸合成途径和丙氨酸合成途径对于菌株生长和生物量积累是必需的，但是这两个旁路途径会与目的产物合成途径竞争碳源，会降低目的产物的转化率，所以需要在细胞生长阶段表达和在发酵阶段切断。然而，丙酮酸羧化酶的过表达会竞争进入TCA循环的碳源，阻碍菌株正常生长，但是丙酮酸羧化酶的表达可以积累更多的前体中间代谢物——草酰乙酸，促进苏氨酸生产，提高目的产物的糖酸转化率，所以需要在细胞生长阶段关闭其表达和在发酵阶段开启其表达。以上这些基因表达控制都可以利用该温度开关载体通过温度的改变来实现。
2、基因敲除的方法
CRISPR-Cas9敲除***被用来高效编辑大肠杆菌基因组。编辑质粒pCas首先被电转入苏氨酸生产大肠杆菌中，该质粒包含具有组成型表达的cas9基因和***糖诱导表达的编码lambda Red重组酶的基因，得到含有pCas的菌株。含有pCas的菌株经过过夜培养(8-14h)，保证初始OD 600为0.04，接种到LB培养基，并添加50mg/L的卡那霉素和10mM***糖， 然后在30℃下，200rpm生长直至OD 600达到0.6。收集50mL已诱导培养后的含pCas的菌株培养物，并用冰浴的10％甘油溶液洗涤三次，然后添加2mL冰浴的10％甘油溶液悬浮菌株，分装到1.5mL EP管中于-70℃下保存，得到带有pCas的感受态细胞。
以原始pTargetF载体(pMB1复制子，壮观霉素抗性)为模板，采用嵌有目标基因N 20序列的引物来扩增整个质粒。用DpnI消化PCR产物来消除模板质粒，将其转化到大肠杆菌DH5α中，通过末端重叠序列来产生环状质粒。
根据已有文献报道(Jiang Y,Chen B,Duan C,et al.Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9System)进行基因敲除菌株的构建。例如敲除poxB，将上述PCR产物转化到大肠杆菌DH5α中，挑取转化子，过夜培养，提取质粒pTargetF-poxB(含有靶向poxB基因座的N 20序列)。通过重叠延伸PCR获得具有对应poxB基因座的上下游区域的两个同源臂的敲除模板片段。将pTargetF-poxB与敲除模板片段一起电穿孔转化到菌株TWF001中，该菌株中pCas质粒上的编码lambda Red重组酶的基因已经被诱导表达，挑取转化子，过夜培养。离心浓缩1mL细胞培养物涂布于含有卡那霉素(50mg/L)和壮观霉素(50mg/L)的LB琼脂平板上以进行筛选。使用验证引物进行菌落PCR，获得poxB基因敲除菌株，随后加入0.5mM IPTG去除pTargetF-poxB，42℃孵育去除温度敏感质粒pCas。本发明中的其他敲除菌株的构建与以上步骤相同。
3、比色法测定β-半乳糖苷酶(LacZ)活性，详见参考文献：Li,W.,Zhao,X.,Zou,S.,Ma,Y.,Zhang,K.,Zhang,M..Scanning assay of beta-galactosidase activity。
4、实时定量PCR检测菌株中两个报告基因的相对转录水平，详见参考文献：Livak,K.J.,Schmittgen,T.D..Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))Method。
5、胞外代谢物分析方法
使用UV-1800分光光度计测定OD 600的吸光度来实时监测不同时间点的发酵样品菌体浓度。经过13000×g离心10分钟，取培养物上清液用于分析胞外代谢物含量，主要包括残留葡萄糖、氨基酸和有机酸。用生物传感器分析仪(SBA-40C，山东省科学院生物研究所)测量培养液中的残留葡萄糖浓度。氨基酸通过高效液相色谱在Agilent 1200或1260系列仪器上使用Thermo ODS-2HYPERSIL C18色谱柱(250mm×4.0mm)进行定量。用商品化的邻苯二甲醛试剂溶液(Agilent Technologies)衍生化样品，进样量1μL。氨基酸分析方法程序见参考文献：Koros,A.,Varga,Z.,Molnar-Perl,I..Simultaneous analysis of amino acids and amines as their o-phthalaldehyde-ethanethiol-9-fluorenylmethyl chloroformate derivatives in cheese by  high-performance liquid chromatography。
通过与氨基酸标准品(Sigma)进行比较来测定苏氨酸产量。有机酸的测定采用Aminex HPX-87H色谱柱(300mm×7.8mm；Bio-Rad Laboratories)，色谱柱温度为55℃。将5mM硫酸用作流动相，流速为0.6mL/min。用DAD检测器在发射波长210nm处检测，进样量10μL，扫描各个组分的UV光谱，包括丙酮酸、乙酸根、苹果酸、富马酸根、草酰乙酸等。
6、培养基及发酵生产苏氨酸的条件
STF种子培养基：10g/L蔗糖、20g/L蛋白胨、5g/L酵母提取物、15g/L(NH 4) 2SO 4、1g/L MgSO 4，pH 7.3。
发酵培养基：35g/L葡萄糖、25g/L(NH 4) 2SO 4、7.46g/L KH 2PO 4、2g/L酵母提取物、2g/L柠檬酸、2g/L MgSO 4·7H 2O、5mg/L FeSO 4·7H 2O、5mg/L MnSO 4·4H 2O，pH 7.1。
37℃恒温摇瓶发酵：将包含温度开关载体的菌株接种到50mL灭菌的STF种子培养基，并在37℃、200rpm条件下培养5h，以初始OD 600＝0.2将该种子培养物接种到灭菌的发酵培养基中，发酵培养直至发酵液中葡萄糖全部被消耗完。
变温摇瓶发酵：将包含温度开关载体的菌株接种到50mL灭菌的STF种子培养基，并在37℃、200rpm条件下培养5h，以初始OD 600＝0.2将该种子培养物接种到灭菌的发酵培养基中，发酵6h后，将温度调高至42℃，发酵培养直至发酵液中葡萄糖全部被消耗完。
本发明中涉及的苏氨酸生产平台菌株和温度开关载体的构建方法见表1，基因或蛋白的来源或序列见表2。
表1 苏氨酸生产平台菌株和温度开关载体的构建方法
Figure PCTCN2020138263-appb-000001
Figure PCTCN2020138263-appb-000002
注：Δ表示敲除，(M)、(L)、(H)表示扩增目的基因时，使用的引物不同，如P RL::tetR,MCS1,P LtetO1::pta(M)表示扩增pta时，使用的引物对为pta-Mrbs-F和pta-R；P RL::tetR,MCS1,P LtetO1::pta(LAA)(H)表示扩增pta时，使用的引物对为pta-Hrbs-F和pta-LAA-R，具体见表3。
表2 基因或蛋白的来源或序列
Figure PCTCN2020138263-appb-000003
Figure PCTCN2020138263-appb-000004
通过下述实施例对本发明进行具体描述。
实施例1 温度开关载体构建和表征
(1)温度开关载体的构建
以质粒pFW001(含三氯生抗性和高拷贝数的pMB1复制子)为基础，使用中等拷贝数复制子p15A替换pMB1，使用温度开关回路替换PJ23101启动子，得到温度开关载体。所述的质粒pFW001来源于2018年公开的论文：Zhao,H.,Fang,Y.,Wang,X.,Zhao,L.,Wang,J.,Li,Y.,2018.Increasing L-threonine production in Escherichia coli by engineering the glyoxylate shunt and the L-threonine biosynthesis pathway。
温度开关回路包括温敏回路cI ts-p R-p L、TetR抑制子基因tetR和严谨调控启动子P LtetO-1。这些基因由化学合成获得。为了得到独立的遗传调控，将双多重克隆位点(MCS1和MCS2)和不同的转录终止子(来自噬菌体T7RNA聚合酶的T 7和来自大肠杆菌rrnB基因的T 1)分别***温敏回路cI ts-p R-p L、严谨型回路tetR-P LtetO-1两个独立的模块中。得到的温度开关回路是将温敏抑制子基因cI ts、启动子p R-p L、抑制子基因tetR、多克隆位点序列MCS1、终止子 T7、启动子P LtetO-1、多克隆位点序列MCS2、终止子T1依次串联。
使用ClonExpress MultiS一步克隆试剂盒(Vazyme,Jiangsu,China)，将温度开关回路和来源于pSU2718的p15A复制子一起整合到骨架质粒pFW001上，从而得到环状的温度开关载体。为了使温度开关载体更加适应工程菌株中独立于代谢途径的基因表达控制，对原始温度开关载体作进一步优化和修饰，从而产生了一系列温度开关载体，检测这些载体是否能在低温条件(30℃)下不表达LacZ蛋白，且在高温条件(42℃)下能有效启动lacZ基因。经过初步检测筛选，其中pFT22和pFT24具有较好的性能。
当温度开关回路(温敏抑制子基因cI ts、启动子p R-p L、RBS、抑制子基因tetR、多克隆位点序列MCS1、终止子T7、启动子P LtetO-1、多克隆位点序列MCS2、终止子T1依次串联)中的RBS(高强度)的核苷酸序列为TTAAAGAGGAGAAAGGTACC时，得到的温度开关载体为pFT22；当温度开关回路中的RBS(低强度)的核苷酸序列为AAACGAAGCATTGGGATCTT时，得到的温度开关载体为pFT24(见图4)。
(2)温度开关载体的表征
将两个报告基因lacZ和gfp分别***到温度开关载体的MCS1、MCS2上(p R-p L启动子控制MCS1、P LtetO-1启动子控制MCS2)，以评估温度开关***。
使用带有高强度RBS的引物从大肠杆菌基因组上扩增lacZ片段，并使用ClonExpress II一步克隆试剂盒(Vazyme,Jiangsu,China)将其分别***pFT22和pFT24的MCS1的SpeI酶切位点，得到pFT22-lacZ和pFT24-lacZ载体。类似地，将绿色荧光蛋白基因gfp整合到pFT22-lacZ和pFT24-lacZ载体的MCS2的EcoRI酶切位点，得到pFT22-lacZ-gfp和pFT24-lacZ-gfp。本发明还构建了另外一种重组表达载体，用标准SsrA降解肽链(AADENYALAA，“LAA”)标记GFP，产生了pFT22-lacZ-gfp(LAA)和pFT24-lacZ-gfp(LAA)。将pFT22-lacZ-gfp、pFT24-lacZ-gfp、pFT22-lacZ-gfp(LAA)和pFT24-lacZ-gfp(LAA)转化到大肠杆菌TWF001的lacZI簇敲除菌株TWF101中，以去除基因组上lacZ的背景表达。
为了表征温度开关载体调节基因表达的能力，使用SpectraMax M3酶标仪(Molecular Devices,USA)和UV-1800分光光度计(Shimadzu,Japan)对pFT22和pFT24上两个报告基因的蛋白质水平进行定量。将LB琼脂平板上的菌苔(上述转化的过程中产生的转化子)接种到新鲜的STF种子培养基中，30℃下培养过夜(8-14h)。将种子培养物以初始OD 600＝0.4接种量接种至24孔培养板中，每孔含有2mL的发酵培养基，不同的培养板中种子培养物被置于不同培养温度条件(从30℃到42℃)，200rpm培养4h。为了测试温度开关***pFT24的灵敏度，在开关开启后，测量两个报告蛋白的表达水平变化速率。
将上述在30℃下的过夜培养物以1:30的比例(V/V)稀释到含有30mL发酵培养基的500mL带挡板的摇瓶中，并在37℃，200rpm下孵育直到OD 600达到2.0；然后，将生长条件更改为较高的温度(42℃)作为开始时间，并每20min收集1mL细胞培养物。以上涉及到的培养基中均添加0.9mg/L三氯生以维持质粒稳定。将收集的培养物在96孔板上用于荧光定量，使用488±10nm激发波长和525±10nm发射波长。用600nm吸光度检测细胞密度，所测荧光值用OD 600标准化。使用比色法用5mm石英比色皿测定β-半乳糖苷酶(LacZ)活性。
另外，通过实时定量PCR检测菌株中两个报告基因的相对转录水平。将0.2mL细胞培养物样品与0.4mL RNAstore(Tiangen,Beijing,China)混合，并在4℃下暂时保存直至处理。使用RNA提取试剂盒(BioFlux,Beijing,China)提取预处理样品的总RNA，并使用Nanodrop 2000(Thermo Fisher Scientific,Wilmington,MA,USA)测量RNA浓度。接下来，使用HiScript II Q RT SuperMix for qPCR(+gDNA wiper)(Vazyme,Jiangsu,China)去除总RNA中残留的DNA，并逆转录合成cDNA，确保每个反应均包含等量的RNA进行标准化。使用ChamQ Universal SYBR qPCR Master Mix(Vazyme,Jiangsu,China)，在ABI Step One real-time PCR instrument(Applied Biosystems,San Mateo,CA,USA)上进行实时定量PCR，并以大肠杆菌16S rRNA作为内参。按照试剂盒提供的说明书进行mRNA定量。扩增步骤如下：在95℃预变性30s，然后在95℃下10s，60℃下30s进行40次循环，此外在95℃下15s，60℃下60s，95℃下15s进行熔解曲线分析。自动设置阈值循环值，并监控每个样品的C T值，用于计算两个报告基因与TWF101/pFT22或TWF101/pFT24的相对转录水平的倍数差异。
数据显示，在pFT22和pFT24温度开关***中，LacZ的表达水平在37～40℃之间显著增加，LacZ酶活在较高温度下保持在350Miller Units左右。在37℃或更低的温度下，p R-p L启动子被热敏CI蛋白有效抑制(如图5所示)。因此，工程菌在发酵过程中的生物量积累可以设定在37℃(大肠杆菌生长的适宜温度)。在这两个***中，P LtetO-1启动子驱动GFP的表达，当升高温度时会触发其表达水平下调。与其他对照组相比，pFT24-lacZ-gfp(LAA)在30～37℃之间具有较高的荧光值(高于2000a.u.)，而在40℃以上几乎没有GFP蛋白残留。
此外，为了检测pFT24***的开关敏感性，将在37℃培养的对数期菌株转移到42℃，用来量化两个报告蛋白随时间的变化。在pFT24-lacZ-gfp(LAA)中，GFP荧光在80min内几乎完全消失，同时LacZ活性达到相当高的水平(如图6所示)。当gfp基因的转录被关闭时，带降解标签尾巴的残余GFP蛋白被更快地清除。为了表征不同菌株中相应启动子的强度，同步监测报告基因的转录水平。数据表明，细胞内mRNA水平的相对趋势与上述蛋白水平数据一致。
实施例2 应用温度开关***提高苏氨酸菌株生产能力
(1)优化和改造苏氨酸生产菌株：
以TWF001为出发菌株，该菌株的基因组上已过表达吡啶核苷酸转氢酶(pntAB)和涉及苏氨酸生产的相关基因(ppc、aspC、lysC、asd、thrA G433RBC和rhtA)(见图1)。为了提高菌株苏氨酸生产性能并减少碳源浪费，一些与有机酸合成有关的不重要基因(poxB、pflB、ldhA、adhE和pta)和编码苏氨酸转运蛋白的基因(tdcC)分别被敲除(见图2)，从而产生了平台菌株TWF102、TWF103、TWF104、TWF105、TWF106、TWF107、TWF108，如表1所示。这些平台菌株分别在37℃恒温和变温(从37℃到42℃)条件下进行摇瓶发酵生产苏氨酸，来评估温度变化对菌株产苏氨酸能力影响。
结果如图9所示，摇瓶发酵数据显示了不同平台菌株在37℃恒温发酵和变温(从37℃到42℃)发酵，发酵15h内的细胞生长、葡萄糖消耗、苏氨酸产量和有机酸产量的趋势。首先，和37℃恒温发酵条件相比，变温发酵条件对苏氨酸生产菌生产苏氨酸的性能没有明显的负面影响。其次，通过对比各菌株在变温发酵条件下产苏氨酸产量，菌株TWF106/pFT24具有一定的优势。而菌株TWF107/pFT24被敲除了pta，该菌株在发酵前期生长受到明显的抑制，它的苏氨酸生产能力明显下降。
利用温度开关***关闭一些影响菌株生长的碳源竞争途径，包括乙酸合成途径和L-丙氨酸合成途径。为了构建乙酸合成途径关闭***，苏氨酸生产菌基因组上编码磷酸盐乙酰转移酶的pta基因被敲除。将pta基因***到P LtetO-1启动子之后，得到表达载体pFT24t1、pFT24t2、pFT24t3、pFT24t4、pFT24rpt3，使P LtetO-1启动子控制pta基因在细胞生长阶段正常表达，发酵生产阶段关闭其表达，从而节省碳源(如图11所示)。如图12所示，将表达载体pFT24t1、pFT24t2、pFT24t3、pFT24t4、pFT24rpt3转入敲除了pta的菌株TWF108，但相比于TWF106/pFT24，它们的苏氨酸生产能力没有提高。类似地，L-丙氨酸合成途径通过L-丙氨酸合成转氨酶将丙酮酸转化为L-丙氨酸，包括三种主要蛋白质：AvtA，AlaA和AlaC。因此，构建多敲除菌株TWF113(TWF106ΔavtAΔalaAΔalaC)，用以进一步设计用于关闭L-丙氨酸合成的温度开关***。包含不同RBS序列(见表3)的alaA和alaC基因被分别***到P LtetO-1启动子之后，得到pFT24rpa1、pFT24rpa2、pFT24rpa3和pFT24rpa4，使温度开关载体pFT24控制其在菌株生长时期进行基因表达，以恢复菌株正常生长和生物量积累(如图13所示)。发酵时期关闭这些途径，来减少过多的碳源分流到其他途径，从而集中更多的碳源来合成目标产物。
(2)优化和改造温度开关载体：
为了验证温度开关***对中心代谢途径动态调控效果，使用含有高强度的RBS序列的引物从乳酸乳球菌中扩增了异源基因pyc。类似地，从大肠杆菌TWF001基因组上扩增获得含有高强度的RBS序列的rhtC基因，使用的引物见表3。使用一步克隆试剂盒，将密码子优化的合成基因pycmt以及pyc和rhtC分别***温度开关载体pFT24的SpeI位点，该位点位于p R-p L启动子的下游。两个基因rhtC和pycmt在cI ts-p R-p L环路的控制下共表达，得到温度开关载体pFT24rp。
将这些温度开关载体分别电转化导入工程菌株TWF106中，产生一系列重组菌株。为了提高工程微生物中苏氨酸生产效率，尝试对影响细胞生长的竞争途径的几个关键基因从大肠杆菌染色体迁移到温度开关载体上。用附加了“LAA”降解尾巴的引物从大肠杆菌基因组分别扩增出pta，gltA，alaA和alaC，分别克隆到载体pFT24或pFT24rp的EcoRI限制性位点，酶切位点位于P LtetO-1启动子下游。将含有不同强度RBS的目标基因的互补质粒导入到目标菌株中，以恢复细胞生长。
Figure PCTCN2020138263-appb-000005
实施例3 发酵生产苏氨酸
STF种子培养基是以LB培养基为基础，经过优化改良后得到的。对LB培养基中的蔗糖和蛋白胨含量进行调节后，用来培养TWF001菌株，检测该菌株在不同成分的种子培养基中生长状况及苏氨酸产量(如图8所示)。结果显示，采用STF种子培养基作为种子培养基，采用37℃恒温摇瓶发酵，发酵18h后，TWF001的苏氨酸产量为15.37g/L，相比LB作为种子培养基提高了50.54％。
重组菌株TWF106/pFT24r、TWF106/pFT24p、TWF106/pFT24pm和TWF106/pFT24rp，经过变温摇瓶发酵(如图10所示)，其苏氨酸产量分别是17.24g/L、20.15g/L、20.60g/L和23.29g/L，对应的摩尔糖酸转化率是72.44％、81.50％、98.43％和111.78％。
在苏氨酸发酵过程中，乙酸的积累会影响菌株正常生长，同时降低菌株苏氨酸生产能力。通过传统的敲除pta来切断乙酸生产途径，会严重抑制菌株的生长，进而降低菌株生产苏氨酸能力(如图9所示)。我们尝试通过温度开关***来关闭pta的表达，来节约碳源减少乙酸形成。苏氨酸摇瓶发酵数据显示，相比TWF106/pFT24rp，关闭pta表达虽然最终的菌株生物量增多，但并没有进一步提高菌株苏氨酸生产能力。
在高产苏氨酸菌TWF106/pFT24rp基础上，进一步使用温度开关载体关闭丙氨酸合成途径，获得重组菌株TWF113/pFT24rpa1(如图14所示)，具体为：在TWF106基础上，同时敲除avtA、alaA和alaC三个基因，获得菌株TWF113；在温度表达载体pFT24rp基础上，用启动子P LtetO-1控制alaA表达，获得表达载体pFT24rpa1。重组菌株TWF113/pFT24rpa1经过变温摇瓶发酵，苏氨酸产量达25.85g/L，其摩尔糖酸转化率为124.03％(如图15所示)。检测所有菌株在摇瓶发酵过程中有机酸形成情况，发现这几株菌在发酵前期生产的丙酮酸和乙酸，均能在温度开关***开启后被重新吸收利用。
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。

Claims (13)

  1. 一种载体，其特征在于，将温度开关回路连接到载体上得到的；所述温度开关回路含温敏回路cI ts-p R-p L和严谨型回路tetR-P LtetO-1；所述温敏回路cI ts-p R-p L由温敏抑制子基因cI ts和串联启动子p R-p L组成，核苷酸序列如GenBank:AB248919.1所示；所述严谨型回路tetR-P LtetO-1由抑制子基因tetR和启动子P LtetO-1组成，所述抑制子基因tetR核苷酸序列如SEQ ID NO.1所示。
  2. 如权利要求1所述的载体，其特征在于，将质粒pFW001中的pMB1替换为中等拷贝数复制子p15A，并将质粒pFW001中的PJ23101启动子替换为温度开关回路；所述温度开关回路是将温敏抑制子基因cI ts、启动子p R-p L、RBS、抑制子基因tetR、多克隆位点序列MCS1、终止子T7、启动子P LtetO-1、多克隆位点序列MCS2、终止子T1依次串联。
  3. 如权利要求2所述的载体，其特征在于，所述RBS的核苷酸序列如SEQ ID NO.7所示时，载体为pFT22；所述载体包括pFT22或在pFT22的基础上构建的重组载体。
  4. 如权利要求2所述的载体，其特征在于，所述RBS的核苷酸序列如SEQ ID NO.8所示，载体为pFT24；所述载体包括pFT24或在pFT24的基础上构建的重组载体。
  5. 如权利要求2所述的载体，其特征在于，所述载体包括pFT24r、pFT24p、pFT24pm、pFT24rp、pFT24t1、pFT24t2、pFT24t3、pFT24t4、pFT24rpt3、pFT24rpa1、pFT24rpa2、pFT24rpa3或pFT24rpa4，所述载体的RBS的核苷酸序列如SEQ ID NO.8所示；
    所述载体pFT24r是在权利要求2所述载体的MCS1处***了rhtC基因；
    所述载体pFT24r是在权利要求2所述载体的MCS1处***了pyc基因；
    所述载体pFT24pm是在权利要求2所述载体的MCS1处***了pycmt基因；
    所述载体pFT24rp是在权利要求2所述载体的MCS1处依次***了rhtC和pycmt基因；
    所述载体pFT24t1是在权利要求2所述载体的MCS2处***了pta基因；
    所述载体pFT24t2是在权利要求2所述载体的MCS2处***了与核苷酸序列如SEQ ID NO.9所示的RBS序列连接的pta基因；
    所述载体pFT24t3是在权利要求2所述载体的MCS2处***了用标准SsrA降解肽链标记的pta基因；
    所述载体pFT24t4是在权利要求2所述载体的MCS2处***了用标准SsrA降解肽链标记的与核苷酸序列如SEQ ID NO.9所示的RBS序列连接的pta基因；
    所述载体pFT24rpt3是在权利要求2所述载体的MCS1处依次***了rhtC和pycmt基因，在MCS2中***了用标准SsrA降解肽链标记的pta基因；
    所述载体pFT24rpa1是在权利要求2所述载体的MCS1处依次***了rhtC和pycmt基因，在MCS2中***了用标准SsrA降解肽链标记的alaA基因；
    所述载体pFT24rpa2是在权利要求2所述载体的MCS1处依次***了rhtC和pycmt基因，在MCS2中***了用标准SsrA降解肽链标记的与核苷酸序列如SEQ ID NO.9所示的RBS序列连接的alaA基因；
    所述载体pFT24rpa3是在权利要求2所述载体的MCS1处依次***了rhtC和pycmt基因，MCS2中***了用标准SsrA降解肽链标记的alaC基因；
    所述载体pFT24rpa4是在权利要求2所述载体的MCS1处依次***了rhtC和pycmt基因，MCS2中***了用标准SsrA降解肽链标记的与核苷酸序列如SEQ ID NO.9所示的RBS序列连接的alaC基因；
    所述标准SsrA降解肽链的氨基酸序列如SEQ ID NO.10所示。
  6. 一种调节细胞内丙酮酸和草酰乙酸相对水平的方法，其特征在于，所述方法通过权利要求1-5任一所述载体控制丙酮酸羧化酶表达，在发酵的细胞生长阶段关闭丙酮酸羧化酶的表达，来保证菌株生物量积累；当发酵生物量积累足够后，开启丙酮酸羧化酶的表达，为需要合成的目标产物提供充足的草酰乙酸。
  7. 一种生产苏氨酸的菌株，其特征在于，表达权利要求5所述的温度开关载体。
  8. 如权利要求7所述的菌株，其特征在于，将权利要求5所述的温度开关载体转入苏氨酸生产平台菌株中，所述苏氨酸生产平台菌株包括大肠杆菌TWF001、TWF101、TWF102、TWF103、TWF104、TWF105、TWF106、TWF107、TWF108、TWF110、TWF111、TWF112或TWF113。
  9. 一种生产苏氨酸的方法，其特征在于，是以权利要求7或8所述菌株为发酵菌株，生产苏氨酸。
  10. 如权利要求9所述的方法，其特征在于，将初始OD 600为0.2～0.3的发酵菌株种子培养物接种到发酵培养基中，36～38℃发酵培养5～8h，41～43℃继续培养至发酵液中葡萄糖全部被消耗完。
  11. 权利要求1-4任一所述的温度开关载体在生产蛋白中的应用。
  12. 权利要求1-4任一所述的温度开关载体在生产天冬氨酸家族氨基酸及其衍生物的应用。
  13. 如权利要求12所述的应用，其特征在于，所述天冬氨酸家族氨基酸及其衍生物包括天冬氨酸、高丝氨酸、苏氨酸、赖氨酸、甲硫氨酸、异亮氨酸及其衍生物。
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