WO2016184044A1 - 一种聚合级乳酸单体生产菌及其构建方法与乳酸制造技术 - Google Patents

一种聚合级乳酸单体生产菌及其构建方法与乳酸制造技术 Download PDF

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WO2016184044A1
WO2016184044A1 PCT/CN2015/093686 CN2015093686W WO2016184044A1 WO 2016184044 A1 WO2016184044 A1 WO 2016184044A1 CN 2015093686 W CN2015093686 W CN 2015093686W WO 2016184044 A1 WO2016184044 A1 WO 2016184044A1
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lactic acid
fermentation
strain
acid
pldha
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French (fr)
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王正祥
田康明
牛丹丹
路福平
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天津科技大学
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Priority to EP15892420.9A priority patent/EP3299449B1/en
Publication of WO2016184044A1 publication Critical patent/WO2016184044A1/zh
Priority to US15/818,724 priority patent/US10472654B2/en

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    • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus

Definitions

  • the invention relates to the field of microbial application, in particular to a polymerization grade lactic acid monomer producing bacteria, a construction method thereof and a lactic acid manufacturing technology.
  • Biodegradable materials are a class of novel materials whose waste after use can be degraded and utilized by environmental (micro)organisms.
  • a new generation of biodegradable materials, represented by polylactic acid is classified into poly-L-lactic acid and poly-D-lactic acid, which are obtained by polymerizing monomer L-lactic acid or D-lactic acid, respectively. It is estimated that by 2020, the annual global demand for polylactic acid will reach 15 million tons, and it is a polyethylene terephthalate (PET) and polystyrene (PS) with an annual demand of 50 million tons. The most likely alternative.
  • PET polyethylene terephthalate
  • PS polystyrene
  • the poly-D-lactic acid material which is polymerized with high-quality D-lactic acid can replace the fiber, plastic and other products which are polymerized from common chemical products.
  • high-end consumer goods such as diaper internal gaskets, cigarette filters and other materials processing and polymerization
  • its unique biomass material properties, biocompatibility and non-toxic and harmless properties greatly improve the quality of related products,
  • the market space is huge.
  • the new products made of poly D-lactic acid using 3D printing technology have many advantages such as environmental protection, good mechanical performance and safety, and are widely used in automobiles, disposable products, electronics, medical and other fields.
  • PLA poly-lactic acid
  • L-lactic acid copolymerized with the same biodegradable butylene succinate-butylene adipate The blend of PBSA can greatly increase the strength and toughness of biodegradable materials and expand the application fields of related products.
  • BASF and other companies have completed the trial production of PLA and PBSA blended products, and successfully launched a series of new biodegradable materials with excellent performance.
  • the related progress has greatly increased the market demand for extremely high optical pure D-lactic acid and L-lactic acid. .
  • coli with multiple genetic modification can synthesize lactic acid with extremely high optical purity and very high chemical purity, and the cycle of recombinant Escherichia coli for lactic acid fermentation is more reorganized due to the significantly higher culture temperature of E. coli than yeast. Yeast is greatly shortened. Therefore, recombinant E. coli is considered to be the production scale at the industrial scale. High optical purity D-lactic acid and L-lactic acid are the most ideal strains.
  • Escherichia coli has been widely recognized as a D-lactic acid producing strain.
  • E. coli can also be used for the fermentative production of high optical purity L-lactic acid, it is of great significance for the alternate production of D-lactic acid and L-lactic acid on the same production line. And it also contributes to the rapid development of high-quality lactic acid monomer and polylactic acid manufacturing.
  • the technical problem to be solved by the present invention is to provide a polymerization grade lactic acid monomer producing bacterium.
  • Another technical problem to be solved by the present invention is to provide the above-mentioned polymerization grade lactic acid monomer producing bacteria. Construction method.
  • Another technical problem to be solved by the present invention is to provide a lactic acid manufacturing technique using the above-mentioned polymerization grade lactic acid monomer producing bacteria, specifically, a combination of the characteristics of the metabolic growth and acid production process of the strain and the characteristics of industrial scale production.
  • Low-cost and easy-to-implement microbial method Highly efficient optically pure lactic acid monomer manufacturing technology.
  • the technical solution of the present invention is:
  • a polymerization grade lactic acid monomer-producing bacterium for fermentative production of extremely high optical pure D-lactic acid the strain number is CGMCC No. 11059 (the strain having the strain number B0013-090B in the examples).
  • a polymerization grade lactic acid monomer-producing bacterium for fermentative production of extremely high optical pure L-lactic acid the strain number is CGMCC No. 11060 (the strain having the strain number B0013-101J in the examples).
  • the above-mentioned polymerization grade lactic acid monomer producing bacteria for fermenting extremely high optical pure D-lactic acid and the polymerization grade lactic acid monomer producing bacteria for fermenting extremely high optical pure L-lactic acid respectively have the formation of extremely high optical purity D-lactic acid
  • the optical purity of L-lactic acid is extremely high, the optical purity can be higher than 99.9%, and the optical purity characteristics of the D-lactic acid and L-lactic acid can meet the highest requirements for the optical purity of the lactic acid monomer in the high-quality polylactic acid polymerization process. .
  • the above-mentioned polymerization grade lactic acid monomer producing bacteria for fermentative production of extremely high optical pure D-lactic acid and the polymerization grade lactic acid monomer producing bacteria for fermentative production of extremely high optical pure L-lactic acid respectively have high chemical purity D-lactic acid and The chemical purity of high-purity L-lactic acid can be higher than 99%.
  • the chemical purity characteristics of the D-lactic acid and L-lactic acid can be satisfied by a simple subsequent treatment or even no treatment. The highest requirements for body chemical purity.
  • Single or multiple genes are knocked out for preliminary construction of strains including: ldhA, thiE, dld, ackA, pta, pps, pflB, poxB, frdA, adhE, lldD; single or multiple genes are expressed, including :kan-cIts857-pR-pL-ldhA, ldhBcoa, ldhLca, ldhStrb;
  • the temperature-inducible gene transcription method is used to control and regulate the cell growth process and the lactic acid formation process, including: after the initial construction of the strain, the strain completes the cell fermentation culture in stages at 25-50 ° C - induction - acid production, cell growth process
  • the accumulation of cells can be quantitatively controlled under the control of a single fermentation factor, which is a regulator of the transcription process of key enzymes in the central metabolic pathway of the cell, a regulator of key enzyme translation processes, a regulator of key enzyme secretion processes, or a key enzyme. Regulatory process regulatory factors after expression.
  • the quantitative control of the accumulated amount of the strain can be accurately controlled by adding a certain amount of a single fermentation factor under different fermentation systems according to the actual demand of the production process.
  • the growth process of the strain after initial construction of the strain is associated with the regulation of a single fermentation factor, which may be a carbon source such as glucose or glycerin; Is a unit such as yeast extract, peptone, ammonium sulfate, ammonium hydrogen phosphate; or metal ions such as iron ions, magnesium ions, calcium ions, zinc ions, manganese ions, cobalt ions, copper ions, sodium ions, potassium ions; It can be a nutrient element, such as VB1, VB6, VB12, biotin, thiamine hydrochloride, thiamine pyrophosphate, etc., especially one of the single fermentation factors can quantitatively control the accumulation process of cells and ensure the rapid cell Accumulated and highly active; the lactic acid synthesis process can be initiated or shut down in a switch-controlled manner, and this process can be accomplished by pre-quantitatively
  • a lactic acid manufacturing technology is specifically applied to: applying the above-mentioned polymerization grade lactic acid monomer producing bacteria, wherein the host cell growth process of the strain is rapidly carried out in a fully synthetic inorganic salt medium, wherein the fully synthetic inorganic salt medium is A thiamine-containing medium in which the host cells rapidly grow in a total synthetic medium, wherein the amount of the cells accumulated to a dry weight of 11.5 g/L within 8-10 h of the 7 L fermentation system, and the single addition is quantitatively added. The fermentation factor and the dry weight of the cells are higher accumulated.
  • the lactic acid is not formed in a large amount during the cultivation of the host cell, that is, the trace formation of lactic acid does not affect the cell growth, and the host cell rapidly accumulates lactic acid after the growth is completed.
  • the accumulation process of D-lactic acid and L-lactic acid is carried out by switching the corresponding polymerization grade lactic acid monomer producing bacteria under the same production system.
  • the main component of the fully synthetic inorganic salt medium is as follows:
  • Fermentation medium (g/L) for D-lactic acid fermentation production diammonium phosphate 0-25, potassium dihydrogen phosphate 0-5, disodium hydrogen phosphate 0-25, sodium chloride 0-5, MgSO4 0 -0.5, FeSO4 0-1, FeCl3 0-1, CoCl2 0-1, CuCl2 0-1, Na2MoO4 0-1, H3BO3 0-1, MnCl2 0-1, citric acid 0-25, thiamine 0-1 , xylose 0-50, glycerol 0-50, glucose 0-50, sulfuric acid 0-5, pH 6.0-7.5.
  • Fermentation medium (g/L) for L-lactic acid fermentation production diammonium phosphate 0-25, potassium dihydrogen phosphate 0-5, disodium hydrogen phosphate 0-25, sodium chloride 0-5, MgSO4 0 -0.5, FeSO4 0-1, FeCl3 0-1, CoCl2 0-1, CuCl2 0-1, ZnCl2 0-1, Na2MoO4 0-1, H3BO3 0-1, MnCl2 0-1, citric acid 0-25, sulfur Amine 0-1, xylose 0-50, glycerol 0-50, glucose 0-50, sulfuric acid 0-5, pH 6.0-7.5.
  • the fermentation acid production process has a settable automatic start characteristic, and the combination of the culture medium components and the growth characteristics of the strains automatically increases the content of the corresponding regulatory factor components in the medium after the cells grow to a specific stage. Start the acid production process.
  • the growth process of the strain and the lactic acid formation process are all related to the process of the fermentation temperature, and the fermentation acid production process is completed at a non-fixed temperature, and the fermentation temperature is based on the acidogenic characteristics of the production strain.
  • the change shows a trend of increasing gradient, and in a certain combination, the temperature has the most significant effect on the acid production process, and the acid production efficiency is the highest.
  • the fermentation temperature will increase slowly, while the fermentation temperature will decrease very rapidly. It has the characteristics of accumulating highly active cells; the lactic acid will form rapidly when the fermentation temperature is increased, and the lactic acid will slowly form or not form at the fermentation temperature, and it has the characteristics of accumulating lactic acid efficiently.
  • the strain is rapidly grown by using glucose at 25-36 ° C to form a bacterial cell, and then rapidly accumulating lactic acid by using glucose at 37-50 ° C.
  • the transcription of D-lactic acid and L-lactic acid synthesis key enzyme encoding genes ldhA and BcoaLDH is strongly inhibited; at higher temperatures, such as 37-50 ° C, D -
  • the transcription of the key enzyme encoding genes ldhA and BcoaLDH of lactic acid and L-lactic acid synthesis is strongly activated.
  • the temperature at which the strain grows and the temperature at which lactic acid is formed may be a continuous process at a single temperature or a plurality of temperature points. Gradient combination process. A combination of multiple temperature point gradients is preferred.
  • the specific steps are:
  • the acid-producing strains were obtained by dynamic regulation of the expression of the lactate dehydrogenase-encoding gene on the chromosomes of D-lactic acid and L-lactic acid high-yield recombinant strains by genetic engineering techniques;
  • strains were grown aerobic at 25-36 ° C, 200 r / min for 6-10 h, and then the fermented lactic acid was statically cultured at 37-45 ° C, and the cell strain was analyzed by using the starting strain B0013-070 as a control strain. Sugar consumption, lactic acid yield, major intermediates of metabolism and other organic acid products, etc., to determine the timing of lactic acid synthesis induction; strains were added to 0.06-100 ⁇ g / L thiamine, 200r / min for shake flask culture;
  • the above lactic acid manufacturing technology further comprises an extraction method of lactic acid after fermentation, combined with production
  • the characteristics of the strain after genetic engineering, D-lactic acid and L-lactic acid in the fermentation broth are in the form of extremely high optical purity and chemical purity.
  • the use of the whole synthetic medium ensures the simple extraction process, and the final product.
  • the extraction method includes acidification, removing the cells by the plate frame, removing the pigment and the impurity protein by ultrafiltration, removing the interference between the anion and the cation by ion exchange, preparing the product of the corresponding concentration by concentration, and purifying the product by the nanofiltration.
  • D-lactic acid and L-lactic acid are released by low-temperature acidification, and the lactic acid free process is not affected by other residues in the fermentation liquid, and acidification is used.
  • the acid may be sulfuric acid, hydrochloric acid or oxalic acid, and more preferably sulfuric acid.
  • the above-mentioned lactic acid manufacturing technology the process of producing highly optically pure D-lactic acid and L-lactic acid by fermentation is not more than 30-36 hours, and the levels of D-lactic acid and L-lactic acid are 150 g/L and 180 g/L or more, respectively.
  • the optical purity of D-lactic acid and L-lactic acid is above 99.95, and the chemical purity is above 97%.
  • the above lactic acid manufacturing technology also includes other chemicals having a similar reaction process, such as citric acid, formic acid, acetic acid, pyruvic acid, succinic acid, malic acid, ⁇ -ketoglutaric acid, succinic acid, adipic acid, pentane.
  • other chemicals having a similar reaction process such as citric acid, formic acid, acetic acid, pyruvic acid, succinic acid, malic acid, ⁇ -ketoglutaric acid, succinic acid, adipic acid, pentane.
  • organic acids and organic amines such as diamine, hexamethylenediamine, methacrylic acid, isoprene, itaconic acid; or valine, alanine, lysine, methionine, glutamic acid, arginine And a variety of amino acids; thiamine, vitamin B12 and other microorganisms; or short-chain alcohols such as ethanol, propanol; or oligo-isomaltose, oligofructose, galacto-oligosaccharides and other functional sugars.
  • organic acids and organic amines such as diamine, hexamethylenediamine, methacrylic acid, isoprene, itaconic acid; or valine, alanine, lysine, methionine, glutamic acid, arginine And a variety of amino acids; thiamine, vitamin B12 and other microorganisms; or short-chain alcohols such as ethanol, propanol; or oli
  • the present application introduces a temperature regulating element at the level of gene transcription as described in ZL201210102731.8, and cooperates with the fermentation temperature regulation strategy to greatly improve the synthesis efficiency of D-lactic acid, and introduces a quantitative control mechanism for the growth of the cell body to form a mechanism.
  • the double-switching mechanism of the cell growth process and the D-lactic acid synthesis process further improves the D-lactic acid synthesis efficiency; the extremely high optical pure L-lactic acid producing bacteria in the present invention make full use of the exogenous L-lactate dehydrogenase translation After the expression of the enzymatic properties, and introduced the temperature regulation mechanism of its catalytic activity, combined with the regulation mechanism of quantitative control of cell growth, the double-switch control of cell growth and L-lactic acid synthesis was completed.
  • the extremely high optical pure D-lactic acid production process and the extremely high optical pure L-lactic acid production process in the present invention can complete the mass production of two products under the same production system by simple switching of strains. .
  • the recombinant bacteria provided by the present invention have the remarkable ability to efficiently synthesize extremely high optical purity D-lactic acid and L-lactic acid, and the above-mentioned recombinant bacteria have the ability to synthesize high chemical purity D-lactic acid and L-lactic acid.
  • the cells grow rapidly under the condition of 25-50 °C for 8-10 h, and the acid is fermented for 16-18 h, and the D-lactate level is reached. 15% (w/v) or more, the L-lactic acid production level reaches 18% (w/v) or more;
  • the highly efficient synthesis of extremely high optical pure lactic acid monomer genetic engineering bacteria of the invention can be effectively controlled by temperature change during cell growth and fermentation acid production;
  • the highly efficient synthesis of extremely high optical pure lactic acid monomer genetic engineering bacteria of the invention can be quantitatively controlled and controlled by the addition of nutrient elements in the process of cell growth and fermentation acid production.
  • High-efficiency manufacturing process of the extremely high optical pure lactic acid monomer of the invention the cell growth temperature of the cells is rapidly grown by using glucose at 25-36 ° C for 6-12 h to form a bacterial cell; and the rapid synthesis of glucose is performed at 37-50 ° C; High optical purity of D-lactic acid and L-lactic acid. Namely: using the recombinant bacteria of the invention and the preparation process of the extremely high optical pure lactic acid monomer, the production process only needs to change the fermentation temperature control parameter and cooperate with the addition of nutrient elements to realize extremely high optical pure D-lactic acid and L-lactic acid. Efficient preparation process.
  • Figure 1 shows the physical map of the mutant cassette plasmid pUC-ldhAp::kan-cIts857-pR-pL;
  • Figure 2 is a physical map of the mutant cassette plasmid pUC-thiE'::difGm;
  • Figure 3 is a physical map of the mutant cassette plasmid pUC-dld'::difGm;
  • Figure 4 is a physical map of the mutant cassette plasmid pUC-ackA-pta'::difGm;
  • Figure 5 is a physical map of the mutant cassette plasmid pUC-pps'::difGm;
  • Figure 6 is a physical map of the mutant cassette plasmid pSK-pflB'::difGm;
  • Figure 8 is a physical map of the mutant cassette plasmid pSKsym-frdA'::difGm;
  • Figure 10 is a physical map of the mutant cassette plasmid pUC-ldhA::difGm;
  • Figure 11 is a physical map of the mutant cassette plasmid pUC-lldD'::PldhA-ldhBcoa-difGm;
  • Figure 12 is a physical map of the mutant cassette plasmid pUC-lldD'::PldhA-ldhLca-difGm;
  • Figure 13 is a physical map of the mutant cassette plasmid pUC-lldD'::PldhA-ldhStrb-difGm,
  • Figure 14 is a physical map of the mutant cassette plasmid pUC-ldhA'::PldhA-ldhLca-difGm;
  • Figure 15 is a physical map of the mutant cassette plasmid pUC-ldhA'::PldhA-ldhStrb-difGm;
  • Figure 16 is a physical map of the mutant cassette plasmid pUC-thiE'::PldhA-ldhLca-difGm;
  • Figure 17 is a physical map of the mutant cassette plasmid pUC-thiE'::PldhA-ldhStrb-difGm.
  • Figure 18 Physical map of the mutant cassette plasmid pUC-thiE'::PldhA-ldhBcoa-difGm.
  • Figure 21 shows the process of very high optical pure D-lactic acid fermentation
  • Figure 23 is a very high optical pure L-lactic acid fermentation process
  • Classification noun Escherichia coli
  • Classification noun Escherichia coli
  • the invention provides an ultra-high optical pure D-lactic acid and a very high optical pure L-lactic acid high-yield strain and a construction method thereof, and an extremely high optical pure D-lactic acid and an extremely high optical pure L-lactic acid high-efficiency preparation process, and the bacterial body is in 25- Highly active cells were formed by rapid growth of glucose at 36 ° C, and then extremely high optical pure D-lactic acid and extremely high optical pure L-lactic acid were efficiently accumulated at 37-50 ° C, and the chemical purity of the accumulated lactic acid was extremely high.
  • the process characteristics are: under the condition of 25-50 ° C, respectively, the cells are cultured by fermentation, and the temperature is high and the acid production stage is reached, and the D-lactic acid level is 15% (w/v) or more, and the optical purity is over 99.9%, and the chemical purity is obtained. 97% or more; L-lactic acid production level of 18% (w / v) or more, optical purity of 99.9% or more, chemical purity of 98% or more.
  • Chromosomal gene integration technology amplification from E. coli genome using PCR (polymerase chain reaction)
  • the 50-700 bp gene sequences upstream and downstream of the chromosomal target integration site were obtained.
  • the target integrated expression gene is ligated to the resistance gene, and the obtained fragment is cloned between the upstream and downstream gene sequences of the above-mentioned target integration site to form a target gene integration sequence, such as ldhA::kan-cIts857-pR-pL, thiE'::difGm,dld'::difGm, ackA-pta'::difGm,pps'::difGm,pflB'::difGm,poxB'::difGm,frdA'::difGm,adhE'::difGm, ldhA'::difGm,lldD'::PldhA-
  • the above gene integration sequences are transformed into E. coli either alone or in combination or in multiple combinations. Transformants are selected for culture on selective media. The transformant chromosomal DNA was extracted, and the target gene mutation of the transformant was verified by PCR. The fermentation test screens the optimal ultra-high optical pure D-lactic acid high-yield bacteria such as B0013-090B and extremely high optical pure L-lactic acid high-yield bacteria such as B0013-101J.
  • the promoter ldhAp of the lactate dehydrogenase gene in the starting strain obtained in step 1 was replaced with the pR-pL promoter (Love CA et al., Gene, 1996, 176: 49-53). And obtained recombinant E. coli 1 .
  • thiE thienin phosphate synthase
  • the D-lactate dehydrogenase (ldhA)-encoding gene deletion cassette ldhA'::difGm was constructed, and the ldhA gene of the recombinant B0013-070 was deleted to obtain recombinant Escherichia coli 67.
  • the D-lactate dehydrogenase gene (ldhA) encoding gene deletion cassette ldhA'::PldhA-ldhLca-difGm was constructed, and the ldhA gene of the recombinant B0013-070 was deleted to obtain recombinant Escherichia coli 68.
  • the D-lactate dehydrogenase gene (ldhA) encoding gene deletion mutation cassette ldhA'::PldhA-ldhStrb-difGm was constructed, and the ldhA gene of the recombinant B0013-070 was deleted to obtain recombinant Escherichia coli 69.
  • step 14 Using gene integration technology to construct FML coenzyme L-lactate dehydrogenase gene (lldD) encoding gene deletion mutation cassette lldD'::PldhA-ldhBcoa-difGm, delete recombinant bacteria B0013-070 and step 11, step 12 and The lldD gene of the recombinant strain obtained in step 13 was obtained into recombinant Escherichia coli 70-73.
  • lldD FML coenzyme L-lactate dehydrogenase gene
  • step 15 Using gene integration technology to construct FML coenzyme L-lactate dehydrogenase gene (lldD) encoding gene deletion mutation cassette lldD'::PldhA-ldhLca-difGm, delete recombinant bacteria B0013-070 and step 11, step 12 and The lldD gene of the recombinant strain obtained in step 13 was obtained into recombinant Escherichia coli 74-77.
  • lldD FML coenzyme L-lactate dehydrogenase gene
  • step 16 using gene integration technology to construct FML coenzyme L-lactate dehydrogenase gene (lldD) encoding gene deletion mutation cassette lldD'::PldhA-ldhLca-difGm, delete recombinant bacteria B0013-070 and step 11, step 12 and The lldD gene of the recombinant strain obtained in step 13 was obtained into recombinant Escherichia coli 78-81.
  • lldD FML coenzyme L-lactate dehydrogenase gene
  • the thiamine phosphate synthase (thiE) encoding gene deletion mutation cassette thiE'::difGm, deleting step 11, step 12, step 13, step 14, step 15 and step 16 obtained recombinant bacteria
  • the thiE gene was obtained as a recombinant strain 82-96.
  • thiamine phosphate synthase encoding gene deletion cassette thiE'::PldhA-ldhLca-difGm, delete the thiE gene of the recombinant strain obtained in step 11, step 12, step 13, step 14, step 15 and step 16 to obtain recombinant strain 97-111.
  • the thiamine phosphate synthase (thiE) encoding gene deletion mutation cassette thiE'::PldhA-ldhStrb-difGm, deleting step 11, step 12, step 13, step 14, step 15 and step 16
  • the thiE gene of the recombinant strain was obtained, and the recombinant strain 112-126 was obtained.
  • thiamine phosphate synthase encoding gene deletion mutation cassette thiE'::PldhA-ldhBcoa-difGm, deleting step 11, step 12, step 13, step 14, step 15 and step 16 Recombinant thiE gene was obtained, and recombinant strain 127-156 was obtained.
  • step 21 will be step 4, step 5, step 6, step 7, step 8, step 9, step 10, step 11, step 12, step 13, step 14, step 15, step 16, step 17, step 18, step 19 and
  • the recombinant strain obtained in step 20 was shake-cultured at 30 ° C, 45 ° C, and 200 r/min, and the starting strain B0013-070 was used as a control strain to analyze the specific activity of lysine dehydrogenase to identify pR-pL. Promoter and thiE functions, and screen out the optimal strain.
  • step 22 The optimal strains obtained in step 21 were shake-cultured at 25-36 ° C and 200 r/min, respectively, and the starting strain B0013-070 was used as a control strain to analyze cell density, lactic acid, major intermediates of metabolism and Other organic acid products, etc., determine the growth temperature of the cells.
  • step 21 The optimal strain obtained in step 21 was grown at 25-36 ° C, 200 r / min aerobic for 10 h, and then the fermented lactic acid was statically cultured at 37-45 ° C, and the starting strain B0013-070 was used as the control strain. Analysis of cell density, sugar consumption, lactic acid yield, metabolism of major intermediates and other organic acid products, etc., to determine the timing of lactic acid synthesis induction.
  • the optimal strains obtained by the optimal steps were shaken by adding 0.06-100 ⁇ g/L thiamine and 200r/min, and the cell strain and lactic acid were analyzed by using the starting strain B0013-070 as the control strain. Metabolism of major intermediates and other organic acid products, etc., to determine the amount of thiamine added by the cells.
  • the optimal strain obtained in the step 20 was subjected to a lactic acid fermentation test in a 7L-30,000L fermentor. Timed sampling during fermentation, analysis of cell density, sugar consumption, lactic acid yield, metabolism of major intermediates and other organic acid products.
  • the ldhA gene fragment ldhA' on the B0013 chromosome was PCR-amplified with the primers ldhA1 and ldhA2, and cloned into the pUC18 vector to obtain a recombinant plasmid pUC-ldhA'.
  • Plasmid pPL451 with primers PPL1 and PPL2 Reverse PCR amplification was carried out, and the kanamycin resistance gene fragment (the plasmid pSKsymKm was digested with SmaI, and the 666 bp fragment was recovered by gel) was ligated to obtain a recombinant plasmid pPL-Kan.
  • the plasmid pPL-Kan was amplified by PCR with primers PPL3 and PPL4, and the product was digested with EcoRI and EcoRV, and inverted PCR amplification with primers Ec-RlA1 and Ec-RlA2 using pUC-ldhA' as template.
  • the EcoRI was digested with the digested product to obtain the recombinant plasmid pUC-ldhAp::kan-cIts857-pR-pL.
  • the physical map of the resulting recombinant plasmid is shown in Figure 1.
  • the recombinant plasmid pUC-ldhAp::kan-cIts857-pR-pL was digested with KpnI, and the linearized plasmid was recovered by gel, and amplified by PCR with primers ldhA1 and ldhA2 to obtain the ldhAp::kan-cIts857-pR-pL gene fragment. .
  • a partial sequence of the thiE gene with a length of 0.6 kb was obtained by using E. coli B0013 chromosomal DNA as a template and ThiE1p and ThiE2p as primers. This was cloned into pUC18 to obtain the recombinant plasmid pUC-thiE. The 1.2 kb difGm fragment was cloned into the StuI site in the middle of thiE to obtain the recombinant plasmid pUC-thiE::difGm. The physical map of the resulting recombinant plasmid is shown in Figure 2.
  • the recombinant plasmid pUC-thiE::difGm was digested with ApaLI, and the linearized plasmid was recovered by gel, and PCR amplification was carried out with primers ThiE1p and ThiE2p to obtain a thiE::difGm gene fragment.
  • the dld' gene fragment (0.9 kb) was amplified by PCR with primers Dld1 and Dld2, and the PCR product was cloned into the vector pUC18SmaI site to obtain a recombinant plasmid pUC-dld'.
  • the recombinant plasmid was digested with EcoRV, and the 0.4 kb gene fragment of the middle of the dld' gene was removed and cloned into a dif-Gm-dif fragment to obtain a recombinant plasmid pUC-dld'::difGm.
  • the physical map of the resulting recombinant plasmid is shown in Figure 3.
  • the recombinant plasmid pUC-dld'::difGm was digested with EcoRI, and the linearized plasmid was recovered by gel, and PCR amplification was carried out using primers Dld1 and Dld2 to obtain a dld'::difGm gene fragment.
  • the ackA-pta' gene fragment (2.8 kb) was amplified by PCR with primers AckA-Pta1 and AckA-Pta2, and the PCR product was cloned into vector pUC18 to obtain recombinant plasmid pUC-ackA-pta'.
  • the recombinant plasmid was digested with EcoRV, and the 2.6 kb gene fragment of the ackA-pta' gene was removed and cloned into a dif-Gm-dif fragment to obtain a recombinant plasmid pUC-ackA-pta'::difGm.
  • the physical map of the resulting recombinant plasmid is shown in Figure 4.
  • the recombinant plasmid pUC-ackA-pta'::difGm was digested with KpnI, and the linearized plasmid was recovered by gel, and PCR amplification was carried out using primers AckA-Pta1 and AckA-Pta2 to obtain a ackA-pta'::difGm gene fragment.
  • the pps gene fragment (2.3 kb) was amplified by PCR using the primer Pps1 and Pps2, and the PCR product was cloned into the vector pUC18 to obtain the recombinant plasmid pUC-pps'.
  • plasmid pUC-pps' as a template, reverse PCR amplification was performed with primers RPps1 and RPps2, and the 2.1 kb gene fragment of pps' gene was removed and cloned into dif-Gm-dif fragment to obtain recombinant plasmid pUC-pps':: difGm.
  • the physical map of the resulting recombinant plasmid is shown in Figure 5.
  • the recombinant plasmid pUC-pps'::difGm was digested with ApaLI, and the linearized plasmid was recovered by gel, and PCR amplification was carried out using primers Pps1 and Pps2 to obtain a pps'::difGm gene fragment.
  • the physical map of the resulting recombinant plasmid is shown in Figure 6.
  • the recombinant plasmid pSK-pflB'::difGm was digested with ApaLI, and the linearized plasmid was recovered by gel, and PCR amplification was carried out using primers PflB1 and PflB2 to obtain a pflB'::difGm gene fragment.
  • the poxB gene fragment of Escherichia coli was amplified with the primers PoxB1 and PoxB2, and the PCR product was cloned into the SmaI restriction site of the plasmid pUC18 to obtain the recombinant plasmid pUC-poxB'.
  • the recombinant plasmid was digested with EcoRV and cloned into a dif-Gm fragment to obtain a recombinant plasmid pUC-poxB'::difGm.
  • the physical map of the resulting recombinant plasmid is shown in Figure 7.
  • the recombinant plasmid pUC-poxB'::difGm was digested with ApaLI, and the linearized plasmid was recovered by gel, and PCR amplification was carried out with primers PoxB1 and PoxB2 to obtain a poxB'::difGm gene fragment.
  • the frdA' gene fragment (1.6 kb) was amplified by PCR with primers FrdA1 and FrdA2, and the PCR product was cloned into the vector pSKsym SmaI site to obtain a recombinant plasmid pSKsym-frdA'.
  • the recombinant plasmid was digested with PstI to remove the 1.3 kb gene fragment of the frdA' gene, and the sticky end of PstI was smoothed with T4 DNA polymerase, and cloned into the dif-Gm-dif fragment to obtain the recombinant plasmid pSKsym-frdA'::difGm.
  • the physical map of the resulting recombinant plasmid is shown in Figure 8.
  • the recombinant plasmid pSKsym-frdA'::difGm was digested with ApaLI, and the linearized plasmid was recovered by gel, and PCR amplification was carried out using the primers FrdA1 and FrdA2 to obtain a frdA'::difGm gene fragment.
  • adhE' was amplified by PCR with primers AdhE1 and AdhE2.
  • the gene fragment (1.4 kb) was cloned into the vector pUC19SmaI site to obtain a recombinant plasmid pUC-adhE'.
  • the recombinant plasmid was digested with EcoRV, and the 1 kb gene fragment of the middle of the adhE' gene was removed and cloned into a dif-Gm-dif fragment to obtain a recombinant plasmid pUC-adhE'::difGm.
  • the physical map of the resulting recombinant plasmid is shown in Figure 9.
  • the recombinant plasmid pUC-adhE'::difGm was digested with EcoRI and PstI to recombine the recombinant plasmid pUC-adhE'::difGm, and the adhE1-dif-Gm-dif-adhE2 gene fragment was recovered by gel.
  • the chromosomal DNA of Escherichia coli B0013 was used as a template, ldhA3 and ldhA4 were used as primers, and the lactate dehydrogenase gene (ldhA) was obtained by PCR amplification.
  • the PCR product was cloned into the EcoRI site of pUC18 to obtain the recombinant plasmid pUC-ldhA.
  • the 400 bps fragment was digested with PstI and the cohesive ends were filled in with T4 DNA polymerase, and ligated with the difGm fragment to obtain the recombinant plasmid pUC-ldhA::Gmdif.
  • the physical map of the resulting recombinant plasmid is shown in Figure 10.
  • the recombinant plasmid was digested with EcoRI to obtain a gene deletion sequence of lactate dehydrogenase, ldhA'-dif-Gm-dif-ldhA, namely: ldhA::Gmdif.
  • E.coli CICIM B0013 chromosomal DNA as a template, amplify (primer ldhA5 and ldhA6) to obtain all promoters and partial structural region fragments of ldhA gene, and clone ldhA' into subcloning vector pUC18 to obtain recombinant plasmid pUC-ldhA' .
  • Reverse PCR amplification was carried out using primers RldhA1 and RldhA12 with pUC-ldhA' as a template, and the product was self-ligated to obtain a recombinant plasmid pUC-PldhA.
  • the Bclillus coagulans CICIM B1821 chromosomal DNA was used as a template for PCR amplification (primers BcoaLDH1 and BcoaLDH4).
  • the ldhBcoa gene fragment was digested with BamHI and EcoRI and cloned into BglII and EcoRI of plasmid pUC-PldhA' to obtain recombinant plasmid pUC-PldhA- ldhBcoa.
  • the difGm fragment was then cloned into the EcoRV site of the recombinant plasmid pUC-PldhA-ldhBcoa.
  • the recombinant plasmid pUC-PldhA-ldhBcoa-difGm was obtained.
  • the lldD gene fragment was obtained by PCR amplification (primers lldD1 and lldD2), and the fragment was cloned into the subcloning vector pUC18 to obtain a recombinant plasmid pUC-lldD.
  • the recombinant plasmid pUC-PldhA-ldhBcoa-difGm was digested with BamHI to obtain the PldhA-ldhBcoa-difGm fragment cloned into the BamHI site of the recombinant plasmid pUC-lldD.
  • the recombinant plasmid pUC-lldD::PldhA-ldhBcoa-difGm was obtained.
  • the physical map of the resulting recombinant plasmid is shown in Figure 11.
  • the recombinant plasmid pUC-lldD::PldhA-ldhBcoa-difGm was digested with SmaI, and the lldD::PldhA-ldhBcoa-difGm gene fragment was obtained by gel recovery.
  • Amplification of the chromosomal DNA of E.coli CICIM B0013 as a template (primers ldhA5 and ldhA6)
  • the entire promoter and part of the structural region fragment of the ldhA gene were obtained, and the fragment ldhA' was cloned into the subcloning vector pUC18 to obtain the recombinant plasmid pUC-ldhA'.
  • Reverse PCR amplification was carried out using primers RldhA1 and RldhA12 with pUC-ldhA' as a template, and the product was self-ligated to obtain a recombinant plasmid pUC-PldhA.
  • Lactobacillus casei B1192 chromosomal DNA was used as a template for amplification by PCR (primers LcaLDH1 and LcaLDH4).
  • the ldhLca gene fragment was digested with BamHI and EcoRI and cloned into BglII and PstI of plasmid pUC-PldhA' to obtain recombinant plasmid pUC-PldhA-ldhLca. .
  • the difGm fragment was then cloned into the EcoRV site of the recombinant plasmid pUC-PldhA-ldhLca.
  • the recombinant plasmid pUC-PldhA-ldhLca-difGm was obtained.
  • the lldD gene fragment was obtained by PCR amplification (primers lldD1 and lldD2), and the fragment was cloned into the subcloning vector pUC18 to obtain a recombinant plasmid pUC-lldD.
  • the recombinant plasmid pUC-PldhA-ldhLca-difGm was digested with BamHI to obtain the PldhA-ldhLca-difGm fragment cloned into the BamHI site of the recombinant plasmid pUC-lldD.
  • the recombinant plasmid pUC-lldD::PldhA-ldhLca-difGm was obtained.
  • the physical map of the resulting recombinant plasmid is shown in Figure 12.
  • the recombinant plasmid pUC-lldD::PldhA-ldhLca-difGm was digested with SmaI, and the lldD::PldhA-ldhLca-difGm gene fragment was obtained by gel recovery.
  • E.coli CICIM B0013 chromosomal DNA as a template, amplify (primer ldhA5 and ldhA6) to obtain all promoters and partial structural region fragments of ldhA gene, and clone ldhA' into subcloning vector pUC18 to obtain recombinant plasmid pUC-ldhA' .
  • Reverse PCR amplification was carried out using primers RldhA1 and RldhA12 with pUC-ldhA' as a template, and the product was self-ligated to obtain a recombinant plasmid pUC-PldhA.
  • PCR was performed using Steptococcus.bovis 1.1624 chromosomal DNA as a template (primer StrbLDH1 and StrbLDH2).
  • the ldhStrb gene fragment was digested with BamHI and EcoRI and cloned into BglII and EcoRI of plasmid pUC-PldhA' to obtain recombinant plasmid pUC-PldhA- ldhStrb.
  • the difGm fragment was then cloned into the EcoRV site of the recombinant plasmid pUC-PldhA-ldhStrb.
  • the recombinant plasmid pUC-PldhA-ldhBcoa-difGm was obtained.
  • the lldD gene fragment was obtained by PCR amplification (primers lldD1 and lldD2), and the fragment was cloned into the subcloning vector pUC18 to obtain a recombinant plasmid pUC-lldD.
  • the recombinant plasmid pUC-PldhA-ldhBcoa-difGm was digested with BamHI to obtain the PldhA-ldhStrb-difGm fragment cloned into the BamHI site of the recombinant plasmid pUC-lldD.
  • the recombinant plasmid pUC-lldD::PldhA-ldhStrb-difGm was obtained.
  • the physical map of the resulting recombinant plasmid is shown in Figure 13.
  • the recombinant plasmid pUC-lldD::PldhA-ldhStrb-difGm was digested with SmaI and recovered by gel. lldD::PldhA-ldhStrb-difGm gene fragment.
  • the recombinant plasmid pUC-ldhA in Example 10 was digested with PstI to remove the 400 bps fragment and the viscous ends were filled in with pfu polymerase, and the recombinant plasmid pUC-PldhA-ldhLca-difGm in Example 12 was digested with BamHI.
  • the pfu polymerase ligated the cohesive end-filling fragment PldhA-ldhLca-difGm to obtain the recombinant plasmid pUC-ldhA::PldhA-ldhLca-difGm.
  • the physical map of the resulting recombinant plasmid is shown in Figure 14.
  • the recombinant plasmid pUC-ldhA::PldhA-ldhLca-difGm was digested with ApaLI, and the linearized plasmid was recovered by gel, and amplified by PCR with primers ldhA3 and ldhA4 to obtain a ldhA::PldhA-ldhLca-difGm gene fragment.
  • the recombinant plasmid pUC-ldhA in Example 10 was digested with PstI to remove the 400 bp fragment thereof and the viscous ends were filled in with pfu polymerase, and the recombinant plasmid pUC-PldhA-ldhStrb-difGm in Example 13 was digested with BamHI.
  • the pfu polymerase ligated the cohesive end-blunt fragment PldhA-ldhStrb-difGm to obtain the recombinant plasmid pUC-ldhA::PldhA-ldhStrb-difGm.
  • the physical map of the resulting recombinant plasmid is shown in Figure 15.
  • the recombinant plasmid pUC-ldhA::PldhA-ldhStrb-difGm was digested with ApaLI, and the linearized plasmid was recovered by gel, and amplified by PCR with primers ldhA3 and ldhA4 to obtain a ldhA::PldhA-ldhStrb-difGm gene fragment.
  • the recombinant plasmid pUC-PldhA-ldhLca-difGm in Example 12 was digested with BamHI to obtain a PldhA-ldhLca-difGm fragment, and the DNA polymerase pfu was used to fill the fragment PldhA-ldhLca-difGm and cloned into the recombination in Example 2.
  • StuI site of plasmid pUC-thiE The recombinant plasmid pUC-thiE::PldhA-ldhLca-difGm was obtained.
  • the physical map of the resulting recombinant plasmid is shown in Figure 16.
  • the recombinant plasmid pUC-thiE::PldhA-ldhLca-difGm was digested with ApaLI, and the linearized plasmid was recovered by gel, and PCR amplification was carried out with primers ThiE1p and ThiE2p to obtain a thiE::PldhA-ldhLca-difGm gene fragment.
  • the recombinant plasmid pUC-PldhA-ldhStrb-difGm in Example 13 was digested with BamHI to obtain a PldhA-ldhStrb-difGm fragment, and the DNA polymerase pfu was used to fill the fragment ldhA-ldhStrb-difGm and cloned into the recombination of Example 2.
  • StuI site of plasmid pUC-thiE The recombinant plasmid pUC-thiE::PldhA-ldhStrb-difGm was obtained.
  • the physical map of the resulting recombinant plasmid is shown in Figure 17.
  • the recombinant plasmid pUC-thiE::PldhA-ldhStrb-difGm was digested with ApaLI, and the linearized plasmid was recovered by gel. PCR amplification was carried out using the primers ThiE1p and ThiE2p to obtain a thiE::PldhA-ldhStrb-difGm gene fragment.
  • the recombinant plasmid pUC-PldhA-ldhBcoa-difGm in Example 11 was digested with BamHI to obtain a PldhA-ldhBcoa-difGm fragment, and the DNA polymerase pfu was used to fill the fragment PldhA-ldhBcoa-difGm and cloned into the recombination in Example 2.
  • StuI site of plasmid pUC-thiE The recombinant plasmid pUC-thiE::PldhA-ldhBcoa-difGm was obtained.
  • the physical map of the resulting recombinant plasmid is shown in Figure 18.
  • the recombinant plasmid pUC-thiE::PldhA-ldhBcoa-difGm was digested with ApaLI, and the linearized plasmid was recovered by gel, and PCR amplification was carried out with primers ThiE1p and ThiE2p to obtain a thiE::PldhA-ldhBcoa-difGm gene fragment.
  • Example 19 Construction of a temperature-controlled ultra-high optical pure D-lactic acid producing strain
  • Example 20 Construction of a highly quantitative optically pure D-lactic acid producing strain capable of quantitatively regulating growth
  • thiE thienin phosphate synthase
  • Example 21 Construction of D-lactic acid metabolism utilization pathway blocking type extremely high optical pure D-lactic acid producing strain
  • the FAD-dependent D-lactate dehydrogenase (dld) encoding gene deletion mutation cassette dld'::difGm was constructed, and the dld gene of the recombinant strain obtained in step 3 was deleted to obtain the recombinant strain Escherichia coli B0013-030B. (B0013-020B dld::dif).
  • Example 22 Construction of a anaerobic acetic acid synthesis pathway blocking type extremely high optical pure D-lactic acid producing strain
  • ackA-pta'::difGm mutation cassette for the deletion of the acetate kinase (ackA) and phosphotransacetylase (pta) encoding gene, and delete the ackA-pta gene of the recombinant strain obtained in step 4 to obtain the recombinant strain.
  • Escherichia coli B0013-040B B0013-030B ackA-pta::dif).
  • Example 23 Construction of a very high optical pure D-lactic acid producing strain blocked by pyruvate reflux pathway
  • the mutation cassette pps'::difGm for the deletion of the phosphoenolpyruvate synthase (pps) gene, and delete the pps gene of the recombinant strain obtained in steps 4 and 5 to obtain the recombinant strain Escherichia coli B0013. -040C(B0013-030B pps::dif) and B0013-050B(B0013-040B Pps::dif).
  • Example 24 Construction of a very high optical pure D-lactic acid producing strain for blocking anaerobic synthesis pathway of formic acid
  • pflB pyruvate formate lyase
  • pflB'::difGm delete the pflB gene of the recombinant strain obtained in step 4, step 5 and step 6, and obtain the recombinant strain Escherichia coli B0013 -040D(B0013-030B pflB::dif), B0013-050C(B0013-040B pflB::dif), B0013-050D(B0013-040C pflB::dif) and B0013-060B(B0013-050B pflB::dif) .
  • Example 25 Construction of a very high optical pure D-lactic acid producing strain blocked by an aerobic synthesis pathway of acetic acid
  • Example 26 Construction of a succinic acid synthesis pathway blocking type extremely high optical pure D-lactic acid producing strain
  • Example 27 Construction of a very high optical pure D-lactic acid producing strain blocked by an ethanol synthesis pathway
  • Example 29 Construction of a D-lactic acid synthesis pathway blocking type extremely high optical pure L-lactic acid producing strain
  • Example 30 Construction of a D-lactic acid synthesis pathway blocking type extremely high optical pure L-lactic acid producing strain
  • Example 31 Construction of a very high optical pure L-lactic acid producing strain blocked by L-lactic acid metabolism utilization pathway
  • the lldD gene of the recombinant strains B0013-070, B0013-080H, B0013-080I and B0013-080J was deleted by deleting the mutant cassette lldD'::PldhA-ldhBcoa-difGm, and the recombinant strain Escherichia coli B0013-080K (B0013-070lldD) was obtained.
  • Example 32 Construction of a very high optical pure L-lactic acid producing strain blocked by L-lactic acid metabolism utilization pathway
  • the L-lactate dehydrogenase gene (lldD) encoding the gene encoding FMN was cloned into a mutant cassette lldD'::PldhA-ldhLca-difGm, and the recombinant bacteria B0013-070, B0013-080H, B0013-080I were deleted.
  • B0013-080J obtained the recombinant strain Escherichia coli B0013-080L (B0013-070lldD::PldhA-ldhLca-dif), B0013-090F (B0013-080H lldD::PldhA-ldhLca-dif), B0013-090G (B0013-080I lldD::PldhA-ldhLca-dif) and B0013-090H (B0013-080J lldD::PldhA-ldhLca-dif).
  • Example 33 Construction of a very high optical pure L-lactic acid producing strain blocked by L-lactic acid metabolism utilization pathway
  • the L-lactate dehydrogenase gene (lldD) encoding the gene encoding FMN was cloned into a mutant cassette lldD'::PldhA-ldhLca-difGm, and the recombinant bacteria B0013-070, B0013-080H, B0013-080I were deleted.
  • B0013-080J obtained the recombinant strain Escherichia coli B0013-080M (B0013-070lldD::PldhA-ldhStrb-dif), B0013-090I (B0013-080H lldD::PldhA-ldhStrb-dif), B0013-090J (B0013-080I lldD::PldhA-ldhStrb-dif) and B0013-090K (B0013-080J lldD::PldhA-ldhStrb-dif).
  • Example 34 Construction of a highly quantitative optically pure L-lactic acid producing strain capable of quantitatively regulating growth
  • thiE thiamine phosphate synthase
  • Recombinant thiE gene obtained recombinant strain B0013-090P (B0013-080H thiE::PldhA-ldhLca-dif), B0013-090Q (B0013-080I thiE::PldhA-ldhLca-dif), B0013-090R (B0013- 080J thiE::PldhA-ldhLca-dif), B0013-090S (B0013-080K thiE::PldhA-ldhLca-dif), B0013-100K (B0013-090C thiE::PldhA-ldhLca-dif), B0013-100L (B0013 -090
  • Example 36 Construction of a Growth-Quantitatively Regulated Very High Optical Pure L-Lactic Acid Producing Strain
  • Recombinant thiE gene obtained recombinant strain B0013-090U (B0013-080H thiE::PldhA-ldhStrb-dif), B0013-090V (B0013-080I thiE::PldhA-ldhStrb-dif), B0013-090W (B0013- 080J thiE::PldhA-ldhStrb-dif), B0013-090X(B0013-080K thiE::PldhA-ldhStrb-dif), B0013-100T(B0013-090C thiE::PldhA-ldhStrb-dif),B0013-100U(B0013 -090
  • the recombinant strains obtained in steps 19-27 were separately grown with 0.06-100 ⁇ g/L thiamine, 25-36 ° C, 200 r/min aerobic growth for -10 h, and then the fermented lactic acid was statically cultured at 37-50 ° C, and Using the starting strain B0013-070 as the control strain, the D-lactic acid synthesis level, the optical purity and chemical purity of the produced D-lactic acid were analyzed, and the optimal strain was screened. The screening results are shown in Figure 19 and Table 1.
  • the recombinant strains obtained in steps 28-37 were separately grown with 0.06-100 ⁇ g/L thiamine, 25-36 ° C, 200 r/min aerobic growth for -10 h, and then the fermented lactic acid was statically cultured at 37-50 ° C, and Using the starting strain B0013-070 as the control strain, the L-lactic acid synthesis level, the optical purity and chemical purity of the produced L-lactic acid were analyzed, and the optimal strain was screened. The screening results are shown in Figures 20 and 2.
  • Example 38 The optimum strain obtained in Example 38 was subjected to a lactic acid fermentation test in a 7L-30,000L fermentor. Timed sampling during fermentation, analysis of cell density, sugar consumption, lactic acid yield, metabolism of major intermediates and other organic acid products. Perform aerobic culture at 25-36 °C until the OD600 value is about 15-40, set the temperature of the fermenter to 37-50 °C, continue aerobic culture for 0-120 min, and then set the aeration amount to 0-0.2 vvm for oxygen limitation.
  • Fermentation, oxygen-limited stage 0-3h fermentation temperature is 33-39 ° C, 3-6h fermentation temperature is 37-42 ° C, 6-10h fermentation temperature is 38-45 ° C, 10-16h fermentation temperature is 40-48 ° C, 16 The fermentation temperature of -24h is 45-50 °C.
  • the fermentation medium is (g/L): diammonium phosphate 0-25, potassium dihydrogen phosphate 0-5, disodium hydrogen phosphate, 0-25, sodium chloride 0-5, MgSO4 0-0.5, FeSO4 0 -1, FeCl3 0-1, CoCl2 0-1, CuCl2 0-1, Na2MoO4 0-1, H3BO3 0-1, MnCl2 0-1, citric acid 0-25, thiamine 0-1, xylose 0- 50, glycerol 0-50, glucose 0-50, sulfuric acid 0-5, pH 6.0-7.5.
  • Example 39 The optimum strain obtained in Example 39 was subjected to a lactic acid fermentation test in a 7L-30,000L fermentor. Timed sampling during fermentation, analysis of cell density, sugar consumption, lactic acid yield, metabolism of major intermediates and other organic acid products. Perform aerobic culture at 25-36 °C until the OD600 value is about 15-40, set the temperature of the fermenter to 37-50 °C, continue aerobic culture for 0-120 min, and then set the aeration amount to 0-0.2 vvm for oxygen limitation. In the fermentation, the fermentation temperature in the oxygen-limited stage is 37-50 °C.
  • the fermentation medium is (g/L): diammonium phosphate 0-25, potassium dihydrogen phosphate 0-5, disodium hydrogen phosphate, 0-25, sodium chloride 0-5, MgSO4 0-0.5, FeSO4 0 -1, FeCl3 0-1, CoCl2 0-1, CuCl2 0-1, ZnCl2 0-1, Na2MoO4 0-1, H3BO3 0-1, MnCl2 0-1, citric acid 0-25, thiamine 0-1 , xylose 0-50, glycerol 0-50, glucose 0-50, sulfuric acid 0-5, pH 6.0-7.5.
  • Example 38 The extremely high-purity D-lactic acid optimal production strain obtained in Example 38 was subjected to continuous production of 5 batches in a 10 ton fermentor.
  • a total of 12.6 g of the ingredients of the ingredient 1 was dissolved in water to 700 mL, 121 ° C, and sterilized for 20 minutes.
  • the ingredients of the ingredients 2 were 18 g in total, dissolved in 150 mL of water and sterilized separately, and added at the time of inoculation.
  • a total of 5 g of the ingredients of the ingredients 3 was dissolved in 150 mL of water and sterilized separately, and added at the time of inoculation.
  • the composition of ZT1 medium includes (g/L): diammonium phosphate 0-25, potassium dihydrogen phosphate 0-5, disodium hydrogen phosphate, 0-25, sodium chloride 0-5, MgSO4 0-0.5, FeSO4 0-1, FeCl3 0-1, CoCl2 0-1, CuCl2 0-1, ZnCl2 0-1, Na2MoO4 0-1, H3BO3 0-1, MnCl2 0-1, citric acid 0-25, thiamine 0- 1, xylose 0-50, glycerol 0-50, glucose 0-50, sulfuric acid 0-5, pH 6.0-7.5.
  • One seed of the above seed solution was inoculated with a 100 L secondary seed tank, and the liquid volume was 50 L. Incubate at 33-37 °C for 10-13 h in low intensity ventilation.
  • a total of 570 g of the ingredients of the ingredient 1 was dissolved in water to 30 L, 121 ° C, and sterilized for 20 minutes.
  • the composition of ZT1 medium includes (g/L): diammonium phosphate 0-25, potassium dihydrogen phosphate 0-5, disodium hydrogen phosphate, 0-25, sodium chloride 0-5, MgSO4 0-0.5, FeSO4 0-1, FeCl3 0-1, CoCl2 0-1, CuCl2 0-1, ZnCl2 0-1, Na2MoO4 0-1, H3BO3 0-1, MnCl2 0-1, citric acid 0-25, thiamine 0- 1, xylose 0-50, glycerol 0-50, glucose 0-50, sulfuric acid 0-5, pH 6.0-7.5.
  • the above-mentioned two-stage expanded seed liquid 50L was transferred to a 10m3 fermenter, the initial working volume was 5m3, and the fermentation medium was based on: ZT2 medium.
  • Ca(OH)2 was added to maintain pH 7.0. Transfer to non-ventilated fermentation acid production stage, fermentation temperature is 40-45 ° C, Ca (OH) 2 maintains pH 7.0.
  • Glucose was added to a final concentration of 50 g/L (calculated according to the initial fermentation volume), and was added in four batches at intervals of 3 h. Fermentation for 20-22h, determination of residual glucose below 0.1%, can be put into the tank. At the end of the fermentation, five batches of fermentation production were repeated in accordance with the above procedure. The fermentation results are shown in Table 3.
  • Example 42 Following the fermentation procedure in Example 42, the production strain was replaced with the optimal strain obtained in Example 39 for 5 batches of continuous production in a 10 ton fermentor.
  • the fermentation results are shown in Table 4.
  • the sulfuric acid is directly added to the fermentation broth at a concentration of 1% to 50% of sulfuric acid.
  • Plate and frame filtration The acidified solution is filtered by a plate and frame filter. After filter press, wash the filter cake with hot water at 85 ° C, clean The concentration of lactic acid in the wash solution is less than 0.1%. After the end of the washing, it is dried with compressed air, and the washing liquid is incorporated into the clear liquid. Bacteria and solid calcium salts are used as cement or concrete ingredients.
  • Ultrafiltration Ultrafiltration to remove pigments, proteins, amino acids and residual bacteria.
  • the filtrate was subjected to evaporation concentration before ultrafiltration, and the concentration ratio was 4:1, and the concentrate was subjected to ultrafiltration.
  • Ion exchange The filtrate obtained after ultrafiltration is ion exchanged.
  • Cation exchange 732 crosslinked to a 7% styrene-diethylene copolymer with a sulfonic acid group (-SO3H) cation exchange resin ( ⁇ 001 ⁇ 7 (732) strong acid styrene cation exchange resin);
  • the feed liquid should be cooled to room temperature before the exchange.
  • Anion exchange 330 (701) weakly basic anion exchange resin (weakly basic epoxy anion exchange resin, mainly used in water treatment to remove Cl-, SO42- plasma, remove inorganic acid, extract organic acid and decolorize, and Recovery of copper and silver ions.); Regeneration treatment: 5% W/v NaOH solution; exchange flow rate is 300 ml/min (depending on the test results). Intermediate inspection: Cl- does not exceed 3ppm; SO42- does not exceed 5ppm.
  • Product Concentration Concentrate to the desired lactic acid concentration of the product. This product is a polymeric grade D-lactic acid/L-lactic acid.
  • the present invention dynamically regulates the expression of the lactate dehydrogenase-encoding gene on the chromosomes of D-lactic acid and L-lactic acid high-yield recombinant strains by genetic engineering technology, thereby realizing the efficient single-reform of recombinant bacteria from glucose.
  • the technology of the present invention can be used for other industrially important microbial metabolites after simple modification, but is not limited to, for example, citric acid, formic acid, acetic acid, pyruvic acid, succinic acid, malic acid, ⁇ -ketoglutaric acid, Various organic acids and organic amines such as succinic acid, adipic acid, pentanediamine, hexamethylenediamine, methacrylic acid, isoprene, itaconic acid; or valine, alanine, lysine, Various amino acids such as methionine, glutamic acid, and arginine; various microorganisms such as thiamine and vitamin B12; or short-chain alcohols such as ethanol and propanol; or oligomeric isomaltose, oligofructose, galactooligosaccharide, etc.
  • citric acid formic acid, acetic acid, pyruvic acid
  • succinic acid malic
  • the nucleotide sequence of ldhA1 is the sequence shown in the sequence ⁇ 400>1
  • the nucleotide sequence of ldhA2 is the sequence shown in the sequence ⁇ 400>2
  • the nucleotide sequence of PPL1 is the sequence table ⁇ 400>3.
  • the sequence of the PPL2 is the sequence shown in the sequence ⁇ 400>4
  • the nucleotide sequence of PPL3 is the sequence shown in the sequence ⁇ 400>5
  • the nucleotide sequence of PPL4 is the sequence shown in the sequence ⁇ 400>6.
  • the nucleotide sequence of Ec-RlA1 is the sequence shown in the sequence ⁇ 400>7
  • the nucleotide sequence of Ec-RlA2 is the sequence shown in the sequence ⁇ 400>8
  • the nucleotide sequence of ThiE1p is the sequence table ⁇ 400>9
  • the sequence of the ThiE2p nucleotide sequence is the sequence shown in the sequence ⁇ 400>10
  • the nucleotide sequence of Dld1 is the sequence shown in the sequence ⁇ 400>11
  • the nucleotide sequence of Dld2 is the sequence table.
  • the sequence represented by ⁇ 400>12, the AckA-Pta1 nucleotide sequence is the sequence shown in the sequence ⁇ 400>13, the AckA-Pta2 nucleotide sequence is the sequence shown in the sequence ⁇ 400>14, and the Pps1 nucleotide sequence is The sequence shown in the sequence ⁇ 400>15, the Pps2 nucleotide sequence is the sequence shown in the sequence ⁇ 400>16, the RPps1 nucleotide sequence is the sequence shown in the sequence ⁇ 400>17, and the RPps2 nucleotide sequence is the sequence table.
  • the PflB1 nucleotide sequence is the sequence shown in the sequence ⁇ 400>19
  • the PflB2 nucleotide sequence is the sequence shown in the sequence ⁇ 400>20
  • the PoxB1 nucleotide sequence is the sequence table ⁇ 400. >21
  • the sequence of the PoxB2 nucleotide sequence is the sequence shown in the sequence ⁇ 400>22
  • the nucleotide sequence of the FrdA1 is the sequence shown in the sequence ⁇ 400>23
  • the nucleotide sequence of the FrdA2 is the sequence table ⁇ 400>24.
  • the AdhE1 nucleotide sequence is the sequence shown in the sequence ⁇ 400>25
  • the AdhE2 nucleotide sequence is the sequence shown in the sequence ⁇ 400>26
  • the ldhA3 nucleotide sequence is shown in the sequence table ⁇ 400>27.
  • the sequence of the ldhA4 nucleotide sequence is the sequence shown in the sequence ⁇ 400>28
  • the ldhA5 nucleotide sequence is the sequence shown in the sequence ⁇ 400>29
  • the ldhA6 nucleotide sequence is the sequence shown in the sequence table ⁇ 400>30.
  • the nucleotide sequence of RldhA1 is the sequence shown in the sequence ⁇ 400>31
  • the nucleotide sequence of RldhA2 is the sequence shown in the sequence ⁇ 400>32
  • the nucleotide sequence of BcoaLDH1 is the sequence shown in the sequence ⁇ 400>33
  • the BcoaLDH4 core is the sequence shown in the sequence ⁇ 400>34
  • the nucleotide sequence of LldD1 is the sequence shown in the sequence ⁇ 400>35
  • the nucleotide sequence of LldD2 is the sequence shown in the sequence ⁇ 400>36
  • the sequence is the sequence shown in the sequence ⁇ 400>37
  • the nucleotide sequence of LcaLDH4 is the sequence shown in the sequence ⁇ 400>38
  • the nucleotide sequence of StrbLDH1 is the sequence shown in the sequence ⁇ 400>39
  • the nucleotide sequence of StrbLDH2 is Sequence shown in the sequence listing ⁇ 400>40. Specific information is as follows:
  • ldhA1 TCCGGTACCCAGCCCGAGCGTCATCAG; KpnI
  • ldhA2 GTCAAGGTCGACGTTATTGAAACCG;
  • PPL1 AGCTTGGCTGCAGGTGATGATTATCAGC;
  • PPL2 ATCGCCGGCAATTCGTAATCATGG; EcoRI
  • PPL3 TAAGATATCCCATGATTACGAATTGCCGGC; EcoRV
  • PPL4 TAAGAATTCAGTTAACCTCCTTAGGATCCCAATGCTT EcoRI
  • Ec-RlA2 AAGACTTTCTCCAGTGATGTTGAATCACAT;
  • Dld1 AGTACGTCTTGATACCTTCGAAGCGG;
  • Dld2 GGATTCATGCTGTTGGTCGGATC;
  • AckA-Pta1 TGAACATCATCACCTGCCACCTG;
  • AckA-Pta2 CAGCGCAAAGCTGCGGATG
  • RPps2 GTCCGACCACGAAGACTTTGCC
  • PflB1 TTCAGACTTCGGACCAACCTGCA
  • PflB2 CCGCGAACTGGATCCGATGA;
  • PoxB1 CAAACGGTTGCAGCTTATATCGCC
  • AdhE1 ATCTGATCGGCTGGATCGATCAAC
  • AdhE2 GAACCAGGTTGGCGTCGACAAT
  • ldhA6 GCTGCCGGAAATCATCATTTTTT
  • RldhA1 GGAAGATCTTCCGCGAGTTTCATAAGACTT BglII
  • RldhA2 CGGAATTCCGAACGAACTGGTTTA EcoRI
  • LldD2 GGCCCGGGCAGGCAACTCTTTACCCAGCCC SmaI

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Abstract

极高光学纯D-乳酸和L-乳酸发酵生产菌种及其构建方法和用该菌株制备极高光学纯D-乳酸和L-乳酸的方法，其中，D-乳酸发酵生产菌种的保藏号为CGMCC No.11059，L-乳酸发酵生产菌种的保藏号为CGMCC No.11060。

Description

一种聚合级乳酸单体生产菌及其构建方法与乳酸制造技术 技术领域
本发明涉及微生物应用领域，尤其是一种聚合级乳酸单体生产菌及其构建方法与乳酸制造技术。
背景技术
生物可降解材料是指其使用后的废弃物能够被环境(微)生物所降解和利用的一类新颖材料。新一代生物可降解材料以聚乳酸为代表，分为聚L-乳酸和聚D-乳酸，分别通过其单体L-乳酸或D-乳酸聚合而得。预计到2020年，全球聚乳酸的年需求量将达到1500万吨，并且是年需求达5,000万吨的聚对苯二甲酸乙二醇酯(Polyethylene terephthalate,PET)和聚苯乙烯(Polystyrene,PS)的最可能的替代品。
以高品质D-乳酸为原材料聚合而成的聚D-乳酸材料可以很好的替代普通化工产品聚合而成的纤维、塑料等制品。特别是在高端消费品领域，如尿不湿内置垫片，香烟过滤头等材料加工聚合方面，其独特的生物质材料特性、生物体相容性和无毒无害特性大幅度提升相关产品的品质，市场空间巨大。以聚D-乳酸为原料采用3D打印技术制造的新产品具备环保、机械性能良好、安全性等多重优点，被广泛应用于汽车、一次性用品、电子、医疗等领域。更加值得关注的是极高光学纯D-乳酸和L-乳酸加工聚合而成的高品质聚乳酸(PLA)与同样生物可降解的丁二酸丁二醇酯-己二酸丁二醇酯共聚物(PBSA)混纺可以大幅提高生物可降解材料的强度和韧性，拓展相关产品的应用领域。目前巴斯夫等公司已经完成了PLA与PBSA混纺产品的试制，并成功推出了一系列性能优良的新型生物可降解材料，相关进展大幅度增加了极高光学纯D-乳酸和L-乳酸的市场需求。
米根霉，嗜热乳杆菌等传统乳酸生产菌株由于其自身特性和所需培养基复杂等特点导致所产乳酸光学纯度无法满足聚合级的要求而不能用于极高光学纯D-乳酸和L-乳酸的规模化生产。重组酵母和重组大肠杆菌具备极高光学纯D-乳酸和L-乳酸合成能力，且具备营养要求低，易于高密度培养，工业化规模推广容易的优点，成为极高光学纯D-乳酸和L-乳酸生产菌株的研究热点。与重组酵母，经过多重遗传修饰过的大肠杆菌，能够合成极高光学纯度和极高化学纯度的乳酸，并且由于大肠杆菌培养温度显著高于酵母，重组大肠杆菌用于乳酸发酵生产的周期较重组酵母大为缩短。因此，重组大肠杆菌被认为是工业化规模下生产极 高光学纯度D-乳酸和L-乳酸最理想的菌株。
特别是近年来，大肠杆菌作为D-乳酸生产菌种得到广泛的认可。当大肠杆菌同样可以用于高光学纯度L-乳酸的发酵生产时，对于D-乳酸和L-乳酸在同一条生产线上的交替生产意义重大。而且这也有助于高品质乳酸单体及聚乳酸制造业的快速发展。
围绕重组大肠杆菌作为生产菌株用于极高光学纯D-乳酸的发酵生产已经进行了多项成效显著的研究(Zhou L.et al.,Current Microbiology,2011,62:981-989；Zhu Y.et al.,Applied Environmental Microbiology,2007,73:456-464；Zhou S.et al.,Applied Environmental Microbiology,2003,69:399-407；Zhu J.et al.,Applied Microbiology and Biotechnology,2004,64:367-375；Zhu J.et al.,Metabolic Engineering,2005,7:104-115；Bunch P.K.et al.,Microbiology,1997,143:187-195)。如，1)严格厌氧发酵提高产酸效率(Li et al.,Applied Microbiology and Biotechnology,2002,60:101-106)；2)停止通风条件下的低搅拌限氧发酵提高产酸效率(Zhou L.et al.,Current Microbiology,2011,62:981-989)；3)维持微好氧发酵条件提高产酸效率(Tian K et al.,2012
African Journal of Biotechnology,11(21):4860-4867；Zhou L et al.,Biotechnology letters 2012,34:1123–1130)；4)采用适宜培养温度菌体生长和亚适宜生长温度限制菌体生长提高发酵产酸效率(Niu D et al.,Microbial Cell Factories 2014,13:78-88；Zhou L et al.,Metabolic Engineering,2012,14:560–568)；5)通过菌体生长和发酵产酸采用不同碳源的方式提高发酵产酸效率等(Zhu L et al.,Applied Environmental Microbiology,2007,73:456-464).
发明人前期获得的授权专利(中国专利，专利号：ZL201210102731.8)则通过基因转录水平上引入温度调控元件，并配合发酵温度调控策略大幅度提高了D-乳酸的合成效率。本发明在此基础上通过引入菌体生长定量控制调控机制，形成了菌体生长过程和D-乳酸合成过程的双开关机制，进一步提高了D-乳酸合成效率。
发明内容
本发明所要解决的技术问题在于提供了一种聚合级乳酸单体生产菌。
本发明所要解决的另一技术问题在于提供了上述聚合级乳酸单体生产菌的 构建方法。
本发明所要解决的另一技术问题在于提供了应用上述聚合级乳酸单体生产菌的乳酸制造技术，具体来说，是结合菌种代谢生长及产酸过程的特性和工业化规模生产的特点形成的低成本易实施的微生物法极高光学纯乳酸单体高效制造技术。
为解决上述技术问题，本发明的技术方案是：
一种用于发酵生产极高光学纯D-乳酸的聚合级乳酸单体生产菌，菌株保藏号为CGMCC No.11059(是实施例中菌株编号为B0013-090B的菌株)。
一种用于发酵生产极高光学纯L-乳酸的聚合级乳酸单体生产菌，菌株保藏号为CGMCC No.11060(是实施例中菌株编号为B0013-101J的菌株)。
上述用于发酵生产极高光学纯D-乳酸的聚合级乳酸单体生产菌和用于发酵生产极高光学纯L-乳酸的聚合级乳酸单体生产菌分别具备形成极高光学纯度D-乳酸和极高光学纯度L-乳酸的能力，光学纯度可以高于99.9％，所述D-乳酸和L-乳酸的光学纯度特征均可以满足高品质聚乳酸聚合过程对乳酸单体光学纯度的最高要求。
上述用于发酵生产极高光学纯D-乳酸的聚合级乳酸单体生产菌和用于发酵生产极高光学纯L-乳酸的聚合级乳酸单体生产菌分别具备形成高化学纯度D-乳酸和高化学纯度L-乳酸的能力，化学纯度可以高于99％，所述D-乳酸的和L-乳酸的化学纯度特征经过简单的后续处理甚至不处理可以满足高品质聚乳酸聚合过程对乳酸单体化学纯度的最高要求。
上述用于发酵生产极高光学纯D-乳酸的聚合级乳酸单体生产菌和用于发酵生产极高光学纯L-乳酸的聚合级乳酸单体生产菌的构建方法为：
单个或多个基因被敲除进行菌株初步构建，这些基因包括：ldhA，thiE，dld，ackA，pta，pps，pflB，poxB，frdA，adhE，lldD；单个或多个基因被表达，这些基因包括：kan-cIts857-pR-pL-ldhA,ldhBcoa，ldhLca，ldhStrb；
使用了温度诱导型基因转录方式来控制和调节细胞生长过程和乳酸形成过程，包括：菌株初步构建后，菌株在25-50℃条件下分阶段完成细胞发酵培养-诱导-产酸，细胞生长过程在单一发酵因子调控下可以定量控制细胞的积累量，所述单一发酵因子是细胞中央代谢途径关键酶转录过程的调控因子、关键酶翻译过程的调控因子、关键酶分泌过程的调控因子或关键酶表达后催化过程调控因子。
优选的，上述聚合级乳酸单体生产菌的构建方法，所述菌株积累量的定量控制可以通过预先计算，在不同的发酵体系下添加一定量的单一发酵因子按照生产过程的实际需求精确控制。
优选的，上述聚合级乳酸单体生产菌的构建方法，菌株初步构建后菌株的生长过程都与单一发酵因素的调控相关联，所述单一发酵因素可以是碳源，如葡萄糖、甘油；也可以是单元如酵母膏、蛋白胨、硫酸铵、磷酸氢铵；也可以是金属离子，如铁离子、镁离子、钙离子、锌离子、锰离子、钴离子、铜离子、钠离子、钾离子；也可以是营养元素，如VB1、VB6、VB12、生物素、盐酸硫胺素、焦磷酸硫胺素，等等，特别是其中一种单一发酵因素可以定量的控制细胞的积累过程，并保证细胞快速积累且具备高活性；可以以开关控制的方式启动或关闭乳酸的合成过程，并且这一过程可以通过预先定量添加对应的发酵因素完成上述调控过程。
一种乳酸制造技术，具体方法为：应用上述聚合级乳酸单体生产菌，所述菌株的宿主细胞生长过程在全合成的无机盐培养基中快速进行，所述全合成的无机盐培养基为含硫胺素的培养基，所述宿主细胞在全合成培养基中快速生长，其中，7L发酵体系下于8-10h内菌体量积累到细胞干重11.5g/L，通过定量补加单一发酵因子，细胞干重进行更高积累，宿主细胞培养过程中不大量形成乳酸，即乳酸的痕量形成不影响细胞生长，宿主细胞在生长完成后快速积累乳酸。
优选的，上述乳酸制造技术，D-乳酸和L-乳酸的积累过程通过更换相应聚合级乳酸单体生产菌在同一套生产体系下切换式进行。
上述全合成培养基中有机物的添加不影响上述细胞高密度培养的过程，但不添加有机物更有利于上述细胞高密度培养过程的进行。
优选的，上述乳酸制造技术，所述全合成的无机盐培养基主体成分如下：
用于D-乳酸发酵生产的发酵培养基(g/L)：磷酸氢二铵0-25，磷酸二氢钾0-5，磷酸氢二钠0-25，氯化钠0-5，MgSO4 0-0.5，FeSO4 0-1，FeCl3 0-1，CoCl2 0-1，CuCl2 0-1，Na2MoO4 0-1，H3BO3 0-1，MnCl2 0-1，柠檬酸0-25，硫胺素0-1，木糖0-50，甘油0-50，葡萄糖0-50，硫酸0-5，pH 6.0-7.5。
用于L-乳酸发酵生产的发酵培养基(g/L)：磷酸氢二铵0-25，磷酸二氢钾0-5，磷酸氢二钠0-25，氯化钠0-5，MgSO4 0-0.5，FeSO4 0-1，FeCl3 0-1，CoCl2 0-1，CuCl2 0-1，ZnCl2 0-1，Na2MoO4 0-1，H3BO3 0-1，MnCl2 0-1，柠檬酸0-25，硫胺素0-1，木糖0-50，甘油0-50，葡萄糖0-50，硫酸0-5，pH  6.0-7.5。
所述的发酵产酸过程具备可设定型的自动启动特征，通过培养基组分和菌种自身生长特性的组合，细胞生长至特定阶段后由于培养基中对应调控因子组分的含量变化自动启动产酸过程。
优选的，上述乳酸制造技术，所述菌株的生长过程以及乳酸形成过程都与发酵温度的过程相关联，发酵产酸过程在非固定温度下完成的，根据生产菌种产酸特性，其发酵温度的变化呈现梯度上升的趋势，且存在一定组合方式下，温度对产酸过程的影响最显著，产酸效率最高，如：发酵温度升高生长会变缓慢，而发酵温度降低生长会非常迅速，具备积累高活性细胞的特点；发酵温度升高乳酸会快速形成，而发酵温度降低乳酸会缓慢形成，甚至不形成，具备高效积累乳酸的特点。
优选的，上述乳酸制造技术，所述菌株先在25-36℃下利用葡萄糖快速生长形成菌体，然后在37-50℃下利用葡萄糖快速积累乳酸。
由于菌株在较低的温度下，如25-36℃，D-乳酸和L-乳酸合成关键酶编码基因ldhA和BcoaLDH的转录被强烈抑制；而在较高温度下，如37-50℃，D-乳酸和L-乳酸合成关键酶编码基因ldhA和BcoaLDH的转录被强烈启动，由此可见，菌株在生长的温度和形成乳酸的温度可以是单一温度下的持续过程，也可以是多个温度点梯度式的组合过程。更优先使用多个温度点梯度式的组合过程。
优选的，上述乳酸制造技术，具体步骤为：
(1)通过基因工程技术对D-乳酸和L-乳酸高产重组菌染色体上的乳酸脱氢酶编码基因的表达进行简易条件下的动态调控获得产酸菌株；
(2)将菌株在25-36℃、200r/min好氧生长6-10h，再在37-45℃静置培养发酵乳酸，并以出发菌种B0013-070作为对照菌种，分析细胞密度、糖耗、乳酸产率、代谢主要中间产物及其它有机酸产物等，确定乳酸合成诱导时机；菌株分别在添加0.06-100μg/L硫胺素、200r/min进行摇瓶培养；
(3)发酵生产极高光学纯D-乳酸时，限氧阶段0-3h发酵温度为33-39℃，3-6h发酵温度为37-42℃，6-10h发酵温度为38-45℃，10-16h发酵温度为40-48℃，16-24h发酵温度为45-50℃。
(3’)发酵生产极高光学纯L-乳酸时，限氧阶段发酵温度为37-50℃，其余同步骤(3)。
优选的，上述乳酸制造技术，还包括发酵结束后乳酸的提取方法，结合生产 菌种基因工程改造后的特征，发酵液中D-乳酸和L-乳酸均以极高光学纯度和化学纯度的形式存在，全合成培养基的使用保证了后提取过程的简洁型，其最终产品的提取方式包括酸化，板框去除菌体，超滤去除色素和杂蛋白，离子交换去除阴阳离子干扰，浓缩制备相应浓度的产品，并纳滤精制产品等环节。
优选的，上述乳酸制造技术，所述乳酸形成过程结束后通过低温酸化的方式将D-乳酸和L-乳酸游离出来，且上述乳酸游离的过程不受发酵液中其他残余物的影响，酸化使用的酸可以是硫酸，也可以是盐酸或草酸，更优选使用硫酸。
上述乳酸制造技术，所发酵生产的极高光学纯D-乳酸和L-乳酸过程耗时不多于30-36h，产D-乳酸和L-乳酸水平分别达到150g/L和180g/L或以上，D-乳酸和L-乳酸光学纯度均在99.95以上，化学纯度均在97％以上。
上述乳酸制造技术，还包括具有类似反应过程的其他化学品，如柠檬酸、甲酸、乙酸、丙酮酸、丁二酸、苹果酸、α-酮戊二酸、丁二酸、己二酸、戊二胺、己二胺、甲基丙烯酸、异戊二烯、衣康酸等多种有机酸和有机胺；或脯氨酸、丙氨酸、赖氨酸、蛋氨酸、谷氨酸、精氨酸等多种氨基酸；硫胺素、维生素B12等多种微生物；或乙醇、丙醇等短链醇；或低聚异麦芽糖、低聚果糖、低聚半乳糖等多种功能糖等等。
本发明的有益效果是：
本申请在ZL201210102731.8所述通过基因转录水平上引入温度调控元件，并配合发酵温度调控策略大幅度提高了D-乳酸的合成效率的基础上，通过引入菌体生长定量控制调控机制，形成了菌体生长过程和D-乳酸合成过程的双开关机制，进一步提高了D-乳酸合成效率；本发明中所述极高光学纯L-乳酸产生菌充分利用了外源L-乳酸脱氢酶翻译表达后的酶学特性，并引入了其催化活性的温度调控机制，配合菌体生长定量控制的调控机制，完成了菌体生长和L-乳酸合成的双开关控制。
更为突出的是，本发明中所述极高光学纯D-乳酸生产过程和极高光学纯L-乳酸生产过程可以通过菌种的简单切换在同一生产体系下完成两个产品的规模化生产。
具体有益效果如下：
1、本发明提供的重组菌具有明显的高效合成极高光学纯D-乳酸和L-乳酸的能力，同时上述重组菌具备的合成高化学纯度D-乳酸和L-乳酸的能力。菌种在25-50℃的条件下细胞快速生长8-10h，发酵产酸16-18h，产D-乳酸水平达到 15％(w/v)或以上，产L-乳酸水平达到18％(w/v)或以上；
2、本发明的高效合成极高光学纯乳酸单体基因工程菌，在菌体培养过程中，采用全合成培养基，培养液澄清，有利于后续产品分离提取；
3、本发明的高效合成极高光学纯乳酸单体基因工程菌，在菌体生长和发酵产酸过程可以通过温度变化有效控制；
4、本发明的高效合成极高光学纯乳酸单体基因工程菌，在菌体生长和发酵产酸过程可以通过营养元素的添加定量控制和开关控制
5、本发明的极高光学纯乳酸单体高效制造过程：菌体细胞生长温度在25-36℃下利用葡萄糖快速生长6-12h，形成菌体；在37-50℃下利用葡萄糖快速合成极高光学纯D-乳酸和L-乳酸。即：运用本发明的重组菌及其极高光学纯乳酸单体制备工艺，生产过程仅需改变发酵温度控制参数并配合营养元素的添加，即可实现极高光学纯D-乳酸和L-乳酸的高效制备过程。
附图说明
图1突变盒质粒pUC-ldhAp::kan-cIts857-pR-pL的物理图谱；
图2突变盒质粒pUC-thiE’::difGm的物理图谱；
图3突变盒质粒pUC-dld’::difGm的物理图谱；
图4突变盒质粒pUC-ackA-pta’::difGm的物理图谱；
图5突变盒质粒pUC-pps’::difGm的物理图谱；
图6突变盒质粒pSK-pflB’::difGm的物理图谱；
图7突变盒质粒pUC-poxB’::difGm的物理图谱；
图8突变盒质粒pSKsym-frdA’::difGm的物理图谱；
图9突变盒质粒pUC-adhE’::difGm的物理图谱；
图10突变盒质粒pUC-ldhA::difGm的物理图谱；
图11突变盒质粒pUC-lldD’::PldhA-ldhBcoa-difGm的物理图谱；
图12突变盒质粒pUC-lldD’::PldhA-ldhLca-difGm的物理图谱；
图13突变盒质粒pUC-lldD’::PldhA-ldhStrb-difGm的物理图谱，
图14突变盒质粒pUC-ldhA’::PldhA-ldhLca-difGm的物理图谱；
图15突变盒质粒pUC-ldhA’::PldhA-ldhStrb-difGm的物理图谱；
图16突变盒质粒pUC-thiE’::PldhA-ldhLca-difGm的物理图谱；
图17突变盒质粒pUC-thiE’::PldhA-ldhStrb-difGm的物理图谱。
图18突变盒质粒pUC-thiE’::PldhA–ldhBcoa-difGm的物理图谱。
图19极高光学纯D-乳酸产生菌株筛选结果；
图20极高光学纯L-乳酸产生菌株筛选结果；
图21极高光学纯D-乳酸发酵产生进程；
图22D-乳酸光学纯度检测图谱；
图23极高光学纯L-乳酸发酵产生进程；
图24L-乳酸光学纯度检测图谱。
保藏信息
分类名词：大肠埃希氏菌Escherichia coli
保藏单位名称：中国微生物菌种保藏管理委员会普通微生物中心
保藏单位地址：北京市朝阳区北辰西路1号院3号
保藏日期：2015年7月7日
保藏号：CGMCC No.11059
参据的生物材料(株)：B0013-090B
分类名词：大肠埃希氏菌Escherichia coli
保藏单位名称：中国微生物菌种保藏管理委员会普通微生物中心
保藏单位地址：北京市朝阳区北辰西路1号院3号
保藏日期：2015年7月7日
保藏号：CGMCC No.11060
参据的生物材料(株)：B0013-101J
具体实施方式
下面结合具体实施例对本发明所述技术方案作进一步的说明。
本发明提供一种极高光学纯D-乳酸和极高光学纯L-乳酸高产菌株与构建方法及极高光学纯D-乳酸和极高光学纯L-乳酸高效制备工艺，菌体在25-36℃下利用葡萄糖快速生长形成高活性细胞，然后在37-50℃下高效积累极高光学纯D-乳酸和极高光学纯L-乳酸，且所积累乳酸的化学纯度极高。其工艺特征是：25-50℃的条件下，分别经过发酵培养菌体，变温高效产酸阶段，产D-乳酸水平达到15％(w/v)或以上，光学纯度99.9％以上，化学纯度97％以上；产L-乳酸水平达到18％(w/v)或以上，光学纯度99.9％以上，化学纯度98％以上。
本发明涉及的具体方法有：
染色体基因整合技术：运用PCR(多聚酶链反应)从大肠杆菌基因组中扩增 获得染色体目标整合位点的上游及下游各50-700bp基因序列。将目的整合表达基因与抗性基因进行连接，所获得的片段克隆入上述目标整合位点的上游和下游基因序列之间，形成目的基因整合序列，如ldhA::kan-cIts857-pR-pL,thiE’::difGm,dld’::difGm,ackA-pta’::difGm,pps’::difGm,pflB’::difGm,poxB’::difGm,frdA’::difGm,adhE’::difGm,ldhA’::difGm,lldD’::PldhA-ldhBcoa-difGm,lldD’::PldhA-ldhLca-difGm,lldD’::PldhA-ldhStrb-difGm,ldhA’::PldhA-ldhLca-difGm,ldhA’::PldhA-ldhStrb-difGm,thiE’::PldhA-ldhLca-difGm，thiE’::PldhA-ldhStrb-difGm，thiE’::PldhA-ldhBcoa-difGm。将上述基因整合序列单独或两两组合或多个组合转化入大肠杆菌。在选择性培养基上选择培养出转化子。提取转化子染色体DNA，用PCR对转化子的目的基因突变进行验证。发酵试验筛选最优的极高光学纯D-乳酸高产菌如B0013-090B和极高光学纯L-乳酸高产菌如B0013-101J。
利用上述重组方法，按照以下步骤完成对动态调控D-乳酸重组菌的构建。
1研究菌种：大肠杆菌B0013(Zhou L.et al.,Curr Microbiol,2011,62:981-989)。
2利用基因整合技术，将步骤1所获得的出发菌种中的乳酸脱氢酶基因的启动子ldhAp替换为pR-pL启动子(Love C.A.et al.,Gene,1996,176:49-53)，并获得重组大肠杆菌1。
3利用基因整合技术，构建硫胺素磷酸合成酶(thiE)编码基因删除用突变盒thiE’::difGm，删除步骤2所获重组菌的thiE基因，获得重组大肠杆菌2。
4利用基因整合技术，构建FAD依赖型D-乳酸脱氢酶(dld)编码基因删除用突变盒dld’::difGm，删除步骤3所获重组菌的dld基因，获得重组大肠杆菌3。
5利用基因整合技术，构建乙酸激酶(ackA)和磷酸转乙酰酶(pta)编码基因删除用突变盒ackA-pta’::difGm，删除步骤4所获重组菌的ackA-pta基因，获得重组大肠杆菌4。
6利用基因整合技术，构建磷酸烯醇式丙酮酸合酶(pps)编码基因删除用突变盒pps’::difGm，删除步骤4和步骤5所获重组菌的pps基因，获得重组大肠杆菌5和6。
7利用基因整合技术，构建丙酮酸甲酸裂解酶(pflB)编码基因删除用突变盒pflB’::difGm，删除步骤4，步骤5和步骤6所获重组菌的pflB基因，获得重 组大肠杆菌7-10。
8利用基因整合技术，构建丙酮酸氧化酶(poxB)编码基因删除用突变盒poxB’::difGm，删除步骤4，步骤5，步骤6和步骤7所获重组菌的pflB基因，获得重组大肠杆菌11-18。
9利用基因整合技术，构建富马酸还原酶(frdA)编码基因删除用突变盒frdA’::difGm，删除步骤4，步骤5，步骤6，步骤7和步骤8所获重组菌的frdA基因，获得重组大肠杆菌19-34。
10利用基因整合技术，构建乙醇脱氢酶(adhE)编码基因删除用突变盒adhE’::difGm，删除步骤4，步骤5，步骤6，步骤7，步骤8和步骤9所获重组菌的adhE基因，获得重组大肠杆菌35-66。
11利用基因整合技术，构建D-乳酸脱氢酶(ldhA)编码基因删除用突变盒ldhA’::difGm，删除重组菌B0013-070的ldhA基因，获得重组大肠杆菌67。
12利用基因整合技术，构建D-乳酸脱氢酶基因(ldhA)编码基因删除用突变盒ldhA’::PldhA-ldhLca-difGm，删除重组菌B0013-070的ldhA基因，获得重组大肠杆菌68。
13利用基因整合技术，构建D-乳酸脱氢酶基因(ldhA)编码基因删除用突变盒ldhA’::PldhA-ldhStrb-difGm，删除重组菌B0013-070的ldhA基因，获得重组大肠杆菌69。
14利用基因整合技术，构建FMN为辅酶的L-乳酸脱氢酶基因(lldD)编码基因删除用突变盒lldD’::PldhA-ldhBcoa-difGm，删除重组菌B0013-070和步骤11，步骤12以及步骤13所获重组菌的lldD基因，获得重组大肠杆菌70-73。
15利用基因整合技术，构建FMN为辅酶的L-乳酸脱氢酶基因(lldD)编码基因删除用突变盒lldD’::PldhA-ldhLca-difGm，删除重组菌B0013-070和步骤11，步骤12以及步骤13所获重组菌的lldD基因，获得重组大肠杆菌74-77。
16利用基因整合技术，构建FMN为辅酶的L-乳酸脱氢酶基因(lldD)编码基因删除用突变盒lldD’::PldhA-ldhLca-difGm，删除重组菌B0013-070和步骤11，步骤12以及步骤13所获重组菌的lldD基因，获得重组大肠杆菌78-81。
17利用基因整合技术，硫胺素磷酸合成酶(thiE)编码基因删除用突变盒thiE’::difGm，删除步骤11，步骤12，步骤13，步骤14，步骤15和步骤16所获重组菌的thiE基因，获得重组菌种82-96。
18利用基因整合技术，硫胺素磷酸合成酶(thiE)编码基因删除用突变盒 thiE’::PldhA-ldhLca-difGm，删除步骤11，步骤12，步骤13，步骤14，步骤15和步骤16所获重组菌的thiE基因，获得重组菌种97-111。
19利用基因整合技术，硫胺素磷酸合成酶(thiE)编码基因删除用突变盒thiE’::PldhA-ldhStrb-difGm，删除步骤11，步骤12，步骤13，步骤14，步骤15和步骤16所获重组菌的thiE基因，获得重组菌种112-126。
20利用基因整合技术，硫胺素磷酸合成酶(thiE)编码基因删除用突变盒thiE’::PldhA-ldhBcoa-difGm，删除步骤11，步骤12，步骤13，步骤14，步骤15和步骤16所获重组菌的thiE基因，获得重组菌种127-156
21将步骤4，步骤5，步骤6，步骤7，步骤8，步骤9，步骤10，步骤11，步骤12，步骤13，步骤14，步骤15，步骤16，步骤17，步骤18，步骤19和步骤20所获得的重组菌种在30℃和45℃、200r/min进行摇瓶培养，并以出发菌种B0013-070作为对照菌种，分析赖氨酸脱氢酶比活性，鉴定pR-pL启动子和thiE的功能，并筛选出最优菌种。
22将步骤21所获得的最优菌种分别在25-36℃、200r/min进行摇瓶培养，并以出发菌种B0013-070作为对照菌种，分析细胞密度、乳酸、代谢主要中间产物及其它有机酸产物等，确定菌体生长培养温度。
23将步骤21所获得的最优菌种在25-36℃、200r/min好氧生长-10h，再在37-45℃静置培养发酵乳酸，并以出发菌种B0013-070作为对照菌种，分析细胞密度、糖耗、乳酸产率、代谢主要中间产物及其它有机酸产物等，确定乳酸合成诱导时机。
24将步骤最优所获得的最优菌种分别在添加0.06-100μg/L硫胺素、200r/min进行摇瓶培养，并以出发菌种B0013-070作为对照菌种，分析细胞密度、乳酸、代谢主要中间产物及其它有机酸产物等，确定菌体生长硫胺素添加量。
25将步骤20所获得的最优菌种在7L-30,000L发酵罐中进行乳酸发酵试验。发酵过程中定时取样，分析细胞密度、糖耗、乳酸产率、代谢主要中间产物及其它有机酸产物等。
下面以具体实例对本发明技术方案作进一步详细说明。
实施例1——突变盒ldhA::kan-cIts857-pR-pL的构建
用引物ldhA1和ldhA2PCR扩增B0013染色体上的ldhA基因片段ldhA’，克隆入pUC18载体，获得重组质粒pUC-ldhA’。用引物PPL1和PPL2对质粒pPL451 进行反向PCR扩增，并与卡那霉素抗性基因片段(质粒pSKsymKm用SmaI酶切，胶回收966bp片段)进行连接，获得重组质粒pPL-Kan。用引物PPL3和PPL4对质粒pPL-Kan进行PCR扩增，产物用EcoRI和EcoRV进行双酶切，并与以pUC-ldhA’为模板用引物Ec-RlA1和Ec-RlA2进行反向PCR扩增并用EcoRI进行酶切的产物连接，从而获得重组质粒pUC-ldhAp::kan-cIts857-pR-pL。所得重组质粒的物理图谱如附图1所示。将重组质粒pUC-ldhAp::kan-cIts857-pR-pL用KpnI酶切，并胶回收线性化质粒，用引物ldhA1和ldhA2进行PCR扩增，获得ldhAp::kan-cIts857-pR-pL基因片段。
实施例2——突变盒thiE::difGm的构建
以E.coli B0013染色体DNA为模板，ThiE1p和ThiE2p为引物扩增获得长度为0.6kb的thiE基因部分序列。将此克隆入pUC18，获得重组质粒pUC-thiE。将长度为1.2kb的difGm片段克隆入thiE中间的StuI位点，获得重组质粒pUC-thiE::difGm。所得重组质粒的物理图谱如附图2所示。将重组质粒pUC-thiE::difGm用ApaLI酶切，并胶回收线性化质粒，用引物ThiE1p和ThiE2p进行PCR扩增，获得thiE::difGm基因片段。
实施例3——突变盒dld::difGm的构建
以菌株B0013染色体DNA为模板，用引物Dld1和Dld2PCR扩增dld’基因片段(0.9kb)，PCR产物克隆于载体pUC18SmaI位点，获得重组质粒pUC-dld’。用EcoRV酶切该重组质粒，去除dld’基因中部0.4kb基因片段，并克隆入dif-Gm-dif片段，获得重组质粒pUC-dld’::difGm。所得重组质粒的物理图谱如附图3所示。将重组质粒pUC-dld’::difGm用EcoRI酶切，并胶回收线性化质粒，用引物Dld1和Dld2进行PCR扩增，获得dld’::difGm基因片段。
实施例4——突变盒ackA-pta::difGm的构建
以菌株B0013染色体DNA为模板，用引物AckA-Pta1和AckA-Pta2PCR扩增ackA-pta’基因片段(2.8kb)，PCR产物克隆于载体pUC18中，获得重组质粒pUC-ackA-pta’。用EcoRV酶切该重组质粒，去除ackA-pta’基因中部2.6kb基因片段，并克隆入dif-Gm-dif片段，获得重组质粒pUC-ackA-pta’::difGm。所得重组质粒的物理图谱如附图4所示。将重组质粒pUC-ackA-pta’::difGm用KpnI酶切，并胶回收线性化质粒，用引物AckA-Pta1和AckA-Pta2进行PCR扩增，获得ackA-pta’::difGm基因片段。
实施例5——突变盒pps::difGm的构建
以菌株B0013染色体DNA为模板，用引物Pps1和Pps2PCR扩增pps基因片段(2.3kb)，PCR产物克隆于载体pUC18中，获得重组质粒pUC-pps’。以质粒pUC-pps’为模板，用引物RPps1和RPps2进行反向PCR扩增，去除pps’基因中部2.1kb基因片段，并克隆入dif-Gm-dif片段，获得重组质粒pUC-pps’::difGm。所得重组质粒的物理图谱如附图5所示。将重组质粒pUC-pps’::difGm用ApaLI酶切，并胶回收线性化质粒，用引物Pps1和Pps2进行PCR扩增，获得pps’::difGm基因片段。
实施例6——突变盒pflB::difGm的构建
以大肠杆菌B0013染色体DNA为模板，PflB1和PflB2为引物进行PCR扩增，其扩增产物PflB’克隆于载体pSK的EcoRV和SmaI位点(在这过程中PstI和EcoRI消失)，获得重组质粒pSK-pflB’。PstI酶切重组质粒pSK-pflB’，补平并与pSK-EcdifGm中的difGm连接获得重组质粒pSK-pflB’::difGm。所得重组质粒的物理图谱如附图6所示。重组质粒pSK-pflB’::difGm用ApaLI酶切，并胶回收线性化质粒，用引物PflB1和PflB2进行PCR扩增，获得pflB’::difGm基因片段。
实施例7——突变盒poxB::difGm的构建
用引物PoxB1和PoxB2扩增大肠杆菌的poxB基因片段，PCR产物克隆入质粒pUC18的SmaI酶切位点获得重组质粒pUC-poxB’。该重组质粒用EcoRⅤ酶切，克隆入dif-Gm片段获得重组质粒pUC-poxB’::difGm。所得重组质粒的物理图谱如附图7所示。将重组质粒pUC-poxB’::difGm用ApaLI酶切，并胶回收线性化质粒，用引物PoxB1和PoxB2进行PCR扩增，获得poxB’::difGm基因片段。
实施例8——突变盒frdA::difGm的构建
以菌株B0013染色体DNA为模板，用引物FrdA1和FrdA2PCR扩增frdA’基因片段(1.6kb)，PCR产物克隆于载体pSKsym SmaI位点，获得重组质粒pSKsym-frdA’。用PstI酶切该重组质粒，去除frdA’基因中部1.3kb基因片段，用T4DNA polymerase使PstI粘性末端平滑化，并克隆入dif-Gm-dif片段，获得重组质粒pSKsym-frdA’::difGm。所得重组质粒的物理图谱如附图8所示。将重组质粒pSKsym-frdA’::difGm用ApaLI酶切，并胶回收线性化质粒，用引物FrdA1和FrdA2进行PCR扩增，获得frdA’::difGm基因片段。
实施例9——突变盒adhE::difGm的构建
以菌株B0013染色体DNA为模板，用引物AdhE1和AdhE2PCR扩增adhE’ 基因片段(1.4kb)，PCR产物克隆于载体pUC19SmaI位点，获得重组质粒pUC-adhE’。用EcoRV酶切该重组质粒，去除adhE’基因中部1kb基因片段，并克隆入dif-Gm-dif片段，获得重组质粒pUC-adhE’::difGm。所得重组质粒的物理图谱如附图9所示。重组质粒pUC-adhE’::difGm用EcoRI和PstI双酶切重组质粒pUC-adhE’::difGm，胶回收adhE1-dif-Gm-dif-adhE2基因片段。
实施例10——突变盒ldhA::difGm的构建
以大肠杆菌B0013染色体DNA为模板，ldhA3和ldhA4为引物，运用PCR扩增获得了乳酸脱氢酶基因(ldhA)，将此PCR产物克隆入pUC18的EcoRI位点中，获得重组质粒pUC-ldhA。用PstI酶切去除其中的400bps的片段并用T4DNA多聚酶将粘性末端补平，再与difGm片段连接，获得重组质粒pUC-ldhA::Gmdif。所得重组质粒的物理图谱如附图10所示。用EcoRI酶切该重组质粒，获得乳酸脱氢酶的基因删除序列，ldhA'-dif-Gm-dif-ldhA，即：ldhA::Gmdif。
实施例11——突变盒lldD::PldhA-ldhBcoa-difGm的构建
以E.coli CICIM B0013染色体DNA为模板，扩增(引物ldhA5和ldhA6)获得ldhA基因的全部启动子和部分结构区片段，并将片段ldhA’克隆入亚克隆载体pUC18获得重组质粒pUC-ldhA’。以pUC-ldhA’为模板用引物RldhA1和RldhA12进行反向PCR扩增并进行产物自连接，从而获得重组质粒pUC-PldhA。以Bacillus coagulans CICIM B1821染色体DNA为模板运用PCR技术扩增(引物BcoaLDH1和BcoaLDH4)获得ldhBcoa基因片段经BamHI和EcoRI酶切后克隆入质粒pUC-PldhA’的BglII和EcoRI，获得重组质粒pUC-PldhA-ldhBcoa。然后将difGm片段克隆入重组质粒pUC-PldhA-ldhBcoa的EcoRV位点。获得重组质粒pUC-PldhA-ldhBcoa-difGm。
通过PCR扩增(引物lldD1和lldD2)获得lldD基因片段，并将该片段克隆入亚克隆载体pUC18获得重组质粒pUC-lldD。重组质粒pUC-PldhA-ldhBcoa-difGm经BamHI酶切获得PldhA-ldhBcoa-difGm片段克隆入重组质粒pUC-lldD的BamHI位点。获得重组质粒pUC-lldD::PldhA-ldhBcoa-difGm。所得重组质粒的物理图谱如附图11所示。将重组质粒pUC-lldD::PldhA-ldhBcoa-difGm用SmaI酶切，并胶回收获得lldD::PldhA-ldhBcoa-difGm基因片段。
实施例12——突变盒lldD::PldhA-ldhLca-difGm的构建
以E.coli CICIM B0013染色体DNA为模板，扩增(引物ldhA5和ldhA6)获 得ldhA基因的全部启动子和部分结构区片段，并将片段ldhA’克隆入亚克隆载体pUC18获得重组质粒pUC-ldhA’。以pUC-ldhA’为模板用引物RldhA1和RldhA12进行反向PCR扩增并进行产物自连接，从而获得重组质粒pUC-PldhA。以Lactobacillus casei B1192染色体DNA为模板运用PCR技术扩增(引物LcaLDH1和LcaLDH4)获得ldhLca基因片段经BamHI和EcoRI酶切后克隆入质粒pUC-PldhA’的BglII和PstI，获得重组质粒pUC-PldhA-ldhLca。然后将difGm片段克隆入重组质粒pUC-PldhA-ldhLca的EcoRV位点。获得重组质粒pUC-PldhA-ldhLca-difGm。
通过PCR扩增(引物lldD1和lldD2)获得lldD基因片段，并将该片段克隆入亚克隆载体pUC18获得重组质粒pUC-lldD。重组质粒pUC-PldhA-ldhLca-difGm经BamHI酶切获得PldhA-ldhLca-difGm片段克隆入重组质粒pUC-lldD的BamHI位点。获得重组质粒pUC-lldD::PldhA-ldhLca-difGm。所得重组质粒的物理图谱如附图12所示。将重组质粒pUC-lldD::PldhA-ldhLca-difGm用SmaI酶切，并胶回收获得lldD::PldhA-ldhLca-difGm基因片段。
实施例13——突变盒lldD::PldhA-ldhStrb-difGm的构建
以E.coli CICIM B0013染色体DNA为模板，扩增(引物ldhA5和ldhA6)获得ldhA基因的全部启动子和部分结构区片段，并将片段ldhA’克隆入亚克隆载体pUC18获得重组质粒pUC-ldhA’。以pUC-ldhA’为模板用引物RldhA1和RldhA12进行反向PCR扩增并进行产物自连接，从而获得重组质粒pUC-PldhA。以Steptococcus.bovis 1.1624染色体DNA为模板运用PCR技术扩增(引物StrbLDH1和StrbLDH2)获得ldhStrb基因片段经BamHI和EcoRI酶切后克隆入质粒pUC-PldhA’的BglII和EcoRI，获得重组质粒pUC-PldhA-ldhStrb。然后将difGm片段克隆入重组质粒pUC-PldhA-ldhStrb的EcoRV位点。获得重组质粒pUC-PldhA-ldhBcoa-difGm。
通过PCR扩增(引物lldD1和lldD2)获得lldD基因片段，并将该片段克隆入亚克隆载体pUC18获得重组质粒pUC-lldD。重组质粒pUC-PldhA-ldhBcoa-difGm经BamHI酶切获得PldhA-ldhStrb-difGm片段克隆入重组质粒pUC-lldD的BamHI位点。获得重组质粒pUC-lldD::PldhA-ldhStrb-difGm。所得重组质粒的物理图谱如附图13所示。将重组质粒pUC-lldD::PldhA-ldhStrb-difGm用SmaI酶切，并胶回收获得 lldD::PldhA-ldhStrb-difGm基因片段。
实施例14——突变盒ldhA::PldhA-ldhLca-difGm的构建
以实施例10中的重组质粒pUC-ldhA用PstI酶切去除其中的400bps的片段并用pfu多聚酶将粘性末端补平，与实施例12中的重组质粒pUC-PldhA-ldhLca-difGm经BamHI酶切经pfu多聚酶将粘性末端补平片段PldhA-ldhLca-difGm连接，获得重组质粒pUC-ldhA::PldhA-ldhLca-difGm。所得重组质粒的物理图谱如附图14所示。将重组质粒pUC-ldhA::PldhA-ldhLca-difGm用ApaLI酶切，并胶回收线性化质粒，用引物ldhA3和ldhA4进行PCR扩增，获得ldhA::PldhA-ldhLca-difGm基因片段。
实施例15——突变盒ldhA::PldhA-ldhStrb-difGm的构建
以实施例10中的重组质粒pUC-ldhA用PstI酶切去除其中的400bps的片段并用pfu多聚酶将粘性末端补平，与实施例13中的重组质粒pUC-PldhA-ldhStrb-difGm经BamHI酶切经pfu多聚酶将粘性末端补平片段PldhA-ldhStrb-difGm连接，获得重组质粒pUC-ldhA::PldhA-ldhStrb-difGm。所得重组质粒的物理图谱如附图15所示。将重组质粒pUC-ldhA::PldhA-ldhStrb-difGm用ApaLI酶切，并胶回收线性化质粒，用引物ldhA3和ldhA4进行PCR扩增，获得ldhA::PldhA-ldhStrb-difGm基因片段。
实施例16——突变盒thiE::PldhA-ldhLca-difGm的构建
将实施例12中的重组质粒pUC-PldhA-ldhLca-difGm经BamHI酶切获得PldhA-ldhLca-difGm片段，DNA聚合酶pfu作用下补平片段PldhA-ldhLca-difGm并克隆入实施例2中的重组质粒pUC-thiE的StuI位点。获得重组质粒pUC-thiE::PldhA-ldhLca-difGm。所得重组质粒的物理图谱如附图16所示。将重组质粒pUC-thiE::PldhA-ldhLca-difGm用ApaLI酶切，并胶回收线性化质粒，用引物ThiE1p和ThiE2p进行PCR扩增，获得thiE::PldhA-ldhLca-difGm基因片段。
实施例17——突变盒thiE::PldhA-ldhStrb-difGm的构建
将实施例13中的重组质粒pUC-PldhA-ldhStrb-difGm经BamHI酶切获得PldhA-ldhStrb-difGm片段，DNA聚合酶pfu作用下补平片段ldhA-ldhStrb-difGm并克隆入实施例2中的重组质粒pUC-thiE的StuI位点。获得重组质粒pUC-thiE::PldhA-ldhStrb-difGm。所得重组质粒的物理图谱如附图17所示。将重组质粒pUC-thiE::PldhA-ldhStrb-difGm用ApaLI酶切，并胶回收线性化质粒， 用引物ThiE1p和ThiE2p进行PCR扩增，获得thiE::PldhA-ldhStrb-difGm基因片段。
实施例18——突变盒thiE::PldhA-ldhBcoa-difGm的构建
将实施例11中的重组质粒pUC-PldhA-ldhBcoa-difGm经BamHI酶切获得PldhA-ldhBcoa-difGm片段，DNA聚合酶pfu作用下补平片段PldhA-ldhBcoa-difGm并克隆入实施例2中的重组质粒pUC-thiE的StuI位点。获得重组质粒pUC-thiE::PldhA-ldhBcoa-difGm。所得重组质粒的物理图谱如附图18所示。将重组质粒pUC-thiE::PldhA-ldhBcoa-difGm用ApaLI酶切，并胶回收线性化质粒，用引物ThiE1p和ThiE2p进行PCR扩增，获得thiE::PldhA-ldhBcoa-difGm基因片段。
实施例19——温控型极高光学纯D-乳酸产生菌株的构建
利用基因整合技术，将出发菌株B0013中的乳酸脱氢酶基因的启动子ldhAp替换为pR-pL启动子(Love C.A.et al.,Gene,1996,176:49-53)，并获得重组菌种大肠杆菌B0013-010B(B0013ldhA::kan-cIts857-pR-pL)。
实施例20——生长可定量调控型极高光学纯D-乳酸产生菌株的构建
利用基因整合技术，构建硫胺素磷酸合成酶(thiE)编码基因删除用突变盒thiE’::difGm，删除步骤2所获重组菌的thiE基因，获得重组菌种大肠杆菌B0013-020B(B0013-010B thiE::dif)。
实施例21——D-乳酸代谢利用途径阻断型极高光学纯D-乳酸产生菌株的构建
利用基因整合技术，构建FAD依赖型D-乳酸脱氢酶(dld)编码基因删除用突变盒dld’::difGm，删除步骤3所获重组菌的dld基因，获得重组菌种大肠杆菌B0013-030B(B0013-020B dld::dif)。
实施例22——厌氧乙酸合成途径阻断型极高光学纯D-乳酸产生菌株的构建
利用基因整合技术，构建乙酸激酶(ackA)和磷酸转乙酰酶(pta)编码基因删除用突变盒ackA-pta’::difGm，删除步骤4所获重组菌的ackA-pta基因，获得重组菌种大肠杆菌B0013-040B(B0013-030B ackA-pta::dif)。
实施例23——丙酮酸回流途径阻断型极高光学纯D-乳酸产生菌株的构建
利用基因整合技术，构建磷酸烯醇式丙酮酸合酶(pps)编码基因删除用突变盒pps’::difGm，删除步骤4和步骤5所获重组菌的pps基因，获得重组菌种大肠杆菌B0013-040C(B0013-030B pps::dif)和B0013-050B(B0013-040B  pps::dif)。
实施例24——甲酸厌氧合成途径阻断型极高光学纯D-乳酸产生菌株的构建
利用基因整合技术，构建丙酮酸甲酸裂解酶(pflB)编码基因删除用突变盒pflB’::difGm，删除步骤4，步骤5和步骤6所获重组菌的pflB基因，获得重组菌种大肠杆菌B0013-040D(B0013-030B pflB::dif)，B0013-050C(B0013-040B pflB::dif)，B0013-050D(B0013-040C pflB::dif)和B0013-060B(B0013-050B pflB::dif)。
实施例25——乙酸好氧合成途径阻断型极高光学纯D-乳酸产生菌株的构建
利用基因整合技术，构建丙酮酸氧化酶(poxB)编码基因删除用突变盒poxB’::difGm，删除步骤4，步骤5，步骤6和步骤7所获重组菌的pflB基因，获得重组菌种大肠杆菌B0013-040E(B0013-030B poxB::dif)，B0013-050E(B0013-040B poxB::dif)，B0013-050F(B0013-040C poxB::dif)，B0013-060C(B0013-050B poxB::dif)，B0013-050G(B0013-040D poxB::dif)，B0013-060D(B0013-050C poxB::dif)，B0013-060F(B0013-050D poxB::dif)和B0013-070B(B0013-060B poxB::dif)。
实施例26——丁二酸合成途径阻断型极高光学纯D-乳酸产生菌株的构建
利用基因整合技术，构建富马酸还原酶(frdA)编码基因删除用突变盒frdA’::difGm，删除步骤4，步骤5，步骤6，步骤7和步骤8所获重组菌的frdA基因，获得重组菌种大肠杆菌B0013-040F(B0013-030B frdA::dif)，B0013-050H(B0013-040B frdA::dif)，B0013-050I(B0013-040C frdA::dif)，B0013-060G(B0013-050B frdA::dif)，B0013-050J(B0013-040D frdA::dif)，B0013-060H(B0013-050C frdA::dif)，B0013-060I(B0013-050D frdA::dif)，B0013-070C(B0013-060B frdA::dif)，B0013-050K(B0013-040E frdA::dif)，B0013-060J(B0013-050E frdA::dif)，B0013-060K(B0013-050F frdA::dif)，B0013-070D(B0013-060C frdA::dif)，B0013-060L(B0013-050G frdA::dif)，B0013-070E(B0013-060D frdA::dif)，B0013-070F(B0013-060F frdA::dif)和B0013-080B(B0013-070B frdA::dif)。
实施例27——乙醇合成途径阻断型极高光学纯D-乳酸产生菌株的构建
利用基因整合技术，构建乙醇脱氢酶(adhE)编码基因删除用突变盒adhE’::difGm，删除步骤4，步骤5，步骤6，步骤7，步骤8和步骤9所获重组菌的adhE基因，获得重组菌种大肠杆菌B0013-040G(B0013-030B adhE::dif)，B0013-050L (B0013-040B adhE::dif)，B0013-050M(B0013-040C adhE::dif)，B0013-060M(B0013-050B adhE::dif)，B0013-050N(B0013-040D adhE::dif)，B0013-060N(B0013-050C adhE::dif)，B0013-060O(B0013-050D adhE::dif)，B0013-070G(B0013-060B adhE::dif)，B0013-050O(B0013-040E adhE::dif)，B0013-060P(B0013-050E adhE::dif)，B0013-060Q(B0013-050F adhE::dif)，B0013-070H(B0013-060C adhE::dif)，B0013-060R(B0013-050G adhE::dif)，B0013-070I(B0013-060D adhE::dif)，B0013-070J(B0013-060F adhE::dif)，B0013-080C(B0013-070B adhE::dif)。B0013-050P(B0013-040F adhE::dif)，B0013-060S(B0013-050H adhE::dif)，B0013-060T(B0013-050I adhE::dif)，B0013-070K(B0013-060G adhE::dif)，B0013-060U(B0013-050J adhE::dif)，B0013-070L(B0013-060H adhE::dif)，B0013-070M(B0013-060I adhE::dif)，B0013-080D(B0013-070C adhE::dif)，B0013-060V(B0013-050K adhE::dif)，B0013-070N(B0013-060J adhE::dif)，B0013-070O(B0013-060K frdA::dif)，B0013-080E(B0013-070D adhE::dif)，B0013-070P(B0013-060L adhE::dif)，B0013-080F(B0013-070E adhE::dif)，B0013-080G(B0013-070F adhE::dif)和B0013-090B(B0013-080B adhE::dif)。
实施例28——D-乳酸合成途径阻断型菌株的构建
利用基因整合技术，构建D-乳酸脱氢酶(ldhA)编码基因删除用突变盒ldhA’::difGm，删除重组菌B0013-070的ldhA基因，获得重组菌种大肠杆菌B0013-080H(B0013-070ldhA::dif)。
实施例29——D-乳酸合成途径阻断型极高光学纯L-乳酸产生菌株的构建
利用基因整合技术，构建D-乳酸脱氢酶基因(ldhA)编码基因删除用突变盒ldhA’::PldhA-ldhLca-difGm，删除重组菌B0013-070的ldhA基因，获得重组菌种大肠杆菌B0013-080I(B0013-070ldhA::PldhA-ldhLca-dif)。
实施例30——D-乳酸合成途径阻断型极高光学纯L-乳酸产生菌株的构建
利用基因整合技术，构建D-乳酸脱氢酶基因(ldhA)编码基因删除用突变盒ldhA’::PldhA-ldhStrb-difGm，删除重组菌B0013-070的ldhA基因，获得重组菌种大肠杆菌B0013-080J(B0013-070ldhA::PldhA-ldhStrb-dif)。
实施例31——L-乳酸代谢利用途径阻断型极高光学纯L-乳酸产生菌株的构建
利用基因整合技术，构建FMN为辅酶的L-乳酸脱氢酶基因(lldD)编码基 因删除用突变盒lldD’::PldhA-ldhBcoa-difGm，删除重组菌B0013-070，B0013-080H，B0013-080I和B0013-080J的lldD基因，获得重组菌种大肠杆菌B0013-080K(B0013-070lldD::PldhA-ldhBcoa-dif)，B0013-090C(B0013-080H lldD::PldhA-ldhBcoa-dif)，B0013-090D(B0013-080I lldD::PldhA-ldhBcoa-dif)和B0013-090E(B0013-080J lldD::PldhA-ldhBcoa-dif)。
实施例32——L-乳酸代谢利用途径阻断型极高光学纯L-乳酸产生菌株的构建
利用基因整合技术，构建FMN为辅酶的L-乳酸脱氢酶基因(lldD)编码基因删除用突变盒lldD’::PldhA-ldhLca-difGm，删除重组菌B0013-070，B0013-080H，B0013-080I和B0013-080J的lldD基因，获得重组菌种大肠杆菌B0013-080L(B0013-070lldD::PldhA-ldhLca-dif)，B0013-090F(B0013-080H lldD::PldhA-ldhLca-dif)，B0013-090G(B0013-080I lldD::PldhA-ldhLca-dif)和B0013-090H(B0013-080J lldD::PldhA-ldhLca-dif)。
实施例33——L-乳酸代谢利用途径阻断型极高光学纯L-乳酸产生菌株的构建
利用基因整合技术，构建FMN为辅酶的L-乳酸脱氢酶基因(lldD)编码基因删除用突变盒lldD’::PldhA-ldhLca-difGm，删除重组菌B0013-070，B0013-080H，B0013-080I和B0013-080J的lldD基因，获得重组菌种大肠杆菌B0013-080M(B0013-070lldD::PldhA-ldhStrb-dif)，B0013-090I(B0013-080H lldD::PldhA-ldhStrb-dif)，B0013-090J(B0013-080I lldD::PldhA-ldhStrb-dif)和B0013-090K(B0013-080J lldD::PldhA-ldhStrb-dif)。
实施例34——生长可定量调控型极高光学纯L-乳酸产生菌株的构建
利用基因整合技术，硫胺素磷酸合成酶(thiE)编码基因删除用突变盒thiE’::difGm，删除步骤11，步骤12，步骤13，步骤14，步骤15和步骤16所获重组菌的thiE基因，获得重组菌种B0013-090L(B0013-080H thiE::dif)，B0013-090M(B0013-080I thiE::dif)，B0013-090N(B0013-080J thiE::dif)，B0013-090O(B0013-080K thiE::dif)，B0013-100B(B0013-090C thiE::dif)，B0013-100C(B0013-090D thiE::dif)，B0013-100D(B0013-090E thiE::dif)，B0013-090E(B0013-080L thiE::dif)，B0013-100E(B0013-090F thiE::dif)，B0013-100F(B0013-090G thiE::dif)，B0013-100G(B0013-090H thiE::dif)， B0013-080N(B0013-070thiE::dif)，B0013-100H(B0013-090I thiE::dif)，B0013-100I(B0013-090J thiE::dif)和B0013-100J(B0013-090K thiE::dif)。
实施例35——生长可定量调控型极高光学纯L-乳酸产生菌株的构建
利用基因整合技术，硫胺素磷酸合成酶(thiE)编码基因删除用突变盒thiE’::PldhA-ldhLca-difGm，删除步骤11，步骤12，步骤13，步骤14，步骤15和步骤16所获重组菌的thiE基因，获得重组菌种B0013-090P(B0013-080H thiE::PldhA-ldhLca-dif)，B0013-090Q(B0013-080I thiE::PldhA-ldhLca-dif)，B0013-090R(B0013-080J thiE::PldhA-ldhLca-dif)，B0013-090S(B0013-080K thiE::PldhA-ldhLca-dif)，B0013-100K(B0013-090C thiE::PldhA-ldhLca-dif)，B0013-100L(B0013-090D thiE::PldhA-ldhLca-dif)，B0013-100M(B0013-090E thiE::PldhA-ldhLca-dif)，B0013-090T(B0013-080L thiE::PldhA-ldhLca-dif)，B0013-100N(B0013-090F thiE::PldhA-ldhLca-dif)，B0013-100O(B0013-090G thiE::PldhA-ldhLca-dif)，B0013-100P(B0013-090H thiE::PldhA-ldhLca-dif)，B0013-080O(B0013-070thiE::PldhA-ldhLca-dif)，B0013-100Q(B0013-090I thiE::PldhA-ldhLca-dif)，B0013-100R(B0013-090J thiE::PldhA-ldhLca-dif)和B0013-100S(B0013-090K thiE::PldhA-ldhLca-dif)。
实施例36——生长可定量调控型极高光学纯L-乳酸产生菌株的构建
利用基因整合技术，硫胺素磷酸合成酶(thiE)编码基因删除用突变盒thiE’::PldhA-ldhStrb-difGm，删除步骤11，步骤12，步骤13，步骤14，步骤15和步骤16所获重组菌的thiE基因，获得重组菌种B0013-090U(B0013-080H thiE::PldhA-ldhStrb-dif)，B0013-090V(B0013-080I thiE::PldhA-ldhStrb-dif)，B0013-090W(B0013-080J thiE::PldhA-ldhStrb-dif)，B0013-090X(B0013-080K thiE::PldhA-ldhStrb-dif)，B0013-100T(B0013-090C thiE::PldhA-ldhStrb-dif)，B0013-100U(B0013-090D thiE::PldhA-ldhStrb-dif)，B0013-100V(B0013-090E thiE::PldhA-ldhStrb-dif)，B0013-090Y(B0013-080L  thiE::PldhA-ldhStrb-dif)，B0013-100W(B0013-090F thiE::PldhA-ldhStrb-dif)，B0013-100X(B0013-090G thiE::PldhA-ldhStrb-dif)，B0013-100Y(B0013-090H thiE::PldhA-ldhStrb-dif)，B0013-080P(B0013-070 thiE::PldhA-ldhStrb-dif)，B0013-100Z(B0013-090I thiE::PldhA-ldhStrb-dif)，B0013-100BB(B0013-090J thiE::PldhA-ldhStrb-dif)和B0013-100BC(B0013-090K thiE::PldhA-ldhStrb-dif)。
实施例37——生长可定量调控型极高光学纯L-乳酸产生菌株的构建
利用基因整合技术，硫胺素磷酸合成酶(thiE)编码基因删除用突变盒thiE’::PldhA-ldhBcoa-difGm，删除步骤11，步骤12，步骤13，步骤14，步骤15和步骤16所获重组菌的thiE基因，获得重组菌种B0013-090Z(B0013-080H thiE::PldhA-ldhBcoa-dif)，B0013-090BB(B0013-080I thiE::PldhA-ldhBcoa-dif)，B0013-090BC(B0013-080J thiE::PldhA-ldhBcoa-dif)，B0013-090BD(B0013-080K thiE::PldhA-ldhBcoa-dif)，B0013-100BD(B0013-090C thiE::PldhA-ldhBcoa-dif)，B0013-100BE(B0013-090D thiE::PldhA-ldhBcoa-dif)，B0013-100BF(B0013-090E thiE::PldhA-ldhBcoa-dif)，B0013-090BE(B0013-080L thiE::PldhA-ldhBcoa-dif)，B0013-100BG(B0013-090F thiE::PldhA-ldhBcoa-dif)，B0013-100BH(B0013-090G thiE::PldhA-ldhBcoa-dif)，B0013-100BI(B0013-090H thiE::PldhA-ldhBcoa-dif)，B0013-080Q(B0013-070 thiE::PldhA-ldhBcoa-dif)，B0013-100BJ(B0013-090I thiE::PldhA-ldhBcoa-dif)，B0013-100BK(B0013-090J thiE::PldhA-ldhBcoa-dif)，B0013-100BL(B0013-090K thiE::PldhA-ldhBcoa-dif)，B0013-100BM(B0013-090U thiE::PldhA-ldhBcoa-dif)，B0013-100BN(B0013-090V thiE::PldhA-ldhBcoa-dif)，B0013-100BO(B0013-090W thiE::PldhA-ldhBcoa-dif)，B0013-100BP(B0013-090X thiE::PldhA-ldhBcoa-dif)，B0013-101B(B0013-100T  thiE::PldhA-ldhBcoa-dif)，B0013-101C(B0013-100U thiE::PldhA-ldhBcoa-dif)，B0013-101D(B0013-100V thiE::PldhA-ldhBcoa-dif)，B0013-100BQ(B0013-090Y thiE::PldhA-ldhBcoa-dif)，B0013-101E(B0013-100W thiE::PldhA-ldhBcoa-dif)，B0013-101F(B0013-100X thiE::PldhA-ldhBcoa-dif)，B0013-101G(B0013-100Y thiE::PldhA-ldhBcoa-dif)，B0013-090BF(B0013-080P thiE::PldhA-ldhBcoa-dif)，B0013-101H(B0013-100Z thiE::thiE::PldhA-ldhBcoa-dif)，B0013-101I(B0013-100BB thiE::PldhA-ldhBcoa-dif)和B0013-101J(B0013-100BC thiE::PldhA-ldhBcoa-dif)。
实施例38——极高光学纯D-乳酸高产菌株的筛选
将步骤19-27所获得的重组菌种分别在添加0.06-100μg/L硫胺素，25-36℃、200r/min好氧生长-10h，再在37-50℃静置培养发酵乳酸，并以出发菌种B0013-070作为对照菌种，分析D-乳酸合成水平，所产D-乳酸光学纯度和化学纯度，并筛选出最优菌种。筛选结果如附图19和表1所示。
表1：极高光学纯D-乳酸高产菌株的筛选结果
Figure PCTCN2015093686-appb-000001
Figure PCTCN2015093686-appb-000002
Figure PCTCN2015093686-appb-000003
实施例39——极高光学纯L-乳酸高产菌株的筛选
将步骤28-37所获得的重组菌种分别在添加0.06-100μg/L硫胺素，25-36℃、200r/min好氧生长-10h，再在37-50℃静置培养发酵乳酸，并以出发菌种B0013-070作为对照菌种，分析L-乳酸合成水平，所产L-乳酸光学纯度和化学纯度，并筛选出最优菌种。筛选结果如附图20和表2所示。
表2：极高光学纯L-乳酸高产菌株的筛选结果
Figure PCTCN2015093686-appb-000004
Figure PCTCN2015093686-appb-000005
Figure PCTCN2015093686-appb-000006
Figure PCTCN2015093686-appb-000007
实施例40——极高光学纯D-乳酸的发酵生产
将实施例38所获得的最优菌种在7L-30,000L发酵罐中进行乳酸发酵试验。发酵过程中定时取样，分析细胞密度、糖耗、乳酸产率、代谢主要中间产物及其它有机酸产物等。在25-36℃进行好氧培养至OD600值约为15-40，将发酵罐温度设定为37-50℃继续好氧培养0-120min，再将通气量设为0-0.2vvm进行限氧发酵，限氧阶段0-3h发酵温度为33-39℃，3-6h发酵温度为37-42℃，6-10h发酵温度为38-45℃，10-16h发酵温度为40-48℃，16-24h发酵温度为45-50℃。其发酵培养基为(g/L)：磷酸氢二铵0-25，磷酸二氢钾0-5，磷酸氢二钠，0-25，氯化钠0-5，MgSO4 0-0.5，FeSO4 0-1，FeCl3 0-1，CoCl2 0-1，CuCl2 0-1，Na2MoO4 0-1，H3BO3 0-1，MnCl2 0-1，柠檬酸0-25，硫胺素0-1，木糖0-50，甘油0-50，葡萄糖0-50，硫酸0-5，pH 6.0-7.5。
详细发酵结果见附图21和附图22。
实施例41——极高光学纯L-乳酸高产菌株的发酵生产
将实施例39所获得的最优菌种在7L-30,000L发酵罐中进行乳酸发酵试验。发酵过程中定时取样，分析细胞密度、糖耗、乳酸产率、代谢主要中间产物及其它有机酸产物等。在25-36℃进行好氧培养至OD600值约为15-40，将发酵罐温度设定为37-50℃继续好氧培养0-120min，再将通气量设为0-0.2vvm进行限氧发酵，限氧阶段发酵温度为37-50℃。其发酵培养基为(g/L)：磷酸氢二铵0-25，磷酸二氢钾0-5，磷酸氢二钠，0-25，氯化钠0-5，MgSO4 0-0.5，FeSO4 0-1，FeCl3 0-1，CoCl2 0-1，CuCl2 0-1，ZnCl2 0-1，Na2MoO4 0-1，H3BO3 0-1，MnCl2 0-1，柠檬酸0-25，硫胺素0-1，木糖0-50，甘油0-50，葡萄糖0-50，硫酸0-5，pH 6.0-7.5。
详细发酵结果见附图23和附图24。
实施例42——菌种10吨发酵罐中5批次发酵生产极高光学纯D-乳酸
将实施例38获得的极高纯D-乳酸最优生产菌株在10吨发酵罐中进行5批次的连续生产。
(1)一级种子扩培
取出1ml超低温甘油管保藏菌种，接种专用液体培养基(ZT1培养基)l L/5 L瓶。37℃，200r/min培养10-13h。
ZT1培养基配制
配料1组分共计12.6g，加水溶解至700mL，121℃，灭菌20min。
配料2组分共计18g，加水溶解至150mL单独灭菌，接种时添加。
配料3组分共计5g，加水溶解至150mL单独灭菌，接种时添加。
其中ZT1培养基组成包括(g/L)：磷酸氢二铵0-25，磷酸二氢钾0-5，磷酸氢二钠，0-25，氯化钠0-5，MgSO4 0-0.5，FeSO4 0-1，FeCl3 0-1，CoCl2 0-1，CuCl2 0-1，ZnCl2 0-1，Na2MoO4 0-1，H3BO3 0-1，MnCl2 0-1，柠檬酸0-25，硫胺素0-1，木糖0-50，甘油0-50，葡萄糖0-50，硫酸0-5，pH 6.0-7.5。
(2)二级种子扩培
上述种子液1L接种100L二级种子罐，装液量50L。33-37℃低强度通气培养10-13h。
二级种子罐ZT2培养基配制
配料1组分共计570g，加水溶解至30L，121℃，灭菌20min。
配料2组分共计810g，加水溶解至5L单独灭菌，接种时添加。
配料3组分共计1350g，加水溶解至5L单独灭菌，接种时添加。
其中ZT1培养基组成包括(g/L)：磷酸氢二铵0-25，磷酸二氢钾0-5，磷酸氢二钠，0-25，氯化钠0-5，MgSO4 0-0.5，FeSO4 0-1，FeCl3 0-1，CoCl2 0-1，CuCl2 0-1，ZnCl2 0-1，Na2MoO4 0-1，H3BO3 0-1，MnCl2 0-1，柠檬酸0-25，硫胺素0-1，木糖0-50，甘油0-50，葡萄糖0-50，硫酸0-5，pH 6.0-7.5。
(3)发酵
将上述二级扩培的种子液50L转入10m3发酵罐，工作初始体积为5m3，发酵培养基基础为：ZT2培养基。33-37℃低强度通气培养8-9h，起始通风比1：0.3(料：风)，培养过程中提高转速和通气量控制溶氧不低于20％。加Ca(OH)2维持pH 7.0。转入非通风发酵产酸阶段，发酵温度为40-45℃，Ca(OH)2维持pH 7.0。补加葡萄糖至终浓度50g/L(按照起始发酵体积计算)，分四批补加，间隔时间3h。发酵20-22h，测定残余葡萄糖低于0.1％，即可放罐。发酵结束按照上述流程重复进行五批次发酵生产。发酵结果见表3。
表3：五批次10吨罐发酵生产极高光学纯D-乳酸结果
Figure PCTCN2015093686-appb-000008
实施例43——菌种10吨发酵罐中5批次发酵生产极高光学纯L-乳酸
按照实施例42中的发酵操作流程，将生产菌株替换为实施例39所获得的最优菌种在10吨发酵罐中进行5批次的连续生产。发酵结果见表4。
表4：五批次10吨罐连续转化生产极高光学纯L-乳酸结果
Figure PCTCN2015093686-appb-000009
实施例44——极高光学纯D-乳酸和L-乳酸的产品制备
发酵结束直接在发酵液中缓慢加入浓度为1％-50％的硫酸进行酸化。边搅拌边慢慢流加硫酸，先按体积的1％-18％加入；以BaCl2和草酸铵试剂分别检测，偏酸性时，停止加酸搅拌反应6小时，搅拌时间全程；反应终点以BaCl2和草酸铵试剂分别校验。
板框过滤：酸化液经板框过滤机压滤。压滤后以85℃热水洗涤滤饼，清洗 至洗液中乳酸浓度低于0.1％。清洗结束用压缩空气压干，洗液并入清液中。菌体和固体钙盐用作水泥或混凝土配料。
超滤滤：经超滤去除色素、蛋白质、氨基酸和残留的菌体等。为了提高超滤效率，超滤前对滤过液进行蒸发浓缩，浓缩比为4:1，浓缩液进行超滤。
离子交换：超滤后获得的滤清液进行离子交换。
阳离子交换：732交联为7％的苯乙烯·二乙烯共聚体上带有磺酸基(-SO3H)的阳离子交换树脂(·001×7(732)强酸性苯乙烯系阳离子交换树脂)；离交前料液要冷却到室温。再生处理5％W/v的HCl溶液；中间检查：Fe2+不超过1ppm，交换流速为300ml/min(视检测结果定)。
阴离子交换：330(701)弱碱性阴离子交换树脂(弱碱性环氧系阴离子交换树脂，主要用于水处理中除去Cl-、SO42-等离子，除去无机酸，提取有机酸和脱色，并可回收铜、银离子。)；再生处理：5％W/v NaOH溶液；交换流速为300ml/min(视检测结果定)。中间检查：Cl-不超过3ppm；SO42-不超过5ppm.
产品浓缩：浓缩至产品要求的乳酸浓度。该产品为聚合级D-乳酸/L-乳酸。
综上所述，本发明通过基因工程技术对D-乳酸和L-乳酸高产重组菌染色体上的乳酸脱氢酶编码基因的表达进行简易条件下的动态调控，从而实现了重组菌从葡萄糖高效单一生产D-乳酸和高效单一生产L-乳酸的简洁发酵工艺。本发明技术经过简单修改后，同样可以用于其它工业上重要的微生物代谢产物，但不限于，如柠檬酸、甲酸、乙酸、丙酮酸、丁二酸、苹果酸、α-酮戊二酸、丁二酸、己二酸、戊二胺、己二胺、甲基丙烯酸、异戊二烯、衣康酸等多种有机酸和有机胺；或脯氨酸、丙氨酸、赖氨酸、蛋氨酸、谷氨酸、精氨酸等多种氨基酸；硫胺素、维生素B12等多种微生物；或乙醇、丙醇等短链醇；或低聚异麦芽糖、低聚果糖、低聚半乳糖等多种功能糖的菌种构建、发酵生产和新工艺技术的建立与应用。
上述实施例中，ldhA1核苷酸序列为序列表<400>1所示序列，ldhA2核苷酸序列为序列表<400>2所示序列，PPL1核苷酸序列为序列表<400>3所示序列，PPL2核苷酸序列为序列表<400>4所示序列，PPL3核苷酸序列为序列表<400>5所示序列，PPL4核苷酸序列为序列表<400>6所示序列，Ec-RlA1核苷酸序列为序列表<400>7所示序列，Ec-RlA2核苷酸序列为序列表<400>8所示序列，ThiE1p核苷酸序列为序列表<400>9所示序列，ThiE2p核苷酸序列为序列表<400>10所示序列，Dld1核苷酸序列为序列表<400>11所示序列，Dld2核苷酸序列为序列表 <400>12所示序列，AckA-Pta1核苷酸序列为序列表<400>13所示序列，AckA-Pta2核苷酸序列为序列表<400>14所示序列，Pps1核苷酸序列为序列表<400>15所示序列，Pps2核苷酸序列为序列表<400>16所示序列，RPps1核苷酸序列为序列表<400>17所示序列，RPps2核苷酸序列为序列表<400>18所示序列，PflB1核苷酸序列为序列表<400>19所示序列，PflB2核苷酸序列为序列表<400>20所示序列，PoxB1核苷酸序列为序列表<400>21所示序列，PoxB2核苷酸序列为序列表<400>22所示序列，FrdA1核苷酸序列为序列表<400>23所示序列，FrdA2核苷酸序列为序列表<400>24所示序列，AdhE1核苷酸序列为序列表<400>25所示序列，AdhE2核苷酸序列为序列表<400>26所示序列，ldhA3核苷酸序列为序列表<400>27所示序列，ldhA4核苷酸序列为序列表<400>28所示序列，ldhA5核苷酸序列为序列表<400>29所示序列，ldhA6核苷酸序列为序列表<400>30所示序列，RldhA1核苷酸序列为序列表<400>31所示序列，RldhA2核苷酸序列为序列表<400>32所示序列，BcoaLDH1核苷酸序列为序列表<400>33所示序列，BcoaLDH4核苷酸序列为序列表<400>34所示序列，LldD1核苷酸序列为序列表<400>35所示序列，LldD2核苷酸序列为序列表<400>36所示序列，LcaLDH1核苷酸序列为序列表<400>37所示序列，LcaLDH4核苷酸序列为序列表<400>38所示序列，StrbLDH1核苷酸序列为序列表<400>39所示序列，StrbLDH2核苷酸序列为序列表<400>40所示序列。具体信息如下：
SEQ ID No.1的信息：
(i)序列特征：
(A)长度：27bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.1
ldhA1：TCCGGTACCCAGCCCGAGCGTCATCAG；KpnI
SEQ ID No.2的信息：
(i)序列特征：
(A)长度：25bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.2
ldhA2：GTCAAGGTCGACGTTATTGAAACCG；
SEQ ID No.3的信息：
(i)序列特征：
(A)长度：28bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.3
PPL1：AGCTTGGCTGCAGGTGATGATTATCAGC；
SEQ ID No.4的信息：
(i)序列特征：
(A)长度：24bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.4
PPL2：ATCGCCGGCAATTCGTAATCATGG；EcoRI
SEQ ID No.5的信息：
(i)序列特征：
(A)长度：30bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.5
PPL3：TAAGATATCCCATGATTACGAATTGCCGGC；EcoRV
SEQ ID No.6的信息：
(i)序列特征：
(A)长度：37bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.6
PPL4：TAAGAATTCAGTTAACCTCCTTAGGATCCCAATGCTT EcoRI
SEQ ID No.7的信息：
(i)序列特征：
(A)长度：39bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.7
Ec-RlA1：TAAGAATTCATGAAACTCGCCGTTTATAGCACAAAACAG；EcoRI
SEQ ID No.8的信息：
(i)序列特征：
(A)长度：30bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.8
Ec-RlA2:AAGACTTTCTCCAGTGATGTTGAATCACAT；
SEQ ID No.9的信息：
(i)序列特征：
(A)长度：29bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.9
ThiE1p：AGAGAATTCATTCATCGCCAACTCCTGCA
SEQ ID No.10的信息：
(i)序列特征：
(A)长度：32bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.10
ThiE2p：AGAGAATTCGGTGGACAGCGTACAGTGGATCG；
SEQ ID No.11的信息：
(i)序列特征：
(A)长度：26bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.11
Dld1：AGTACGTCTTGATACCTTCGAAGCGG；
SEQ ID No.12的信息：
(i)序列特征：
(A)长度：23bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.12
Dld2：GGATTCATGCTGTTGGTCGGATC；
SEQ ID No.13的信息：
(i)序列特征：
(A)长度：23bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.13
AckA-Pta1：TGAACATCATCACCTGCCACCTG；
SEQ ID No.14的信息：
(i)序列特征：
(A)长度：19bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.14
AckA-Pta2：CAGCGCAAAGCTGCGGATG
SEQ ID No.15的信息：
(i)序列特征：
(A)长度：25bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.15Pps1：CGGCATGAATGATGTAGACAGGGTT
SEQ ID No.16的信息：
(i)序列特征：
(A)长度：23bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.16
Pps2：TAACCAGGTTTGCACCACGGTGT
SEQ ID No.17的信息：
(i)序列特征：
(A)长度：21bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.17
RPps1：TGTGGCGAAACCATTCGGAAC
SEQ ID No.18的信息：
(i)序列特征：
(A)长度：22bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.18
RPps2:GTCCGACCACGAAGACTTTGCC
SEQ ID No.19的信息：
(i)序列特征：
(A)长度：23bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.19
PflB1：TTCAGACTTCGGACCAACCTGCA
SEQ ID No.20的信息：
(i)序列特征：
(A)长度：20bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.20
PflB2：CCGCGAACTGGATCCGATGA；
SEQ ID No.21的信息：
(i)序列特征：
(A)长度：24bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.21
PoxB1：CAAACGGTTGCAGCTTATATCGCC
SEQ ID No.22的信息：
(i)序列特征：
(A)长度：24bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.22
PoxB2：TGCGGTGGAATGGCTAACTCTTCT
SEQ ID No.23的信息：
(i)序列特征：
(A)长度：23bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.23
FrdA1：CTTTCAAGCCGATCTTGCCATTG
SEQ ID No.24的信息：
(i)序列特征：
(A)长度：24bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.24
FrdA2：ACTCTTTACGTGCCATTGCGGAGT
SEQ ID No.25的信息：
(i)序列特征：
(A)长度：24bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.25
AdhE1：ATCTGATCGGCTGGATCGATCAAC
SEQ ID No.26的信息：
(i)序列特征：
(A)长度：22bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.26
AdhE2：GAACCAGGTTGGCGTCGACAAT
SEQ ID No.27的信息：
(i)序列特征：
(A)长度：35bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.27
ldhA3：TAAGAATTCTTATGAAACTCGCCGTTTATAGCACA EcoRI
SEQ ID No.28的信息：
(i)序列特征：
(A)长度：38bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.28
ldhA4：CTTGAATTCAAGCTTGCTGCCGGAAATCATCATTTTTT EcoRI,HindIII
SEQ ID No.29的信息：
(i)序列特征：
(A)长度：21bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.29
ldhA5：GGGCAGCCCGAGCGTCATCAG
SEQ ID No.30的信息：
(i)序列特征：
(A)长度：23bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.30
ldhA6：GCTGCCGGAAATCATCATTTTTT
SEQ ID No.31的信息：
(i)序列特征：
(A)长度：30bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.31
RldhA1：GGAAGATCTTCCGCGAGTTTCATAAGACTT BglII
SEQ ID No.32的信息：
(i)序列特征：
(A)长度：24bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.32
RldhA2：CGGAATTCCGAACGAACTGGTTTA EcoRⅠ
SEQ ID No.33的信息：
(i)序列特征：
(A)长度：33bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.33
BcoaLDH1CCGGATCCAATCAGGGTGTTGCAGAAGAGCTTG BamHI
SEQ ID No.34的信息：
(i)序列特征：
(A)长度：34bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.34
BcoaLDH4GCGGAATTCTTACAATACAGGTGCCATCGTTTCT EcoRI
SEQ ID No.35的信息：
(i)序列特征：
(A)长度：28bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.35
LldD1GGCCCGGGCATGATTATTTCCGCAGCCA SmaI
SEQ ID No.36的信息：
(i)序列特征：
(A)长度：30bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.36
LldD2：GGCCCGGGCAGGCAACTCTTTACCCAGCCC SmaI
SEQ ID No.37的信息：
(i)序列特征：
(A)长度：30bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.37
LcaLDH1CGCGGATCCAGTATTACGGATAAGGATCAC BamHI
SEQ ID No.38的信息：
(i)序列特征：
(A)长度：25bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.38
LcaLDH4CGCCTGCAGTCCTGTTCTTCGTTTG PstI
SEQ ID No.39的信息：
(i)序列特征：
(A)长度：28bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.39
StrbLDH1CGCGGATCCACTAAACAACACAAAAAAG BamHI
SEQ ID No.40的信息：
(i)序列特征：
(A)长度：28bp
(B)类型：核酸
(C)链性：单链
(D)拓扑结构：线性
(ii)分子类型：寡核苷酸
(iii)序列描述：SEQ ID No.40
StrbLDH2CCGGAATTCTACAGGGATTGTTGCCGCA EcoRI
上述参照实施例对该一种用于构建乳酸的菌株及其制备方法与应用进行的详细描述，是说明性的而不是限定性的，可按照所限定范围列举出若干个实施例，因此在不脱离本发明总体构思下的变化和修改，应属本发明的保护范围之内。

Claims (12)

  1. 一种用于发酵生产极高光学纯D-乳酸的聚合级乳酸单体生产菌，其特征在于：菌株保藏号为CGMCC No.11059。
  2. 一种用于发酵生产极高光学纯L-乳酸的聚合级乳酸单体生产菌，其特征在于：菌株保藏号为CGMCC No.11060。
  3. 权利要求1或2所述聚合级乳酸单体生产菌的构建方法，其特征在于：
    单个或多个基因被敲除进行菌株初步构建，这些基因包括：ldhA，thiE，dld，ackA，pta，pps，pflB，poxB，frdA，adhE，lldD；单个或多个基因被表达，这些基因包括：kan-cIts857-pR-pL-ldhA,ldhBcoa，ldhLca，ldhStrb；
    使用了温度诱导型基因转录方式来控制和调节细胞生长过程和乳酸形成过程，包括：菌株初步构建后，菌株在25-50℃条件下分阶段完成细胞发酵培养-诱导-产酸，细胞生长过程在单一发酵因子调控下可以定量控制细胞的积累量，所述单一发酵因子是细胞中央代谢途径关键酶转录过程的调控因子、关键酶翻译过程的调控因子、关键酶分泌过程的调控因子或关键酶表达后催化过程调控因子。
  4. 根据权利要求3所述的聚合级乳酸单体生产菌的构建方法，所述菌株初步构建后菌株的生长过程都与单一发酵因素的调控相关联，所述单一发酵因素是葡萄糖、甘油、酵母膏、蛋白胨、硫酸铵、磷酸氢铵、铁离子、镁离子、钙离子、锌离子、锰离子、钴离子、铜离子、钠离子、钾离子、VB1、VB6、VB12、生物素、盐酸硫胺素或焦磷酸硫胺素的一种或几种，所述单一发酵因素能够定量的控制细胞的积累过程，并保证细胞快速积累且具备高活性；以开关控制的方式启动或关闭乳酸的合成过程，并且这一过程通过预先定量添加对应的单一发酵因素完成所述调控过程。
  5. 一种乳酸制造技术，其特征在于：具体方法为：应用权利要求1和/或2所述聚合级乳酸单体生产菌，所述菌株的宿主细胞生长过程在全合成的无机盐培养基中快速进行，所述全合成的无机盐培养基为含硫胺素的培养基，所述宿主细胞在全合成培养基中快速生长，其中，7L发酵体系下于8-10h内菌体量积累到细胞干重11.5g/L，通过定量补加单一发酵因子，细胞干重进行更高积累，宿主细胞培养过程中不大量形成乳酸，即乳酸的痕量形成不影响细胞生长，宿主细胞在生长完成后快速积累乳酸。
  6. 根据权利要求5所述的乳酸制造技术，其特征在于：D-乳酸和L-乳酸的 积累过程通过更换相应聚合级乳酸单体生产菌在同一套生产体系下切换式进行；所述的发酵产酸过程具备可设定型的自动启动特征，通过培养基组分和菌种自身生长特性的组合，细胞生长至特定阶段后由于培养基中对应调控因子组分的含量变化自动启动产酸过程。
  7. 根据权利要求5所述的乳酸制造技术，其特征在于：所述全合成的无机盐培养基主体成分如下：
    用于D-乳酸发酵生产的发酵培养基(g/L)：磷酸氢二铵0-25，磷酸二氢钾0-5，磷酸氢二钠0-25，氯化钠0-5，MgSO4 0-0.5，FeSO4 0-1，FeCl3 0-1，CoCl2 0-1，CuCl2 0-1，Na2MoO4 0-1，H3BO3 0-1，MnCl2 0-1，柠檬酸0-25，硫胺素0-1，木糖0-50，甘油0-50，葡萄糖0-50，硫酸0-5，pH 6.0-7.5。
    用于L-乳酸发酵生产的发酵培养基(g/L)：磷酸氢二铵0-25，磷酸二氢钾0-5，磷酸氢二钠0-25，氯化钠0-5，MgSO4 0-0.5，FeSO4 0-1，FeCl3 0-1，CoCl2 0-1，CuCl2 0-1，ZnCl2 0-1，Na2MoO4 0-1，H3BO3 0-1，MnCl2 0-1，柠檬酸0-25，硫胺素0-1，木糖0-50，甘油0-50，葡萄糖0-50，硫酸0-5，pH6.0-7.5。
  8. 根据权利要求5所述的乳酸制造技术，其特征在于：所述菌株的生长过程以及乳酸形成过程都与发酵温度的过程相关联，发酵产酸过程在非固定温度下完成的，根据生产菌种产酸特性，其发酵温度的变化呈现梯度上升的趋势，发酵温度升高生长会变缓慢，而发酵温度降低生长会非常迅速，具备积累高活性细胞的特点；发酵温度升高乳酸会快速形成，而发酵温度降低乳酸会缓慢形成，甚至不形成，具备高效积累乳酸的特点。
  9. 根据权利要求5所述的乳酸制造技术，其特征在于：所述菌株先在25-36℃下利用葡萄糖快速生长形成菌体，然后在37-50℃下利用葡萄糖快速积累乳酸。
  10. 根据权利要求5所述的乳酸制造技术，其特征在于：具体步骤为：
    (1)通过基因工程技术对D-乳酸和L-乳酸高产重组菌染色体上的乳酸脱氢酶编码基因的表达进行简易条件下的动态调控获得产酸菌株；
    (2)将菌株在25-36℃、200r/min好氧生长6-10h，再在37-45℃静置培养发酵乳酸，并以出发菌种B0013-070作为对照菌种，分析细胞密度、糖耗、乳酸产率、代谢主要中间产物及其它有机酸产物等，确定乳酸合成诱导时机；菌株分别在添加0.06-100μg/L硫胺素、200r/min进行摇瓶培养；
    (3)发酵生产极高光学纯D-乳酸时，限氧阶段0-3h发酵温度为33-39℃， 3-6h发酵温度为37-42℃，6-10h发酵温度为38-45℃，10-16h发酵温度为40-48℃，16-24h发酵温度为45-50℃。
    (3’)发酵生产极高光学纯L-乳酸时，限氧阶段发酵温度为37-50℃，其余同步骤(3)。
  11. 根据权利要求5所述的乳酸制造技术，其特征在于：还包括发酵结束后乳酸的提取方法，结合生产菌种基因工程改造后的特征，发酵液中D-乳酸和L-乳酸均以极高光学纯度和化学纯度的形式存在，全合成培养基的使用保证了后提取过程的简洁型，其最终产品的提取方式包括：酸化，板框去除菌体，超滤去除色素和杂蛋白，离子交换去除阴阳离子干扰，浓缩制备相应浓度的产品，并纳滤精制产品。
  12. 根据权利要求5所述的乳酸制造技术，其特征在于：所述乳酸形成过程结束后通过低温酸化的方式将D-乳酸和L-乳酸游离出来，且上述乳酸游离的过程不受发酵液中其他残余物的影响，酸化使用的酸是硫酸、盐酸或草酸。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115011537A (zh) * 2022-06-14 2022-09-06 湖北工业大学 一株双厌氧启动子诱导产高光学纯l-乳酸的工程菌及其制备方法与应用
CN115011537B (zh) * 2022-06-14 2023-06-23 湖北工业大学 一株双厌氧启动子诱导产高光学纯l-乳酸的工程菌及其制备方法与应用

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