途径工程改造大肠杆菌转化甘油合成乳酸
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摘要
当前，石油基化学品作为原材料广泛的应用在聚合材料，纺织品，油漆和有机溶剂等领域。然而在全球气候变暖和石油资源日趋紧缺的大背景下，聚乳酸作为生物可降解材料的典型代表被认为是石油基塑料等材料的主要替代品之一。利用大宗廉价原料，制备聚乳酸加工中需求的极高光学纯度和极高化学纯度的D-乳酸和L-乳酸，是聚乳酸产业发展的必需，也是有机酸工业发展与变革的新动力。与此同时，受油脂水解工业和生物柴油高速发展的推动，甘油这一原来紧缺的多元醇，正逐步成为未来理想的发酵工业大宗原料。为此，本研究以甘油为原料，以乳酸为目标产品，以代谢途径改造获得优良菌种及其发酵新工艺为主要研究内容，以获得高底物转化率、高产物形成率、目标产物的高光学和高化学纯度的乳酸发酵为目标，通过研究，获得如下主要研究结果。
1．对相关代谢途径进行遗传改造，选育获得了代谢甘油为D-乳酸的高产菌株
通过发酵产酸阶段供氧强度的提高建立了甘油高效转化为D-乳酸的发酵工艺，该工艺下，菌种B0013-070在发酵甘油合成D-乳酸的发酵试验中，发酵液中D-乳酸的浓度达到98.5g/L，合成强度为3.45g/L h。在B0013-070基础上，进一步提高D-乳酸脱氢酶的表达强度，则甘油发酵生成D-乳酸的合成强度和转化率分别提高为3.65g/L h和78.0%，发酵液中D-乳酸的浓度达到103.1g/L；采用34℃下菌体生长和42℃发酵产酸的乳酸发酵工艺，此菌发酵甘油为D-乳酸的合成强度和转化率提高到3.66g/L h和82.6%。再通过同源重组技术将温度诱导型启动子pR-pL引入B0013-070的D-乳酸脱氢酶编码基因ldhA的上游并替代其原启动子PldhA，获得D-乳酸高产新菌种B0013-070B，发酵试验表明，其发酵甘油合成D-乳酸的合成强度和转化率提高到4.23g/L h和88.9%。
2.通过基因删除与基因表达改造大肠杆菌相关代谢途径，选育获得了L-乳酸高产菌种
在上述研究获得的B0013-070菌株的基础上，通过基因删除技术，获得ldhA基因删除突变株B0013-080C；对Bacillus coagulans CICIM B1821的L-乳酸脱氢酶编码基因进行克隆，获得L-乳酸脱氢酶基因BcoaLDH。通过基因重组技术，用PldhA引导基因BcoaLDH的转录；通过同源重组技术，将BcoaLDH表达盒插入到菌株B0013-080C的lldD中间，同步突变lldD基因（lldD编码分解L-乳酸的L-乳酸脱氢酶），获得新菌种B0013-090B。发酵试验结果表明，此菌株具有很好的代谢甘油生成L-乳酸的能力，发酵液中L-乳酸浓度达到132.4g/L，甘油到L-乳酸合成强度和转化率分别为4.90g/L h和93.7%。新菌种合成L-乳酸的光学纯度高于99.9%。
3.选育出了硫胺素营养缺陷型突变株，研究了此营养缺陷性表型在细胞生长控制中的应用，通过发酵试验验证了可控菌体生长可进一步改善重组菌代谢甘油合成乳酸的效率
细胞生成是乳酸发酵过程及其控制中的关键因素。为研究细胞生长的简便控制方法，在上述高产菌株B0013-070的基础上，通过同源重组获得了thiE突变的新菌株，B0013-080A，其在不添加硫胺素的培养基中不能生长。进而在液体培养条件下，分析了硫胺素添加量与菌体生成量之间的关系，得出了两者之间的关系式为：y=(4.0×1010)x+1.5902(R~2=0.9987)。采用上述相似的发酵工艺，在发酵体系中添加终浓度2.47×10-7g/mL的盐酸硫胺素，发酵液中D-乳酸浓度可达119.3g/L，甘油到D-乳酸的合成强度和转化率提高到4.06g/L h和87.1%。进一步将上述研究中温度诱导D-乳酸脱氢酶的表达引入，获得新菌种B0013-080B。此菌株在相似的发酵条件下，发酵液中D-乳酸浓度可达129.3g/L，甘油到D-乳酸的合成强度和转化率提高到4.79g/L h和92.1%。
Currently, petrochemicals are used as raw materials in the manufacturing of a variety ofproducts such as polymers, textiles, paints and solvents etc. However, rapid depletion of thepetroleum resource and increase in emission of greenhouse gas encouraged a replacement ofpetroleum with renewable resources. Polylactic acid, as typical biodegradable material, hasshowed a potential to replace petrochemical-based plastics. Manufacturing polylactic acidneeds large amounts of monomer, D-lactic acid and/or L-lactic acid. Therefore,biotechnological production of D-lactic acid and/or L-lactic acid with high optical purity andchemical purity has been increasingly focused in recent years. Production of lactic acid fromcheap available biomass was investigated extensively. Glycerol is the most readily availablerenewable feedstock. With the increasing international biodiesel production, a surplus ofcrude glycerol has been generated resulting in a significant price reduction. In addition to lowcost and abundance, the higher degree of reduction makes glycerol be an excellent potentialcarbon source to produce chemicals with high yield.
In this study, metabolic engineering and process optimization were employed toinvestigate bioconversion of glycerol into D-lactic acid and L-lactic acid by Escherichia coli.The main research findings are as follows:
1. Efficient production of D-lactic acid from glycerol was achieved through geneticmodification of the metabolic pathway and optimization of the fermentation process.
The fermentation process for D-lactate production from glycerol was optimized byincreasing the oxygen supply strength during lactate formation phase. In7-L bioreactor andusing B0013-070strain,98.5g/L of the final concentration and3.45g/L of productivity ofD-lactate were generated, respectively. Furthermore, increasing copy of ldhA gene encodingD-lactate dehydrogenase led to higher production of D-lactic acid, and the final D-lactic acidconcentration and productivity were103.1g/L and3.65g/L h, respectively. The yield reached78.0%.
Moreover, temperature was optimized during cell growth phase and lactate productionphase, respectively. The resulting showed that34°C was optimal for cell growth and42°Cwas optimal for production of lactic acid. Using the temperature-shifting process, engineeredstrain B0013-070produced D-lactic acid with a3.66g/L h of productivity and82.6%of yield.Using another strain E. coli B0013-070B in which the promoter of ldhA was replaced bytemperature-inducible promoter, production performance of D-lactic acid further increased bythis fermentation process. And4.23g/L h of productivity and88.9%of yield were obtained.
2. Metabolic engineering of E. coli for efficient production of L-lactate from glycerol
An exogenous L-lactate dehydrogenase gene (BcoaLDH) was cloned from thermophilicBacillus coagulans CICIM B1821and expressed in E. coli to achieve L-lactic acid producerfrom glycerol. To further increase L-lactic acid production, the BcoaLDH fusing promoter ofldhA was inserted into E. coli chromosome and replacing of the lldD gene that encodes FMN-dependent L-lactate dehydrogenase catalyzing L-lactic acid to pyruvate. Furthermore,the D-lactic acid synthesis pathway was blocked by deleting the ldhA gene to realize extremelyhigh optical purity L-lactic acid synthesis. The resulting strain B0013-090B was used toevaluate the production of L-lactic acid. Using temperature-shifting process,132.4g/L ofL-lactic acid was produced from glycerol, and4.90g/L h of productivity and93.7%of yieldwere obtained, respectively. The optical purity of L-lactic acid reached to99.9%.
3. Fine switch of cell growth and latctic acid production using thiaminehydrochloride through deletion of thiE gene producing thiamine auxotrophy phenotype.And the concise fermentation process of engineered strain was developed for D-lactateproduction.
The thiE gene encodes a critical protein in the synthesis route of thiamine, a cofactor forPDH complex. Therefore, deletion of thiE gene produced thiamine auxotrophy phenotype.We deleted the thiE gene in the D-lactate producer Escherichia coli CICIM B0013-070(ackA, pta, pps, pflB, dld, poxB, adhE, frdA) to generate CICIM B0013-080A.The cell mass of B0013-080A was fine cotrolled by the concentration of thiaminehydrochloride (VB1) added into medium. The equation of the relationship between theamount of VB1and the biomass was obtained as follows: y=(4.0×1010)x+1.5902(R2=0.9987),where y is the obtained biomass and x is the amount of thiamine hydrochloride (VB1) added.When VB1was added into the fermentation medium with a final concentration of2.47×10-7μg/mL, engineered strain performed better for cell growth and D-lactic acid synthesis. Thefinal concentration of D-lactic acid reached119.3g/L. The productivity and yield of D-lactatefrom glycerol increased to4.06g/L h and87.1%, respectively. Furthermore, when theD-lactate dehydrogenase temperature-inducible transcription system was introduced to theauxotrophic strain, efficiency of D-lactate production improved significantly. Usingtemperature-shifting process, the final concentration of D-lactic acid reached129.3g/L. Theproductivity and yield of D-lactate from glycerol increased to4.79g/L h and92.1%,respectively.
1．对相关代谢途径进行遗传改造，选育获得了代谢甘油为D-乳酸的高产菌株
通过发酵产酸阶段供氧强度的提高建立了甘油高效转化为D-乳酸的发酵工艺，该工艺下，菌种B0013-070在发酵甘油合成D-乳酸的发酵试验中，发酵液中D-乳酸的浓度达到98.5g/L，合成强度为3.45g/L h。在B0013-070基础上，进一步提高D-乳酸脱氢酶的表达强度，则甘油发酵生成D-乳酸的合成强度和转化率分别提高为3.65g/L h和78.0%，发酵液中D-乳酸的浓度达到103.1g/L；采用34℃下菌体生长和42℃发酵产酸的乳酸发酵工艺，此菌发酵甘油为D-乳酸的合成强度和转化率提高到3.66g/L h和82.6%。再通过同源重组技术将温度诱导型启动子pR-pL引入B0013-070的D-乳酸脱氢酶编码基因ldhA的上游并替代其原启动子PldhA，获得D-乳酸高产新菌种B0013-070B，发酵试验表明，其发酵甘油合成D-乳酸的合成强度和转化率提高到4.23g/L h和88.9%。
2.通过基因删除与基因表达改造大肠杆菌相关代谢途径，选育获得了L-乳酸高产菌种
在上述研究获得的B0013-070菌株的基础上，通过基因删除技术，获得ldhA基因删除突变株B0013-080C；对Bacillus coagulans CICIM B1821的L-乳酸脱氢酶编码基因进行克隆，获得L-乳酸脱氢酶基因BcoaLDH。通过基因重组技术，用PldhA引导基因BcoaLDH的转录；通过同源重组技术，将BcoaLDH表达盒插入到菌株B0013-080C的lldD中间，同步突变lldD基因（lldD编码分解L-乳酸的L-乳酸脱氢酶），获得新菌种B0013-090B。发酵试验结果表明，此菌株具有很好的代谢甘油生成L-乳酸的能力，发酵液中L-乳酸浓度达到132.4g/L，甘油到L-乳酸合成强度和转化率分别为4.90g/L h和93.7%。新菌种合成L-乳酸的光学纯度高于99.9%。
3.选育出了硫胺素营养缺陷型突变株，研究了此营养缺陷性表型在细胞生长控制中的应用，通过发酵试验验证了可控菌体生长可进一步改善重组菌代谢甘油合成乳酸的效率
细胞生成是乳酸发酵过程及其控制中的关键因素。为研究细胞生长的简便控制方法，在上述高产菌株B0013-070的基础上，通过同源重组获得了thiE突变的新菌株，B0013-080A，其在不添加硫胺素的培养基中不能生长。进而在液体培养条件下，分析了硫胺素添加量与菌体生成量之间的关系，得出了两者之间的关系式为：y=(4.0×1010)x+1.5902(R~2=0.9987)。采用上述相似的发酵工艺，在发酵体系中添加终浓度2.47×10-7g/mL的盐酸硫胺素，发酵液中D-乳酸浓度可达119.3g/L，甘油到D-乳酸的合成强度和转化率提高到4.06g/L h和87.1%。进一步将上述研究中温度诱导D-乳酸脱氢酶的表达引入，获得新菌种B0013-080B。此菌株在相似的发酵条件下，发酵液中D-乳酸浓度可达129.3g/L，甘油到D-乳酸的合成强度和转化率提高到4.79g/L h和92.1%。
Currently, petrochemicals are used as raw materials in the manufacturing of a variety ofproducts such as polymers, textiles, paints and solvents etc. However, rapid depletion of thepetroleum resource and increase in emission of greenhouse gas encouraged a replacement ofpetroleum with renewable resources. Polylactic acid, as typical biodegradable material, hasshowed a potential to replace petrochemical-based plastics. Manufacturing polylactic acidneeds large amounts of monomer, D-lactic acid and/or L-lactic acid. Therefore,biotechnological production of D-lactic acid and/or L-lactic acid with high optical purity andchemical purity has been increasingly focused in recent years. Production of lactic acid fromcheap available biomass was investigated extensively. Glycerol is the most readily availablerenewable feedstock. With the increasing international biodiesel production, a surplus ofcrude glycerol has been generated resulting in a significant price reduction. In addition to lowcost and abundance, the higher degree of reduction makes glycerol be an excellent potentialcarbon source to produce chemicals with high yield.
In this study, metabolic engineering and process optimization were employed toinvestigate bioconversion of glycerol into D-lactic acid and L-lactic acid by Escherichia coli.The main research findings are as follows:
1. Efficient production of D-lactic acid from glycerol was achieved through geneticmodification of the metabolic pathway and optimization of the fermentation process.
The fermentation process for D-lactate production from glycerol was optimized byincreasing the oxygen supply strength during lactate formation phase. In7-L bioreactor andusing B0013-070strain,98.5g/L of the final concentration and3.45g/L of productivity ofD-lactate were generated, respectively. Furthermore, increasing copy of ldhA gene encodingD-lactate dehydrogenase led to higher production of D-lactic acid, and the final D-lactic acidconcentration and productivity were103.1g/L and3.65g/L h, respectively. The yield reached78.0%.
Moreover, temperature was optimized during cell growth phase and lactate productionphase, respectively. The resulting showed that34°C was optimal for cell growth and42°Cwas optimal for production of lactic acid. Using the temperature-shifting process, engineeredstrain B0013-070produced D-lactic acid with a3.66g/L h of productivity and82.6%of yield.Using another strain E. coli B0013-070B in which the promoter of ldhA was replaced bytemperature-inducible promoter, production performance of D-lactic acid further increased bythis fermentation process. And4.23g/L h of productivity and88.9%of yield were obtained.
2. Metabolic engineering of E. coli for efficient production of L-lactate from glycerol
An exogenous L-lactate dehydrogenase gene (BcoaLDH) was cloned from thermophilicBacillus coagulans CICIM B1821and expressed in E. coli to achieve L-lactic acid producerfrom glycerol. To further increase L-lactic acid production, the BcoaLDH fusing promoter ofldhA was inserted into E. coli chromosome and replacing of the lldD gene that encodes FMN-dependent L-lactate dehydrogenase catalyzing L-lactic acid to pyruvate. Furthermore,the D-lactic acid synthesis pathway was blocked by deleting the ldhA gene to realize extremelyhigh optical purity L-lactic acid synthesis. The resulting strain B0013-090B was used toevaluate the production of L-lactic acid. Using temperature-shifting process,132.4g/L ofL-lactic acid was produced from glycerol, and4.90g/L h of productivity and93.7%of yieldwere obtained, respectively. The optical purity of L-lactic acid reached to99.9%.
3. Fine switch of cell growth and latctic acid production using thiaminehydrochloride through deletion of thiE gene producing thiamine auxotrophy phenotype.And the concise fermentation process of engineered strain was developed for D-lactateproduction.
The thiE gene encodes a critical protein in the synthesis route of thiamine, a cofactor forPDH complex. Therefore, deletion of thiE gene produced thiamine auxotrophy phenotype.We deleted the thiE gene in the D-lactate producer Escherichia coli CICIM B0013-070(ackA, pta, pps, pflB, dld, poxB, adhE, frdA) to generate CICIM B0013-080A.The cell mass of B0013-080A was fine cotrolled by the concentration of thiaminehydrochloride (VB1) added into medium. The equation of the relationship between theamount of VB1and the biomass was obtained as follows: y=(4.0×1010)x+1.5902(R2=0.9987),where y is the obtained biomass and x is the amount of thiamine hydrochloride (VB1) added.When VB1was added into the fermentation medium with a final concentration of2.47×10-7μg/mL, engineered strain performed better for cell growth and D-lactic acid synthesis. Thefinal concentration of D-lactic acid reached119.3g/L. The productivity and yield of D-lactatefrom glycerol increased to4.06g/L h and87.1%, respectively. Furthermore, when theD-lactate dehydrogenase temperature-inducible transcription system was introduced to theauxotrophic strain, efficiency of D-lactate production improved significantly. Usingtemperature-shifting process, the final concentration of D-lactic acid reached129.3g/L. Theproductivity and yield of D-lactate from glycerol increased to4.79g/L h and92.1%,respectively.
引文
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42. Ishida N, Saitoh S, Ohnishi T, et al. Metabolic engineering of Saccharomyces cerevisiae for efficientproduction of pure L-(+)-lactic acid[J]. Appl Biochem Biotechnol,2006,131(1-3):795-807
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58. Dien BS, Nichols NN, Bothast RJ. Recombinant Escherichia coli engineered for production of L-lacticacid from hexose and pentose sugars[J]. J Ind Microbiol Biotechnol,2001,27(4):259-264.
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78. Bulut S, Elibol M, Ozer D. Effect of different carbon sources on L(+)-lactic acid production byRhizopus oryzae[J]. Biochem Eng J,2004,21(1):33-37.
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80. Kadam SR, Patil SS, Bastawde KB, et al. Strain improvement of Lactobacillus delbrueckii NCIM2365for lactic acid production[J]. Process Biochem,2006,41(1):120-126.
81.田康明,石贵阳,王正祥.凝结芽孢杆菌CICIM B1821发酵生产L-乳酸的研究.食品工业科技,2011,32(10):246-248.
82. Davidson BE, Llanos RM, Cancilla MR, et al. Current research on the genetics of lactic acidproduction in lactic acid bacteria[J]. Int Dairy J,1995,5(8):763-784.
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35. Zhang ZY, Jin B, Kelly JM, Production of lactic acid from renewable materials by Rhizopus fungi[J].Biochem Eng J,2007,35(3):251-263.
36. Bai DM, Zhao XM, Li XG, et al. Strain improvement of Rhizopus oryzae for over-production of L(+)-lactic acid and metabolic flux analysis of mutants[J]. Biochem Eng J,2004,18(1):41-48.
37. Rhee MS, Kim J, Qian Y, et al. Development of plasmid vector and electroporation condition for genetransfer in sporogenic lactic acid bacterium, Bacillus coagulans[J]. Plasmid,2007,58(1):13-22.
38. Payot T, Chemaly Z, Fick M. Lactic acid production by Bacillus coagulans--kinetic studies andoptimization of culture medium for batch and continuous fermentations[J]. Enzyme Microb Technol,1999,24(3-4):191-199.
39. Ishida N, Suzuki T, Tokuhiro K, et al. D-lactic acid production by metabolically engineeredSaccharomyces cerevisiae[J]. J Biosci Bioeng,2006,101(2):172-177.
40. Ishida N, Saitoh S, Tokuhiro K, et al., Efficient production of L-lactic acid by metabolically engineeredSaccharomyces cerevisiae with a genome-integrated L-lactate dehydrogenase gene[J]. Appl EnvironMicrobiol,2005,71(4):1964-1970.
41. Skory CD. Lactic acid production by Saccharomyces cerevisiae expressing a Rhizopus oryzae lactatedehydrogenase gene[J]. J Ind Microbiol Biotechnol,2003,30(1):22-27.
42. Ishida N, Saitoh S, Ohnishi T, et al. Metabolic engineering of Saccharomyces cerevisiae for efficientproduction of pure L-(+)-lactic acid[J]. Appl Biochem Biotechnol,2006,131(1-3):795-807
43. Chang DE, Jung HC, Rhee JS, et al. Homofermentative production of D-or L-lactate in metabolicallyengineered Escherichia coli RR1[J]. Appl Environ Microbiol,1999,65(4):1384-1389.
44. Zhu Y, Eiteman MA, DeWitt K, et al. Homolactate fermentation by metabolically engineeredEscherichia coli strains[J]. Appl Environ Microbiol,2007,73(2):456-464.
45. Causey TB, Shanmugam KT, Yomano LP, et al. Engineering Escherichia coli for efficient conversionof glucose to pyruvate[J]. Proc Natl Acad Sci U S A,2004,101(8):2235-2240.
46. Jantama K, Zhang X, Moore JC, et al. Eliminating side products and increasing succinate yields inengineered strains of Escherichia coli C[J]. Biotechnol Bioeng,2008,101(5):881-893.
47. Kang Z, Gao C, Wang Q, et al. A novel strategy for succinate and polyhydroxybutyrate co-productionin Escherichia coli[J]. Bioresour Technol,2010,101(19):7675-7678.
48. Zhou L, Zuo ZR, Chen XZ, et al. Evaluation of genetic manipulation strategies on D-lactate productionby Escherichia coli[J]. Curr Microbiol,2011,62(3):981-989.
49. Yang YT, San KY, Bennett GN, Redistribution of metabolic fluxes in Escherichia coli withfermentative lactate dehydrogenase overexpression and deletion[J]. Metab Eng,1999,1:141-152.
50. Wang Q, Ou MS, Kim Y, et al. Metabolic flux control at the pyruvate node in an anaerobicEscherichia coli strain with an active pyruvate dehydrogenase[J]. Appl Environ Microbiol,2010,76(7):2107-2114.
51. Grabar TB, Zhou S, Shanmugam KT, et al. Methylglyoxal bypass identified as source of chiralcontamination in L (+) and D (-)-lactate fermentations by recombinant Escherichia coli[J]. BiotechnolLett,2006,28(19):1527-1535.
52. Subedi KP, Kim I, Kim J, et al. Role of GldA in dihydroxyacetone and methylglyoxal metabolism ofEscherichia coli K12[J]. FEMS Microbial Lett,2008,279(2):180-187.
53. Booth IR, Ferguson GP, Miller S, et al. Bacterial production of methylglyoxal: a survival strategy ordeath by misadventure?[J]. Biochem Soc Trans,2003,31(6):1406-1408.
54. Zhou S, Grabar TB, Shanmugam KT, et al. Betaine tripled the volumetric productivity of D (-)-lactateby Escherichia coli strain SZ132in mineral salts medium[J]. Biotechnol Lett,2006,28(9):671-676.
55. Zhou S, Shanmugam KT, Yomano LP, et al. Fermentation of12%(w/v) glucose to1.2M lactate byEscherichia coli strain SZ194using mineral salts medium[J]. Biotechnol Lett,2006,28(9):663-670.
56. Zhou L, Shen W, Niu DD, et al. Fine tuning the transcription of ldhA for D-lactate production[J]. J IndMicrobiol Biotechnol,2012,39(8):1209-1217.
57. Dien BS, Nichols NN, Bothast RJ. Fermentation of sugar mixtures using Escherichia coli cataboliterepression mutants engineered for production of L-lactic acid[J]. J Ind Microbiol Biotechnol,2002,29(5):221-227.
58. Dien BS, Nichols NN, Bothast RJ. Recombinant Escherichia coli engineered for production of L-lacticacid from hexose and pentose sugars[J]. J Ind Microbiol Biotechnol,2001,27(4):259-264.
59. Tian KM, Chen XZ, Shen W, et al. High-efficiency conversion of glycerol to D-lactic acid withmetabolically engineered Escherichia coli[J]. Afr J Biotechnol,2012,11(21):4860-4867.
60. Zhou L, Tian KM, Niu DD, et al. Improvement of D-lactate productivity in recombinant Escherichiacoli by coupling production with growth[J]. Biotechnol Lett,2012,34(6):1123-1130.
61. Zhou L, Niu DD, Tian KM, et al. Genetically switched D-lactate production in Escherichia coli[J].Metab Eng,2012,14(5):560-568.
62. Joseph S, Russell D W. Molecular cloning: a laboratory manual.2001, Cold Spring Harbor LaboratoryPress: Cold Spring Harbor, NY.
63. Purvis JE, Yomano LP, Ingram LO. Enhanced trehalose production improves growth of Escherichiacoli under osmotic stress[J]. Appl Environ Microbiol,2005,71(7):3761-3769.
64. Singh A, Lynch MD, Gill RT. Genes restoring redox balance in fermentation-deficient E. coliNZN111[J]. Metab Eng,2009,11(6):347-354.
65. Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12using PCR products[J]. Proc Natl Acad Sci U S A,2000,97(12):6640-6645.
66. Hashimoto-Gotoh T, Yamaguchi M, Yasojima K, et al. A set of temperaturesensitive-replication/-segregation and temperature resistant plasmid vectors with different copynumbers and in an isogenic background (chloramphenicol, kanamycin, lacZ, repA, par, polA)[J]. Gene,2000,241(1):185-191.
67. Liu L, Li Y, Zhu Y, et al. Redistribution of carbon flux in Torulopsis glabrata by altering vitamin andcalcium level[J]. Metab Eng,2007,9(1):21-29.
68. Lieph R, Veloso FA, Holmes DS. Thermophiles like hot T[J]. Trends Microbiol,2006,14(10):423-426.
69. Wang Y, Wang Z, Xu Q, et al. Lowering induction temperature for enhanced production ofpolygalacturonate lyase in recombinant Pichia pastoris[J]. Process Biochem,2009,44(9):949-954.
70. Tolia NH, Joshua-Tor L. Strategies for protein coexpression in Escherichia coli[J]. Nat methods,2006,3(1):55-64.
71. Jones PG, VanBogelen RA, Neidhardt FC. Induction of proteins in response to low temperature inEscherichia coli[J]. J Bacteriol,1987,169(5):2092-2095.
72. Choi JH, Keum KC, Lee SY. Production of recombinant proteins by high cell density culture ofEscherichia coli[J]. Chem Eng Sci,2006,61(3):876-885.
73. Michelson T, Kask K, J gi E, et al. L(+)-lactic acid producer Bacillus coagulans SIM-7DSM14043and its comparison with Lactobacillus delbrueckii ssp. lactis DSM20073[J]. Enzyme Microb Technol,2006,39(4):861-867.
74. Ron EZ, Davis BD. Growth rate of Escherichia coli at elevated temperatures: limitation bymethionine[J]. J Bacteriol,1971,107(2):391-396.
75. Millership JS, Fitzpatrick A. Commonly used chiral drugs: a survey[J]. Chirality,2004,5(8):573-576.
76. Garrison AW. Probing the enantioselectivity of chiral pesticides[J]. Environ Sci Technol,2006.40(1):16-23.
77. Aljundi IH, Belovich JM, Talu O. Adsorption of lactic acid from fermentation broth and aqueoussolutions on Zeolite molecular sieves[J]. Chem Eng Sci,2005,60(18):5004-5009.
78. Bulut S, Elibol M, Ozer D. Effect of different carbon sources on L(+)-lactic acid production byRhizopus oryzae[J]. Biochem Eng J,2004,21(1):33-37.
79. John RP, Nampoothiri KM, Pandey A. SoliD-state fermentation for L-lactic acid production from agrowastes using Lactobacillus delbrueckii[J]. Process Biochem,2006,41(4):759-763.
80. Kadam SR, Patil SS, Bastawde KB, et al. Strain improvement of Lactobacillus delbrueckii NCIM2365for lactic acid production[J]. Process Biochem,2006,41(1):120-126.
81.田康明,石贵阳,王正祥.凝结芽孢杆菌CICIM B1821发酵生产L-乳酸的研究.食品工业科技,2011,32(10):246-248.
82. Davidson BE, Llanos RM, Cancilla MR, et al. Current research on the genetics of lactic acidproduction in lactic acid bacteria[J]. Int Dairy J,1995,5(8):763-784.
83. Singh SK, Ahmed SU, Pandey A. Metabolic engineering approaches for lactic acid production[J].Process Biochem,2006,41(5):991-1000.
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