【作者】 田康明；

【导师】 王正祥；

【作者基本信息】 江南大学 ， 发酵工程， 2013， 博士

【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%。更多还原

【Abstract】 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 glycerolAn 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.更多还原