【作者】 李超；

【导师】 许平；

【作者基本信息】 上海交通大学 ， 微生物学， 2016， 博士

【摘要】 D-乳酸作为一种重要的平台化合物,被广泛应用于食品、制药、化妆品等行业。近来对D-乳酸需求量的增加主要是聚D-乳酸可改善以PLA制造产品的机械性能、耐热性和抗水解能力。较L-乳酸来说,D-乳酸生产技术相对落后,因而D-乳酸生产成本比L-乳酸高3～5倍。本文探讨了改造蓝细菌和耐热芽孢杆菌来开发高产、高效的D-乳酸生产菌株,并从甲基化的角度对产乳酸耐热芽孢杆菌的环境嗜热机制进行了研究。构建蓝藻工程菌转化CO₂来生产化合物越来越受到关注,然而与细菌偏好利用NADH为辅酶不同,蓝细菌对NADPH的使用偏好阻碍了异源氧化还原酶的应用。本节首先将来自保加利亚乳杆菌ATCC11842中的D-乳酸脱氢酶改造为以主要依赖NADPH。然后将突变酶基因进行密码子优化,并整合至Synechococcus elongates PCC7942染色体中,所获工程菌株D-乳酸的合成速率提高至少3.6倍。向上述突变株中引入乳酸外排泵和向培养基中通入CO₂均可提高D-乳酸合成。最终应用上述组合策略实现了D-乳酸合成速率的大幅提升。高温发酵具有多重优势,如最小化污染,提高原料转化速率及降低设备能耗。本节首先挖掘到一独特的嗜热D-乳酸脱氢酶,然后将其引入天然产2,3-丁二醇的耐热地衣芽孢杆菌中。由于引入酶和宿主乳酸外排泵的协作产生了一个“陷阱点”,继而形成“碳流陷阱”效应,使得碳流大量（>90%）流向D-乳酸的合成并不可逆地向胞外运转。最优条件发酵后,D-乳酸产量可达226.6 g/L。此外,实验评估了使用玉米浆干粉（CSP）为唯一氮源生产D-乳酸的情况。为进一步降低成本,实验减少CSP用量并采用响应面法优化获得一组更加廉价发酵培养基,最终可获135.4 g/L D-乳酸。耐热凝结芽孢杆菌也是乳酸生产的菌种,但因其缺乏有效的遗传操作体系而在改造为宿主细胞的应用中受到极大的限制。本节从DNA甲基化的视角对该菌的环境耐热机制进行了探讨,使用单分子实时测序法测定了两株凝结芽孢杆菌的甲基化组。分析甲基化基因功能群和高甲基化区域表明,两株菌在高、低温度下均存在巨大差异。进一步分析甲基化修饰的DNA基序发现,??型m⁴C甲基转移酶可能在菌株对热环境的适应中起主要作用。另对菌株进行转录组测序和q PCR验证发现了在高温时转录水平明显上调的甲基转移酶基因。因此,该研究拓宽了对DNA甲基化生理功能的新认识。更多还原

【Abstract】 D-Lactic acid as an important platform chemical is widely used in the food,pharmaceutical,cosmetic,and other industries.Recent demands for D-lactic acid have surged due to the increasing production of biodegradable polylactic acid（PLA）,since a stereocomplex of poly（L-lactic acid）and poly（D-lactic acid）can improve the mechanical performance,thermal resistance,and hydrolysis resistance of PLA-based materials.However,the production technology for D-lactic acid is rather backward compared to that of L-lactic acid,resulting in a 3 to 5-fold higher price of D-lactic acid.This study aims to develop economical and efficient D-lactic acid producers by engineering a cyanobacterium and a thermotolerant Bacillus strain.In addition,methylomes were analyzed to provide mechanism regarding to the thermophilic adaptation of two Bacillus strains.It is increasingly attractive to engineer cyanobacteria for bulk production of chemicals from CO₂.However,cofactor bias of cyanobacteria is different from bacteria that prefer NADH,which hampers cyanobacterial strain engineering.In this study,the key enzyme D-lactate dehydrogenase（Ldh D）from Lactobacillus bulgaricus ATCC11842 was engineered to reverse its favored cofactor from NADH to NADPH.Then,the engineered enzyme was introduced into Synechococcus elongatus PCC7942to construct an efficient light-driven system that produces D-lactic acid from CO₂,the resulting recombinant strain increased D-lactate productivity by over 3.6-fold.We further demonstrated that introduction of a lactic acid transporter and bubbling CO₂-enriched air also enhanced D-lactate productivity.Using this combinational strategy,enhanced D-lactate concentration and productivity were achieved.High-temperature fermentation has multiple advantages,such as minimized the contamination risk,high mass transfer rate and the reductions in equipment requirements and energy consumption.Herein,we hunted and introduced a unique D-lactate dehydrogenase into a thermotolerant 2,3-butanediol-producing strain.A carbon flux trapping effect was observed due to a“trapping point”created by the cooperation between the introduced enzyme and the host-borne efflux pump,which enabled the irreversible transport of D-lactic acid.The overall carbon flux of the engineered strain was significantly enhanced and was dominantly（>90%）redistributed to D-lactic acid.Under optimized conditions,D-lactic acid reached an extremely high titer（226.6 g/L）that was newly recorded to date.In addition,to develop a low-cost process,corn steep powder（CSP）has been evaluated as the sole nitrogen source for D-lactic acid production.After conducting fed-batch fermentation under optimal CSP concentration,a high titer（169.2 g/L）of D-lactic acid was produced.To further reduce the cost,an economical fermentative medium was acquired with the method of response surface analysis.As a result,135.4 g/L of D-lactic acid was achieved.It is reported that thermophilic B.coagulans was also a lactic acid producing strain,however,it was rarely applied to any host cell due to its incompleted genetic manipulation.On the basis of above point and the fact that the role of DNA methylation in regulating bacterial adaptation to high-temperature environment is less understood,this study evaluated the global DNA methylome of two closely related thermotolerant B.coagulans strains using single-molecule real-time（SMRT）sequencing approach.Bioinformatics analysis on distribution of methylated gene function group and hypermethylated regions revealed that wide differences was discovered in the two strains under both high and low growth temperatures.Accordingly,we speculated that m⁴C modification might play a major role in bacterial heat adaptation which attributable to type??DNA methyltransferases（MTases）.The results supported the assumption that the formed relaxed-specificity of DNA MTases in hot environments has increased their recognition sites thus led to better DNA methylation protection.Taken together,the present study shed new light on physiological functions of DNA methylation.更多还原