辅酶A调控丙酮丁醇梭菌利用木薯淀粉高效合成丁醇的机制解析

Cassava is an important and abundant biomass resources in Guangxi province, where contributes to the major production of cassava starch in China. To date, limited research work has been conducted on the resource utilization of cassava starch. Clostridium acetobutylicum, the important industrial strain for producing butanol with cassava starch, has some disadvantages such as the low butanol tolerance and low productivity. CoA and its derivatives are the important cofactors in the industrial strain, and play a vital role on the physiological function. However, the comprehensive understanding of manipulation mechanism of CoA metabolism on butanol production has been globally unexploited. In this project, we attempt to achieve the efficient synthesis of butanol by cofactor engineering and systems metabolic engineering of C. acetobutylicum. The main research work can be summarized as three aspects: (1) constructing the research model, in which different mutants are constructed to precisely regulate intercellular CoA level; (2) some strategies (such as genetic engineering, omics-technology) are applied to investigate the physiological mechanism of CoA metabolism on butanol production from intracellular micro-environment, the profiles of gene and protein expression and metabolic flux distribution; and (3) based on the above results, intracellular CoA metabolism is further modified by metabolic engineering, and the efficient synthesis of butanol is expected to be achieved by the systematic optimization and regulation of the carbon flux of butanol metabolism. The mechanism presented here not only gain improved insight into C. acetobutylicum physiology, but so provide novel strategy to acquire the desired phenotype and increase the added value of cassava starch.

木薯作为广西丰富、开发潜力巨大的生物质资源，其资源化利用研究较为缺乏。而丙酮丁醇梭菌作为高效利用木薯淀粉发酵生产丁醇的工业菌株，仍存在丁醇产量产率偏低、丁醇耐受性弱等问题。辅酶A(CoA)及其衍生物是微生物体内重要的辅因子，参与调控众多代谢途径。然而，CoA代谢如何精确调控丁醇代谢效率的机制尚缺乏全局性的认识。本课题利用辅因子工程和系统代谢工程，通过：(1) 构建能精确调控胞内CoA水平的丙酮丁醇梭菌突变株；(2) 以其为研究模型，借助分子生物学技术、生理生化分析、组学技术等策略，从胞内微环境、转录调控蛋白、代谢流分布、生理特性等方面系统研究CoA代谢调控丁醇代谢流量及其通量的生理机制；(3) 调控胞内CoA水平优化重组和综合调控丁醇代谢网络以实现丁醇的高效合成。本课题研究结果将有助于增加对丙酮丁醇梭菌生理特性的理解，并为改进微生物生理功能，提高木薯淀粉附加产值，具有重要的理论和实践意义。

近年来，生物丁醇发酵作为一种环境友好型、资源节约型的生物转化技术，已然成为未来极具发展和产业化潜力的高新技术。然而，国内外研究报道木薯发酵生产丁醇的水平一般在13～16 g/L，总溶剂产量仅为20～21 g/L，难以满足丁醇发酵的工业化要求。因此，系统改良丙酮丁醇梭菌生产性能已成为生物丁醇研究及其产业化发展的核心问题。为此，本项目利用诱变选育、辅因子工程和系统代谢工程系统研究丙酮丁醇梭菌代谢途径，从而强化丁醇的生产强度，其主要内容包括：（1）以课题组前期筛选的丁醇高产菌株和耐受高浓度丁醇胁迫菌株为出发菌株，利用NTG诱变和基因组重排技术将两出发菌株中优势基因进行随机组合，以丁醇产量和耐受胁迫为筛选指标，筛选获得丁醇高效生产菌株，使其在10 L发酵罐中丁醇产量、产率和总溶剂产量分别提高至20.1 g/L、0. 23 g/g和32.6 g/L；（2）以大肠杆菌为宿主菌株，借助基因表达策略，分别考察乙酰辅酶A合成酶和泛酸激酶的酶学性质，为调控胞内辅酶A 浓度及其形式进一步改善丙酮丁醇梭菌代谢流量分布提供基础；（3）基于氮源浓度、种类等氮源调控方式，从胞内微环境(NADH/NAD+、pH)、酶学水平以及转录水平等多方面解析氮源对丙酮丁醇梭菌高效合成丁醇的影响及其生理机制，研究结果表明：当乙酸铵作为唯一氮源时可缩短丙酮丁醇梭菌ABE发酵时间，但在有机氮存在的情况下，NH4+却可有效抑制细胞对NADH的利用，同时抑制丁酸乙酰乙酸酯CoA转移酶的酶活性且使基因askA、buk、adc、ctfA、adhE以及bdhB表达量下调，进而有效抑制ABE的发酵，降低丙酮丁醇梭菌的发酵性能。因此，本项目研究结果将有助于增加对丙酮丁醇梭菌生理特性的理解，并为改进微生物生理功能和改造丙酮丁醇梭菌生产类似或结构更为复杂的精细化工品奠定坚实的理论基础。