酚类高抗逆Clostridium beijerinckii的选育及酚类物质对其合成丁醇的抑制机理研究

Butanol is used not only as a solvent and a chemical feedstock, but also as a biofuel. Butanol is the main component of acetone/ butanol/ethanol (ABE) fermentation，ABE fermentation has major disadvantages, including the high cost of the substrates（70%）, low productivity and high recovery costs. Using lignocellulosic materials as substrate is an attractive proposition for introducing an economically competitive biological process. Currently, however, no butanol producing clostridia can produce butanol efficiently from lignocellulosic biomass, and a range of toxic compounds,including weak acids, furan derivatives, and soluble total phenolic compounds(TPC), are generated during the hydrolysis of lignocellulosic materials.The cells growth and butanol production of Clostridium beijerinckii were inhibited by TPC. The phenolic compounds in hemicellulosic hydrolysate of bagasse treated with dilute sulfuric acid(SAHHB) was selected as inhibition factor for the growth of C.beijerinckii,C. beijerinckii mutants with high tolerance of phenolic compounds were obtained by ion-beam mutation and metabolic evolution cultivation.At the high concentration of phenolic compounds（TPC≥2.0 g/L）, the mutants could utilize non-detoxified SAHHB grow well, with high or no butanol production were selected.The mechanisims of phenolic compounds on butanol production by C.beijerinckii were tested by RT-qPCR. Furthermore,the medium composition of SAHHB for butanol production by C. beijerinckii mutant with high tolerance and butanol production was optimized using response surface methodology.This project will publish 2-5 articles of SCI paper and apply for 2-4 chinese invention patents.It appears promising for butanol production from lignocellulosic materials.

丁醇作为一种新型生物燃料备受关注，生物法制备丁醇总成本的近70%是原料，因而探索廉价的木质纤维原料制备丁醇成为研究热点。木质纤维在预处理过程中产生的酚类物质严重抑制Clostridium beijerinckii的生长与发酵，因此提高菌株抗逆性是急需攻克的瓶颈问题之一。本项目以甘蔗渣酸解糖液(SAHHB)中的可溶性酚类（TPC）为抑制因子，对C. beijerinckii进行离子束和代谢进化育种，选育高抗逆、高产丁醇的系列突变株。从中选取高抗逆(TPC≥2.0g/L)、丁醇产量差异大的突变样本，对丁醇合成的关键基因进行转录分析；从丁醇合成的角度，解析酚类化合物对C. beijerinckii合成丁醇的抑制机理。以高抗逆、高产丁醇突变株为出发菌株，利用响应面法优化其利用SAHHB制备丁醇的发酵工艺。本项目将发表SCI论文2-5篇，申请专利2-4项，为纤维素丁醇产业化提供优良菌种与技术支撑。

由于木质纤维在预处理过程中产生一毒素物质（有机酸类、糠醛类、酚类化合物等），仍然没有高效利用木质纤维原料制备燃料丁醇的报道，其中酚类物质严重抑制拜氏梭菌的生长与发酵，因此提高菌株抗逆性是急需攻克的瓶颈问题之一。.本课题关注于高抗逆、高产丁醇突变株的选育及其抗逆元件的挖掘与机理机制解析。利用常温等离子ARTP诱变技术，选育得到对酚类化合物高抗逆、高产丁醇的拜氏梭菌突变菌株M11和M17。其中拜氏梭菌突变株M11具有较高的阿魏酸抗逆性，能够直接利用未脱毒甘蔗渣酸解糖液制备燃料丁醇。拜氏梭菌突变株M17，对未脱毒甘蔗渣酸解糖液的毒素物质具有高度耐受性，在可溶性总酚浓度高达2.4 g/L，其总溶剂产量和丁醇产量分别达到9.2 g/L和6.5 g/L。.基于比较基因组学和转录组学研究，挖掘出拜氏梭菌的一些关键基因（Cbei-4110，Cbei-3304，Cbei-4693 和 Cbei-2996）。将拜氏梭菌的Cbei-4110基因编码NADH-泛醌氧还原酶G亚基（nuoG），插入失活后，重组菌株8052-4110表现出明显的性能提升，发酵过程中糖消耗速度、菌体生长繁殖速度明显提高，发酵周期减少一半，丁醇终浓度从7.8g/L提高到9.5g/L，溶剂产量从10.2g/L提高到12.1g/L，丁醇比达78%，溶剂转化率为40.3%。基因Cbei-3304编码一个疑似MFS膜蛋白，在拜氏梭菌NCIMB 8052插入失活Cbei_3304基因后，重组菌株8052-3304对模式酚类化合物（阿魏酸、香兰素等）具有较高抗逆性，并且可直接利用不脱毒的甘蔗渣酸解糖液直接发酵产丁醇；表明基因Cbei_3304与拜氏梭菌的酚类物质抗逆性有重要的相关性。在以上研究的基础上，通过响应面法，优化了甘蔗渣酸解糖液生产丁醇工艺；在毒素浓度TPC>2.3 g/L 的分批发酵中，拜氏梭菌突变株M11的总溶剂产量在12.8 g/L（丁醇9.2 g/L，丙酮3.1 g/L和乙醇0.5 g/L），总溶剂产率为0.39 g /g 总糖。.截至目前，已经发表论文5篇（其中SCI论文4篇），申请中国发明专利3项，授权2项。本项目获得了拜氏梭菌的一些关键抗逆元件，这些关键因子的挖掘为拜氏梭菌对酚类化合物的抗逆机制解析，提供了数据与基础；充实了微生物抗逆机制的理论体系。