氧气对木质纤维素来源抑制物的生物脱毒促进机制研究

A variety of degradation products (including furan derivatives, organic acid and phenolic acid compounds), which have a stong inhibition on the following enzymatic hydrolysis and microbial fermentation, generated in the lignocellulose bioprocessing. Biodetoxification uses microorganisms or their secreted enzymes to metabolize these inhibitors.Compared with other detoxification methods, biodetoxification has many advantages, including complete inhibitors-removal, non-waste water disposal and etc. However, longer than 3 days biodetoxification period restricted its extensive application in the practical bioprocessing. Our previous study showed that the oxygen supply during biodetoxification could greatly improve the inhibitors degradation rates and shorten the biodetoxification period of Amorphotheca resinae ZN1 within 24 hours, but the underlying mechanisms were still unknown. This study aims to uncover the promoting mechanisms of oxygen supply from two aspects, the effect on growth of A. resiane ZN1 and the inhibitors degradation rates. For the former, the newly rapid and accurate biomass assay method for solid-state fermentation will be developed through corelation the quantity of characteristic enzyme encoding gene by qPCR to its colony forming units (CFU) in the pure synthetic medium. Then the biomass of A. resinae ZN1 during solid-state biodetoxification could be monitored by this method, thus to determine the relationship among oxygen supply, biomass and the detoxification rate. For the latter, the degradation pathway of furfural and HMF will be deduced using the sole-carbon source method, then the upregulated key enzyme encoding genes in the presence of oxygen supply will be analyzed by qRT-PCR based on the known whole-genome information of A. resinae ZN1.And further to clarify the effect of oxygen supply on the inhibitors degradation.The implementation of the current study will provide the basic knowledge for development of biodetoxification reactors and provide newly functional genes for constructing consolidated bioprocessing cells (possessing detoxification and fermentation functions).

木质纤维素在生物加工过程中会过度降解生成多种化合物（包括呋喃类、有机酸类和酚酸类物质），它们对后续的酶水解和发酵都有很强的抑制作用。生物脱毒是利用微生物或酶制剂来降解这些抑制物，具有脱毒彻底、无废水排放等优点，但3天以上的脱毒周期严重限制了该方法的应用。前期研究表明，通氧可以明显缩短树脂枝孢霉Amorphotheca resinae ZN1的脱毒时间，但其作用机制尚不清楚。本课题拟从氧气对树脂枝孢霉生长和对抑制物代谢速率的影响两方面入手，通过qPCR的方法建立菌体量与菌体特征性酶编码基因量之间的关联，实时快速监测生物脱毒过程的菌体量变化，确定氧气-菌体量-脱毒速率三者的关系；通过唯一碳源实验对抑制物的降解路径进行表征，用qRT-PCR的方法分析氧气引起的关键酶基因表达差异，阐明氧气在抑制物代谢中的作用。本项目的实施将为脱毒反应器的开发提供依据，为"脱毒-发酵"整合细胞的构建提供良基因来源。

木质纤维素原料在预处理过程中会过度降解生成多种化合物（包括呋喃类、有机酸类和酚酸类物质），它们对后续的酶水解和发酵都有很强的抑制作用。生物脱毒与其它脱毒方法，如“过碱化调节”和水洗相比，具有脱毒彻底、无废水排放等优点。但常规的脱毒周期太长（5-7天），严重限制了该技术的应用。本研究从表观和分子层面分析了氧气促进树脂枝孢霉A. resinae ZN1降解抑制物的规律，即氧气是抑制物降解路径关键酶的辅因子，并用qPCR的方法对这些特征性酶进行了鉴定；两种锌依赖性的醇脱氢酶基因和五种醛/酮还原酶基因负责将糠醛和5-羟甲基糠醛转化为醇的形式；而三种丙醇脱氢酶酶基因，一种NAD(P)+依赖性的醛脱氢酶基因或两种以自由氧为底物的氧化酶基因则负责将呋喃醇氧化为呋喃酸，进而进入三羧酸循环并被完全降解掉；此外，通过合成培养基中供氧降解抑制物的实验，证明了树脂枝孢霉A. resinae ZN1只有在将大部分呋喃类抑制物降解为呋喃醇后，才开始进行菌体的大量增殖，即充足的供氧并不能提高菌体的生物量；接着，我们通过过程工程的方法对脱毒过程的供氧进行了强化，将生物脱毒周期缩短至24-36小时，实现了快速脱毒的目标；并将该快速生物脱毒技术与干法稀酸预处理和高固体含量同步糖化与发酵结合起来，应用于多种不同生物燃料和生物基化学品的生产中（包括纤维素乙醇、纤维素乳酸、纤维素葡萄糖酸钠、纤维素柠檬酸等），其发酵性能（如浓度和得率）以及过程的经济性与淀粉基的生物转化过程相近，使木质纤维素的生物炼制技术真正具备了产业化的条件。