调控里氏木霉胞内槐糖积累促进纤维素酶合成

High cost in cellulase production is one of the bottlenecks for the bioconversion of lignocellulosic biomass. The species Trichoderma reesei is the best cellulase-producer, but its cellulase production requires induction. Although it is expensive, sophorose has been proved to be the most effective inducer so far for cellulase production by the species. However, sophorose is prone to be hydrolyzed by intracellular β-glucosidases synthesized by the species during the fermentation process and needs to be supplemented by feeding to overcome the problem, which is one of the major reasons for high cost with cellulase production. On the other hand, the contribution to cellulose hydrolysis from β-glucosidases produced by T. reesei is negligible, and cellulase produced by the species needs to be formulated by supplementing β-glucosidases.. In previous studies, the mixture of glucose-sophorose (MGD) was proved as the effective inducer for cellulase production, moreover, three kinds of β-glucosidases including Cel1A, Cel1B and Cel3A were mainly produced in T. reesei using MGD as inducer. In this project, single β-glucosidase gene knockout strains would be constructed to address the challenge of sophorose degradation by β-glucosidase to sustain cellulase production. Therefore, glucose would be supplemented as low-cost carbon source to obtain high biomass density for cellulase production. Finally, genome-wide transcriptional response of T. reesei to MGD would be performed using RNA-Seq analysis. No doubt, the progress will lay a solid foundation for more efficient cellulase production by T. reesei, scientifically and technically as well.

纤维素酶成本高是制约木质纤维素类生物质生物转化的主要瓶颈。里氏木霉是纤维素酶高产菌株,但合成纤维素酶需诱导。槐糖是有效的诱导物，可是易被纤维素酶系中的β-葡萄糖苷酶降解，致使胞内槐糖无法维持适宜诱导浓度,需要不断流加补充。并且里氏木霉本底β-葡萄糖苷酶活性太低，对纤维素水解的作用忽略不计。. 以先前开发的葡萄糖-槐糖混合物（MGD）作为有效诱导物,且主要表达Cel1A、Cel1B和Cel3A三种β-葡萄糖苷酶为基础。本课题构建Cel1A、Cel1B和Cel3A单基因敲除里氏木霉菌株，探索其在纤维素酶合成中分解槐糖的能力及相互关系，进而得到可保持胞内槐糖浓度持续诱导纤维素酶合成的基因工程菌株，并利用葡萄糖为碳源高效生产纤维素酶；最后通过转录组数据解析MGD为诱导物合成纤维素酶转录调控及木质纤维素酶系的特异性。预期研究进展为里氏木霉生产纤维素酶创新技术开发奠定科学和技术基础。

先前开发的葡萄糖-二糖混合物（MGD）可诱导里氏木霉高效合成纤维素酶，但发现里氏木霉β-葡萄糖苷酶会MGD中的槐糖降解致使胞内槐糖无法维持适宜诱导浓度，且MGD作为诱导物刺激里氏木霉合成纤维素酶的特点及酶系组成不清晰。.1.首次发现米格列醇可以作为β-葡萄糖苷酶的竞争性抑制剂且其IC50值为2.93 μg/ml。惊喜的是将其添加到里氏木霉发酵培养基中纤维素酶活性得到持续增强，证明了提出假说的正确性：即抑制β-葡萄糖苷酶可维持槐糖胞内适宜的诱导浓度持续诱导纤维素酶合成，减少诱导物用量，降低诱导物的消耗成本。这类似于原核生物中使用菌株不能代谢的IPTG代替乳糖作为诱导物的思想，所以该项目研究成果不但对解决木质纤维素类生物质生物转化中纤维素酶成本高的瓶颈问题具有十分重要的意义，而且对于开发丝状真菌新型蛋白表达系统也同样意义重大；.2.为复配β-葡萄糖苷酶且降低MGD诱导物成本，利用过表达β-葡萄糖苷酶基因的T. reesei PB-3重组菌株，以廉价葡萄糖为碳源通过分批补料发酵使得β-葡萄糖苷酶活性高达78.09 CBU/mL。该酶不但可以用于复配纤维素酶降解木质纤维素，而且其转糖苷活性同样较强。如果讲该酶复配到纤维素酶中可以使5%碱预处理后的玉米秸秆水解效率达到97%，与商业酶效果无显著性差别。如果讲该酶用于转糖苷，测定了动力学常数Km为5.88 mol/L，Vmax为5.31μmol/min·mg，利用该酶制备MGD过程中转糖基化时间由之前的72 h降低到2 h，而不损害其诱导效果。.3. 转录组学对MGD诱导里氏木霉产纤维素酶的转录调控及木质纤维素酶系的特异性进行研究，结果表明，发现该诱导物与乳糖作为诱导物合成纤维素酶有很大差异，所有纤维素酶和木聚糖酶基因表达量均高于乳糖作为诱导物，主要通过Vib1和Xyr1转录激活因子实现，同时可通过MAPK及蛋白质后加工折叠相关基因（TrireC30_6070, Sec62/63, OSTs, GluI, Nef, BiP, Hsp40, Cnx, GlcII, Uggt 等）稳定蛋白质分泌，此外，还发现TrireC30_137795和137001基因具有更强的β-二糖的亲和力和乳糖可能潜在的转运蛋白（TrireC30_26932、124396和91594）。为进一步探究以MGD诱导里氏木霉高产纤维素酶奠定理论基础。