Peng Zhi-Qing, Li Chuang, Lin Yi, Wu Sheng-Shan, Gan Li-Hui, Liu Jian, Yang Shu-Liang, Zeng Xian-Hai, Lin Lu
College of Energy, Xiamen University, Xiamen, 361102, China.
Fujian Engineering and Research Centre of Clean and High-Valued Technologies for Biomass, Xiamen, 361102, China.
Biotechnol Biofuels. 2021 Nov 22;14(1):219. doi: 10.1186/s13068-021-02072-z.
Cellulase plays a key role in converting cellulosic biomass into fermentable sugar to produce chemicals and fuels, which is generally produced by filamentous fungi. However, most of the filamentous fungi obtained by natural breeding have low secretory capacity in cellulase production, which are far from meeting the requirements of industrial production. Random mutagenesis combined with adaptive laboratory evolution (ALE) strategy is an effective method to increase the production of fungal enzymes.
This study obtained a mutant of Trichoderma afroharzianum by exposures to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), Ethyl Methanesulfonate (EMS), Atmospheric and Room Temperature Plasma (ARTP) and ALE with high sugar stress. The T. afroharzianum mutant MEA-12 produced 0.60, 5.47, 0.31 and 2.17 IU/mL FPase, CMCase, pNPCase and pNPGase, respectively. These levels were 4.33, 6.37, 4.92 and 4.15 times higher than those of the parental strain, respectively. Also, it was found that T. afroharzianum had the same carbon catabolite repression (CCR) effect as other Trichoderma in liquid submerged fermentation. In contrast, the mutant MEA-12 can tolerate the inhibition of glucose (up to 20 mM) without affecting enzyme production under inducing conditions. Interestingly, crude enzyme from MEA-12 showed high enzymatic hydrolysis efficiency against three different biomasses (cornstalk, bamboo and reed), when combined with cellulase from T. reesei Rut-C30. In addition, the factors that improved cellulase production by MEA-12 were clarified.
Overall, compound mutagenesis combined with ALE effectively increased the production of fungal cellulase. A super-producing mutant MEA-12 was obtained, and its cellulase could hydrolyze common biomasses efficiently, in combination with enzymes derived from model strain T. reesei, which provides a new choice for processing of bioresources in the future.
纤维素酶在将纤维素生物质转化为可发酵糖以生产化学品和燃料的过程中起着关键作用，纤维素酶通常由丝状真菌产生。然而，通过自然育种获得的大多数丝状真菌在纤维素酶生产中的分泌能力较低，远远不能满足工业生产的需求。随机诱变结合适应性实验室进化（ALE）策略是提高真菌酶产量的有效方法。
本研究通过用N-甲基-N'-硝基-N-亚硝基胍（MNNG）、甲基磺酸乙酯（EMS）、常压室温等离子体（ARTP）处理以及在高糖胁迫下进行ALE，获得了哈茨木霉的一个突变体。哈茨木霉突变体MEA-12分别产生0.60、5.47、0.31和2.17 IU/mL的滤纸酶（FPase）、羧甲基纤维素酶（CMCase）、对硝基苯-β-D-纤维二糖苷酶（pNPCase）和对硝基苯-α-D-葡萄糖苷酶（pNPGase）。这些水平分别比亲本菌株高4.33、6.37、4.92和4.15倍。此外，还发现哈茨木霉在液体深层发酵中与其他木霉具有相同的碳代谢物阻遏（CCR）效应。相比之下，突变体MEA-12能够耐受葡萄糖（高达20 mM）的抑制作用，在诱导条件下不影响酶产量。有趣的是，当与里氏木霉Rut-C30的纤维素酶结合时，MEA-12的粗酶对三种不同的生物质（玉米秸秆、竹子和芦苇）显示出高酶解效率。此外，还阐明了提高MEA-12纤维素酶产量的因素。
总体而言，复合诱变结合ALE有效地提高了真菌纤维素酶的产量。获得了一个高产突变体MEA-12，其纤维素酶与模式菌株里氏木霉的酶结合时能够高效水解常见生物质，这为未来生物资源的加工提供了新的选择。
