过表达内源Trswo1基因提高里氏木霉纤维素酶对玉米秸秆水解效率的研究

Over-expressing of the endogenous Trswo 1 gene in Trichoderma reesei to increase the cellulose degrading capability

利用β-葡萄糖苷酶的转糖苷活性制备廉价葡萄糖-槐糖混合物(mixture of glucose and sophorose, MGD)作为诱导物被成功用于诱导里氏木霉(Trichoderma reesei)高效合成纤维素酶,但蛋白质组学显示相比于纤维素作为诱导物,膨胀素基因(Trswo1)显著性低表达导致纤维素酶水解效率低。因此利用组成型强启动子丙酮酸脱羧酶1(pyruvate decarboxylase1, PDC1)在T.reesei Rut C30中过表达内源Trswo1基因,2个T.reesei阳性转化子OEswo1-1和OEswo1-2的Trswo1基因转录水平分别提高了4.65倍和3.91倍,虽然T.reesei OEswo1-1和OEswo1-2在MGD诱导下合成的纤维素酶活性、内切纤维素酶活性和外切纤维素酶活性与野生菌酶活性均无显著性差异,但所产纤维素酶对玉米秸秆的水解效率分别提高了6.98%和13.93%。进一步通过X射线衍射(X-ray diffraction, XRD)和扫描电镜(scanning electron microscope, SEM)显示水解效率提高主要是由于木质纤维素结晶度的降低,同时纤维结构有不同程度的疏松和崩解。该研究成果对T.reesei利用可溶性诱导物合成纤维素酶系中纤维素降解辅助蛋白含量低的问题具有一定意义。

A mixture of glucose and sophorose(MGD)as developed from glucose through the transglycosylation reaction catalyzed byβ-glucosidase,resulting in the overproduction of cellulase by Trichodema reesei Rut C30.However,proteomics showed that the significantly low expression of the SWO1 gene resulted in a low efficiency of cellulase hydrolysis compared with that of cellulose as an inducer.Therefore,the endogenous Trswo 1 gene was overexpressed in T.reesei Rut C30 using a constitutive promoter pyruvate decarboxylase 1(PDC1),and the SWO1 transcription levels of the two transformants T.reesei OEswo1-1 and OEswo1-2 were increased by 4.65 times and 3.91 times,respectively.There was no significant change in filter paper activity,endoglucanases,or cellobiohydrolases,but the crude enzymes of T.reesei OEswo1-1 and OEswo1-2 were applied to hydrolyze corn stover with a solid loading of 5%,and the hydrolysis efficiencies were increased by 6.98%and 13.93%,respectively.Furthermore,the X-ray diffraction(XRD)and the scanning electron microscope demonstrated that the enhanced hydrolysis efficiency was primarily triggered by the decrease in the crystallinity of lignocellulose.Furthermore,the fiber structure exhibited varying degrees of loosening and disintegration.The results of this study have implications for the low content of auxiliary protein that degrade cellulose in cellulase systems synthesized by T.reesei with MGD as an inducer.