Trichoderma reesei Rut-C30 is widely used in industrial cellulase production, and development of cellulase hyper-producer is of great importance for economic lignocellulosic biorefinery. In this study, T. reesei Rut-C30 was engineered with an artificial zinc finger proteins (AZFPs) library. Two mutants T. reesei M1 and M2 with improved cellulase production were obtained. Compared to the parent strain, the filter paper activity (FPase) of T. reesei M1 and M2 increased 100% and 53%, respectively. In addition, the total amount of extracellular protein from the M1 mutant increased 69%, whereas the endo-β-glucanase (CMCase) activity of the M2 mutant is 64% higher compared to the parental strain. Furthermore, RT-qPCR analysis showed that the major cellulase genes exhibited significantly increased expression in both mutants, but different patterns were observed in the two mutants. On the other hand, the cellulase transcriptional repressor ace1 was down-regulated in both mutants, but the transcription level of the activator xyr1 was only up-regulated in the strain M1. These results demonstrated that different AZFPs exert diverse regulatory mechanisms on cellulase production in T. reesei. Analysis of the target genes of AZFPs from T. reesei M1 and M2 will not only benefit further exploration of the regulatory mechanisms of cellulase biosynthesis in T. reesei, but also enable development of cellulase hyper-producing strains by metabolic engineering.
里氏木霉Trichoderma reesei Rut-C30 是目前研究最广泛的纤维素酶生产菌，选育高产纤维素酶的里氏木霉菌株有助于提高木质纤维素资源生物炼制的经济性。利用人工锌指蛋白文库转化T. reesei Rut-C30，筛选获得了两株高产纤维素酶的突变株T. reesei M1 和M2，与出发菌株比较，突变株M1 和M2 滤纸酶活分别提高100%和53%，且M1 突变株外泌蛋白量提高69%，M2 内切纤维素酶活提高64%。实时定量PCR 分析结果表明，与对照菌株相比，突变株M1 和M2 中主要纤维素酶基因转录均上调，但不同酶基因在两株菌中有不同的变化特征。此外，纤维素酶抑制转录因子基因ace1 在两株突变株中都转录下调，而纤维素酶正调控转录因子基因xyr1 仅在M1 突变株中上调。以上结果表明，不同人工锌指蛋白对纤维素酶活性的影响具有多样性。对这些突变体中人工锌指蛋白靶基因进行深入分析，为进一步深入探究里氏木霉纤维素酶合成调控的机理，以及利用代谢工程选育更高效的产酶菌株提供了基础。.
Keywords: Trichoderma reesei; artificial zinc finger protein; cellulase; metabolic engineering; transcription factor.