Fonseca Lucas Miranda, Parreiras Lucas Salera, Murakami Mario Tyago
Brazilian Biorenewables National Laboratory, Brazilian Center for Research in Energy and Materials, Campinas, São Paulo 13083-100 Brazil.
Biotechnol Biofuels. 2020 May 22;13:93. doi: 10.1186/s13068-020-01732-w. eCollection 2020.
The path for the development of hypersecreting strains of capable of producing industrially relevant enzyme titers remains elusive despite over 70 years of research and industrial utilization. Herein, we describe the rational engineering of the publicly available RUT-C30 strain and a customized process for cellulase production based on agroindustrial by-products.
A CRISPR/Cas9 system was used to introduce six genetic modifications in RUT-C30. Implemented changes included the constitutive expression of a mutated allele of the cellulase master regulator XYR1, the expression of two heterologous enzymes, the β-glucosidase CEL3A from and the invertase SUC1 from , and the deletion of genes encoding the cellulase repressor ACE1 and the extracellular proteases SLP1 and PEP1. These alterations resulted in a remarkable increase of protein secretion rates by RUT-C30 and amended its well described β-glucosidase deficiency while enabling the utilization of sucrose and eliminating the requirement of inducing sugars for enzyme production. With a developed sugarcane molasses-based bioprocess, the engineered strain reached an extracellular protein titer of 80.6 g L (0.24 g L h), which is the highest experimentally supported titer so far reported for . The produced enzyme cocktail displayed increased levels of cellulase and hemicellulase activities, with particularly large increments being observed for the specific activities of β-glucosidase (72-fold) and xylanase (42-fold). Notably, it also exhibited a saccharification efficiency similar to that of a commercially available cellulase preparation in the deconstruction of industrially pretreated sugarcane straw.
This work demonstrates the rational steps for the development of a cellulase hyperproducing strain from a well-characterized genetic background available in the public domain, the RUT-C30, associated with an industrially relevant bioprocess, paving new perspectives for research on cellulase production.
尽管经过70多年的研究和工业应用，但能够产生具有工业相关酶活性的高产菌株的开发途径仍然不明。在此，我们描述了对公开可用的里氏木霉RUT-C30菌株的合理工程改造以及基于农业工业副产品的纤维素酶生产定制工艺。
使用CRISPR/Cas9系统对RUT-C30进行了六处基因改造。实施的改造包括纤维素酶主调节因子XYR1突变等位基因的组成型表达、两种异源酶的表达，即来自黑曲霉的β-葡萄糖苷酶CEL3A和来自酿酒酵母的转化酶SUC1，以及编码纤维素酶阻遏物ACE1和细胞外蛋白酶SLP1和PEP1的基因缺失。这些改变导致RUT-C30的蛋白质分泌率显著提高，改善了其众所周知的β-葡萄糖苷酶缺陷，同时能够利用蔗糖并消除酶生产对诱导糖的需求。通过开发的基于甘蔗 molasses的生物工艺，工程菌株的细胞外蛋白产量达到80.6 g/L（0.24 g/L/h），这是迄今为止报道的里氏木霉实验支持的最高产量。所产生的酶混合物显示纤维素酶和半纤维素酶活性水平增加，β-葡萄糖苷酶（72倍）和木聚糖酶（42倍）的比活性尤其大幅增加。值得注意的是，在工业预处理甘蔗秸秆的解构中，它还表现出与市售纤维素酶制剂相似的糖化效率。
这项工作展示了从公共领域中一个特征明确的遗传背景——里氏木霉RUT-C30开发纤维素酶高产菌株的合理步骤，该菌株与一个与工业相关的生物工艺相关，为纤维素酶生产的里氏木霉研究开辟了新的前景。
