Abstract
BackgroundThe path for the development of hypersecreting strains of Trichoderma reesei 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 T. reesei RUT-C30 strain and a customized process for cellulase production based on agroindustrial by-products.ResultsA 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 Talaromyces emersonii and the invertase SUC1 from Aspergillus niger, 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−1 (0.24 g L−1 h−1), which is the highest experimentally supported titer so far reported for T. reesei. 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.ConclusionThis 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 Trichoderma research on cellulase production.
Highlights
The path for the development of hypersecreting strains of Trichoderma reesei capable of producing industrially relevant enzyme titers remains elusive despite over 70 years of research and industrial utilization
We report the CRISPR/Cas9-based rational engineering of the RUT-C30 strain and the development of an industrially compatible process for cellulase production based on agroindustrial by-products
Target-specific CRISPR/Cas9 plasmids carried a Streptococcus pyogenes Cas9 gene codon-optimized for expression in T. reesei, a ribozyme-mediated guide RNA cassette, an antibiotic resistance selection marker and the AMA1 fungal replicator sequence (Additional file 1: Fig. S3) [53]
Summary
The path for the development of hypersecreting strains of Trichoderma reesei capable of producing industrially relevant enzyme titers remains elusive despite over 70 years of research and industrial utilization. The cost-efficient enzymatic deconstruction of non-edible lignocellulosic materials, mainly agroindustrial residues, is expected to enable the use of this large and mostly untapped source of carbon for the production of biofuels and value-added chemicals [4,5,6,7,8]. As with other classes of enzymes, the industrial production of cellulases is largely controlled by a few longestablished companies [9, 10]. This market dominance arises from, among other factors, the use of proprietary microbial platforms and production processes which have been continuously developed for decades. The formidable action of these enzymes is believed to be favored by the fungus’ diverse membranetrafficking system and clustered genome organization [13]
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