Fig. 1

Genetic engineering of the RUT-C30 strain for improved cellulase production. a A total of six genetic changes were introduced in RUT-C30 through three rounds of engineering using a CRISPR/Cas9 system. In round 1, the T. emersonii cel3a gene under control of the xyn1 promoter was inserted in the slp1 locus. In round 2, the mutated xyr1-V821F allele under control of the pdc1 promoter was inserted in the ace1 locus. In round 3, the A. niger suc1 gene driven by its native regulatory sequences was inserted in the pep1 locus. b The implemented modifications aimed to improve the fungus enzyme production capabilities by (I) enabling the use of sucrose as a carbon source, (II) increasing transcription levels of genes encoding secreted enzymes, (III) decreasing the proteolytic activity of its secretome and (IV) increasing the β-glucosidase activity of the secreted enzyme cocktail