Fusion transcription factors for strong, constitutive expression of cellulases and xylanases in Trichoderma reesei

Abstract

Background: The filamentous ascomycete T. reesei is industrially used to produce cellulases and xylanases. Costeffective production of cellulases is a bottleneck for biofuel production. Previously, different strain and process optimizations were deployed to enhance enzyme production rates. One approach is the overexpression of the main activator Xyr1 and a second is the construction of synthetic transcription factors. Notably, these genetic manipulations were introduced into strains bearing the wild-type xyr1 gene and locus. Results: Here, we constructed a Xyr1-deficient strain expressing a non-functional truncated version of Xyr1. This strain was successfully used as platform strain for overexpression of Xyr1, which enhanced the cellulase and xylanase production rates under inducing conditions, with the exception of lactose-there the cellulase production was severely reduced. Further, we introduced fusion transcription factors consisting of the DNA-binding domain of Xyr1 and the transactivation domain of either Ypr1 or Ypr2 (regulators of the sorbicillinoid biosynthesis gene cluster). The fusion of Xyr1 and Ypr2 yielded a moderately transactivating transcription factor, whereas the fusion of Xyr1 and Ypr1 yielded a highly transactivating transcription factor that induced xylanases and cellulases nearly carbon source independently. Especially, high production levels of xylanases were achieved on glycerol. Conclusion: During this study, we constructed a Xyr1-deficient strain that can be fully reconstituted, which makes it an ideal platform strain for Xyr1-related studies. The mere overexpression of Xyr1 turned out not to be a successful strategy for overall enhancement of the enzyme production rates. We gained new insights into the regulatory properties of transcription factors by constructing respective fusion proteins. The Xyr1-Ypr1-fusion transcription factor could induce xylanase production rates on glycerol to outstanding extents, and hence could be deployed in the future to utilize crude glycerol, the main co-product of the biodiesel production process.

Precision Engineering of the Transcription Factor Cre1 in Hypocrea jecorina (Trichoderma reesei) for Efficient Cellulase Production in the Presence of Glucose

“…The filamentous ascomycete Trichoderma reesei, a clonal derivative of the ascomycete Hypocrea jecorina (Kuhls et al, 1996), is widely used in industrial production of cellulases and xylanases (Derntl et al, 2019). There is a broad range of industrial applications of these cellulolytic enzymes in the food and feed, textile, and pulp and paper industries, as well as in the production of lignocellulosic bioethanol (Wilson, 2009).…”

Penicillium Janthinellum NCIM-1366 Shows Improved Biomass Hydrolysis Performance Over Trichoderma Reesei RUT-C30, and Secretome Analysis Reveals a Larger Number of CAZymes with Higher Induction Levels.

“…Lignocellulose degrading enzymes are critical in biomass conversion to biofuels and lamentous fungi are typically used for the production of these enzymes because of their ability to synthesize and secrete a wide array of plant cell wall degrading enzymes [13]. T. reesei RUT-C30 is the most widely used fungus for cellulase production, in the past several decades, and efforts for improving its enzyme production are still at large [14,15]. P. janthinellum NCIM 1366 is a mutant strain developed at the National Chemical Laboratory, Pune, India through classical mutagenesis and which exhibited enhanced cellulase production compared to the parent strain NCIM 1171 [9].…”

Penicillium janthinellum NCIM1366 shows improved biomass hydrolysis and a larger number of CAZymes with higher induction levels over Trichoderma reesei RUT-C30

“…This is primarily due to the fact that T. reesei produces the highest known titers of enzymes, the extracellular protein concentrations reaching as high 100g/L [19]; and there is a wealth of information accumulated on its genetics and gene regulation through works spanning several decades [20]. However, there are still efforts targeted at improving its enzyme production [21,22] as the cost of cellulases cannot yet be considered as economical for bio-re nery operations. P. janthinellum NCIM 1366 is a mutant strain developed at the National Chemical Laboratory, Pune, India through classical mutagenesis and which exhibited enhanced cellulase production compared to the parent strain NCIM 1171 [9].…”

“…Proteomic approaches have been widely used in lamentous fungi for the identi cation of both intracellular and extracellular proteins [21]. The genome of T. reesei QM6a, which is the parent strain of RUT-C30 was rst sequenced in 2008 giving insight into its CAZyme system [22].…”