Rational engineering of industrial S. cerevisiae: towards xylitol production from sugarcane straw

Abstract

Background Sugarcane hemicellulosic material is a compelling source of usually neglected xylose that could figure as feedstock to produce chemical building blocks of high economic value, such as xylitol. In this context, Saccharomyces cerevisiae strains typically used in the Brazilian bioethanol industry are a robust chassis for genetic engineering, given their robustness towards harsh operational conditions and outstanding fermentation performance. Nevertheless, there are no reports on the use of these strains for xylitol production using sugarcane hydrolysate. Results Potential single-guided RNA off-targets were analyzed in two preeminent industrial strains (PE-2 and SA-1), providing a database of 5′-NGG 20 nucleotide sequences and guidelines for the fast and cost-effective CRISPR editing of such strains. After genomic integration of a NADPH-preferring xylose reductase (XR), FMYX (SA-1 hoΔ::xyl1) and CENPKX (CEN.PK-122 hoΔ::xyl1) were tested in varying cultivation conditions for xylitol productivity to infer influence of the genetic background. Near-theoretical yields were achieved for all strains; however, the industrial consistently outperformed the laboratory strain. Batch fermentation of raw sugarcane straw hydrolysate with remaining solid particles represented a challenge for xylose metabolization, and 3.65 ± 0.16 g/L xylitol titer was achieved by FMYX. Finally, quantification of NADPH — cofactor implied in XR activity — revealed that FMYX has 33% more available cofactors than CENPKX. Conclusions Although widely used in several S. cerevisiae strains, this is the first report of CRISPR-Cas9 editing major yeast of the Brazilian bioethanol industry. Fermentative assays of xylose consumption revealed that NADPH availability is closely related to mutant strains’ performance. We also pioneer the use of sugarcane straw as a substrate for xylitol production. Finally, we demonstrate how industrial background SA-1 is a compelling chassis for the second-generation industry, given its inhibitor tolerance and better redox environment that may favor production of reduced sugars.

Unveiling genetic anchors in Saccharomyces cerevisiae: QTL mapping identifies IRA2 as a key player in ethanol tolerance and beyond

“…To test the influence of IRA2 alleles on the stress response, the gene was knocked out individually and further crossed with the non-deleted remaining parent, rendering strains SA (SA49/CEN.PK113-1A ira2 Δ::hphMX6) and CEN (SA49 ira2 Δ::hphMX6/CEN.PK113-1A). Genetic engineering was performed with a CRISPR-Cas9 system previously described (de Mello et al, 2022b). Overall, a single-guided RNA (sgRNA) with homology to the IRA2 open reading frame was used to insert a hygromycin resistance marker ( hphMX6 ) in the target region, inactivating the endogenous coding sequence.…”

Advances in Environmental Biotechnology with CRISPR/Cas9: Bibliometric Review and Cutting-Edge Applications

“…Emphasis has been placed on enhancing S. cerevisiae, a relevant yeast species in ethanolic fermentation, with efforts directed toward augmenting its activity, tolerance, yield, and efficiency. This pursuit facilitates time reduction and enhanced productivity and streamlines labor requirements, concurrently fostering a more profound comprehension of molecular genetics and metabolism [132,133].…”