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

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Bezerra de Mello, Fellipe da Silveira ; Maneira, Carla ; Lizarazo Suarez, Frank Uriel ; Nagamatsu, Sheila ; Vargas, Beatriz ; Vieira, Carla ; Secches, Thais ; Coradini, Alessando L., V ; de Carvalho Silvello, Maria Augusta ; Goldbeck, Rosana ; Guimaraes Pereira, Goncalo Amarante ; Teixeira, Gleidson Silva

• Document type: Journal article
• Source: JOURNAL OF GENETIC ENGINEERING AND BIOTECHNOLOGY; v. 20, n. 1, p. 16-pg., 2022-05-25.

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 ho Delta::xyl1) and CENPKX (CEN.PK-122 ho Delta::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. (AU)

• FAPESP's process: 15/06677-8 - Metabolic engineering and identification of quantitative traits loci related to HMF tolerance of Saccharomyces cerevisiae strain
• Grantee: Fellipe da Silveira Bezerra de Mello
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• FAPESP's process: 18/03403-2 - Matabolic engineering of Saccharomyces cerevisiae wild strains using the CRISPR/Cas9 system
• Grantee: Carla Maneira da Silva
• Support Opportunities: Scholarships in Brazil - Master
• FAPESP's process: 16/02506-7 - Development of high-throughput approaches for the study of quantitative traits loci related to robustness of industrial yeasts (Saccharomyces cerevisiae)
• Grantee: Gleidson Silva Teixeira
• Support Opportunities: Program for Research on Bioenergy (BIOEN) - Regular Program Grants