Metabolic engineering of Saccharomyces cerevisiae for second-generation ethanol production from xylo-oligosaccharides and acetate

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Procopio, Dielle Pierotti ; Lee, Jae Won ; Shin, Jonghyeok ; Tramontina, Robson ; Avila, Patricia Felix ; Brenelli, Livia Beatriz ; Squina, Fabio Marcio ; Damasio, Andre ; Rabelo, Sarita Candida ; Goldbeck, Rosana ; Franco, Telma Teixeira ; Leak, David ; Jin, Yong-Su ; Basso, Thiago Olitta

• Document type: Journal article
• Source: SCIENTIFIC REPORTS; v. 13, n. 1, p. 14-pg., 2023-11-06.

Abstract

Simultaneous intracellular depolymerization of xylo-oligosaccharides (XOS) and acetate fermentation by engineered Saccharomyces cerevisiae offers significant potential for more cost-effective second-generation (2G) ethanol production. In the present work, the previously engineered S. cerevisiae strain, SR8A6S3, expressing enzymes for xylose assimilation along with an optimized route for acetate reduction, was used as the host for expressing two beta-xylosidases, GH43-2 and GH43-7, and a xylodextrin transporter, CDT-2, from Neurospora crassa, yielding the engineered SR8A6S3-CDT-2-GH34-2/7 strain. Both beta-xylosidases and the transporter were introduced by replacing two endogenous genes, GRE3 and SOR1, that encode aldose reductase and sorbitol (xylitol) dehydrogenase, respectively, and catalyse steps in xylitol production. The engineered strain, SR8A6S3-CDT-2-GH34-2/7 (sor1 Delta gre3 Delta), produced ethanol through simultaneous XOS, xylose, and acetate co-utilization. The mutant strain produced 60% more ethanol and 12% less xylitol than the control strain when a hemicellulosic hydrolysate was used as a mono- and oligosaccharide source. Similarly, the ethanol yield was 84% higher for the engineered strain using hydrolysed xylan, compared with the parental strain. Xylan, a common polysaccharide in lignocellulosic residues, enables recombinant strains to outcompete contaminants in fermentation tanks, as XOS transport and breakdown occur intracellularly. Furthermore, acetic acid is a ubiquitous toxic component in lignocellulosic hydrolysates, deriving from hemicellulose and lignin breakdown. Therefore, the consumption of XOS, xylose, and acetate expands the capabilities of S. cerevisiae for utilization of all of the carbohydrate in lignocellulose, potentially increasing the efficiency of 2G biofuel production. (AU)

• FAPESP's process: 17/15477-8 - Development of a low severity pretreatment process for the production of sugarcane straw oligosaccharides
• Grantee: Lívia Beatriz Brenelli de Paiva
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 20/05784-3 - EMU approved in grant 15 / 50590-4: chromatographic system and detectors for analysis of sugars and lignocellulosic monolignols
• Grantee: Fábio Márcio Squina
• Support Opportunities: Multi-user Equipment Program
• FAPESP's process: 15/50612-8 - An integrated approach to explore a novel paradigm for biofuel production from lignocellulosic feedstocks
• Grantee: Telma Teixeira Franco
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 18/17172-2 - How do fuel ethanol yeasts and contaminating lactic acid bacteria respond toward lignocellulosic-derived inhibitors?
• Grantee: Thiago Olitta Basso
• Support Opportunities: Regular Research Grants
• FAPESP's process: 18/01759-4 - Metabolic engineering of Saccharomyces cerevisiae to produce second generation ethanol from xylooligosaccharides
• Grantee: Dielle Pierotti Procópio
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 22/08958-8 - Development of microorganisms and thermophilic enzymes involved in the degradation and upcycling of fossil plastics
• Grantee: Fábio Márcio Squina
• Support Opportunities: Regular Research Grants
• FAPESP's process: 21/04254-3 - Development of Biorefinery concepts via coumaric acid conversion into high-value compounds
• Grantee: Robson Tramontina
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 15/50590-4 - Lignin valorization in cellulosic ethanol plants: biocatalytic conversion via ferulic acid to high value chemicals
• Grantee: Fábio Márcio Squina
• Support Opportunities: Program for Research on Bioenergy (BIOEN) - Thematic Grants
• FAPESP's process: 22/05731-2 - BEYOND: establishing a fungal cell factory for recombinant protein production
• Grantee: André Ricardo de Lima Damasio
• Support Opportunities: Research Projects - Thematic Grants
• FAPESP's process: 19/18075-3 - Metabolic engineering of Saccharomyces cerevisiae for second generation ethanol from xylo-oligosaccharides and acetate
• Grantee: Dielle Pierotti Procópio
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate