Journal Article

Rational and evolutionary engineering of Saccharomyces cerevisiae for production of dicarboxylic acids from lignocellulosic biomass and exploring genetic mechanisms of the yeast tolerance to the biomass hydrolysate

Lidén, Gunnar, Dato, Laura, Tjosås, Freddy, Schöpping, Marie, Koza, Anna, Borodina, Irina, Ferreira, Bruno Sommer, Forster, Jochen, Stovicek, Vratislav, Almqvist, Henrik, Chekina, Ksenia, Pedersen, Lasse Ebdrup, Figueira, Diogo

2022

Overview

Background Lignosulfonates are significant wood chemicals with a $700 million market, produced by sulfite pulping of wood. During the pulping process, spent sulfite liquor (SSL) is generated, which in addition to lignosulfonates contains hemicellulose-derived sugars—in case of hardwoods primarily the pentose sugar xylose. The pentoses are currently underutilized. If they could be converted into value-added chemicals, overall economic profitability of the process would increase. SSLs are typically very inhibitory to microorganisms, which presents a challenge for a biotechnological process. The aim of the present work was to develop a robust yeast strain able to convert xylose in SSL to carboxylic acids. Results The industrial strain Ethanol Red of the yeast Saccharomyces cerevisiae was engineered for efficient utilization of xylose in a Eucalyptus globulus lignosulfonate stream at low pH using CRISPR/Cas genome editing and adaptive laboratory evolution. The engineered strain grew in synthetic medium with xylose as sole carbon source with maximum specific growth rate (µ max ) of 0.28 1/h. Selected evolved strains utilized all carbon sources in the SSL at pH 3.5 and grew with µ max between 0.05 and 0.1 1/h depending on a nitrogen source supplement. Putative genetic determinants of the increased tolerance to the SSL were revealed by whole genome sequencing of the evolved strains. In particular, four top-candidate genes ( SNG1 , FIT3 , FZF1 and CBP3 ) were identified along with other gene candidates with predicted important roles, based on the type and distribution of the mutations across different strains and especially the best performing ones. The developed strains were further engineered for production of dicarboxylic acids (succinic and malic acid) via overexpression of the reductive branch of the tricarboxylic acid cycle (TCA). The production strain produced 0.2 mol and 0.12 mol of malic acid and succinic acid, respectively, per mol of xylose present in the SSL. Conclusions The combined metabolic engineering and adaptive evolution approach provided a robust SSL-tolerant industrial strain that converts fermentable carbon content of the SSL feedstock into malic and succinic acids at low pH.in production yields reaching 0.1 mol and 0.065 mol per mol of total consumed carbon sources.. Moreover, our work suggests potential genetic background of the tolerance to the SSL stream pointing out potential gene targets for improving the tolerance to inhibitory industrial feedstocks.

• Publisher: BioMed Central,BioMed Central Ltd,Nature Publishing Group
• Subject: Acids / Adaptive evolution / Analysis / Bioenergi / Bioenergy / biofuels / Biologi / Biological Sciences / Biomass / Biorefineries / Biotechnology / Brewer's yeast / Carbon / Carbon content / Carbon sources / Carboxylic acids / chemical reduction / Chemistry / Chemistry and Materials Science / Costs / CRISPR / Dicarboxylic acids / Drug tolerance / Economics / Engineering and Technology / Environmental Engineering/Biotechnology / Ethanol / Eucalyptus / Eucalyptus globulus / Evolution / evolutionary adaptation / feedstocks / Fermentation / Genes / Genetic aspects / genetic background / Genetic engineering / Genomes / Genomics / Hardwood spent sulfite liquor / Hardwoods / Hemicellulose / Hydrolysates / Industrial Biotechnology / Industrial yeast / Industriell bioteknik / Kraft process (woodpulp) / Lignin / Lignocellulose / Lignosulfonates / Malic acid / markets / Metabolic engineering / Metabolism / Microbial genetic engineering / Microbiology / Microorganisms / Mikrobiologi / Monosaccharides / Natural Sciences / Naturvetenskap / Next generation sequencing / nitrogen / Pentose / pentoses / pH effects / Plant biomass / Plant Breeding/Biotechnology / Production processes / profitability / Pulping / Raw materials / Renewable and Green Energy / Robustness / Saccharomyces cerevisiae / specific growth rate / Spent liquors / Strains (organisms) / streams / Succinic acid / Sugar / Sugars / Sulfite / sulfite liquor / Sulfite liquors / Sulfites / Sulfonation / Teknik / Tricarboxylic acid cycle / value added / Whole genome sequencing / wood / Xylose / Yeast / yeasts