Abstract
BackgroundLignosulfonates 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.ResultsThe 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 (µmax) of 0.28 1/h. Selected evolved strains utilized all carbon sources in the SSL at pH 3.5 and grew with µmax 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.ConclusionsThe 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.
Highlights
Lignosulfonates are significant wood chemicals with a $700 million market, produced by sulfite pulping of wood
Engineered industrial strain consumes xylose in complex medium To develop a strain performing in the Eucalyptus spent sulfite liquor containing xylose as the major sugar, the diploid industrial strain Ethanol Red, used in first generation bioethanol plants was engineered for xylose utilization
The CRISPR-Cas9 approach was used for deletion of GRE3 gene with simultaneous integration of two additional copies of the xylose isomerase gene (Fig. 1a)
Summary
Lignosulfonates are significant wood chemicals with a $700 million market, produced by sulfite pulping of wood. S. cerevisiae faces two main challenges in processes using complex agricultural or forestry residues as a substrate for fermentation. This yeast is not naturally able to utilize pentose sugars that are abundantly present in lignocellulosic biomass as hemicellulose polymer. In contrast to first generation feedstocks, lignocellulosic streams need to be pretreated to release sugars from the rigid polymeric substance, releasing a spectrum of compounds negatively affecting growth and fermentation performance of the host organism [11]. Identification of new target genes is desirable for future rational engineering strategies to develop novel robust strains
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