Biotrend (Portugal)

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.

Carboxylic acids are truly central compounds in cellular metabolism. Carbon dioxide is captured from the atmosphere through formation of carboxylic groups and is also released, in part, by decarboxylation reactions. The reactivity of the carboxylic group with amino- or hydroxyl-groups enables the formation of peptide and ester bonds. The functionality of the carboxylic group is also of huge importance in our industrial world for a wide range of applications. The loosely bound hydrogen provides weak acid functionality, much desired for food industry applications in preservatives and flavour compounds. Citric acid is one of our oldest industrial fermentation products. The presence of two carboxylic groups, or a combination of one carboxylic group and another functional group, make the compounds interesting building blocks for polymer production. A number of carboxylic acids, including, e.g., lactic, succinic, 3-hydroxypropionic and itaconic acids, have been identified and recognized as suitable platform chemicals for a foreseen growing carbohydrate based economy. Economic margins are, however, tight when competing with petroleum based production, and production strains, fermentation technology and—not least—downstream processing, all need to be improved to enable viable commercial production.This Special issue will cover current developments within this exciting field. Topics will include: Fermentation physiology of natural carboxylic acid producers; screening and isolation of novel producers; metabolic engineering for improving intrinsic carboxylic acid production; metabolic engineering for expanding product range to non-endogenous carboxylic acids; production from lignocellulosic derived sugars or by-product streams; downstream processing for recovery of carboxylic acids; bioprocess design—including continuous processes and integration.All production organisms—fungi, yeasts, bacteria—are welcome.