Novel Evolutionary Engineering Approach for Accelerated Utilization of Glucose, Xylose, and Arabinose Mixtures by Engineered Saccharomyces cerevisiae Strains

Abstract

Lignocellulosic feedstocks are thought to have great economic and environmental significance for future biotechnological production processes. For cost-effective and efficient industrial processes, complete and fast conversion of all sugars derived from these feedstocks is required. Hence, simultaneous or fast sequential fermentation of sugars would greatly contribute to the efficiency of production processes. One of the main challenges emerging from the use of lignocellulosics for the production of ethanol by the yeast Saccharomyces cerevisiae is efficient fermentation of D-xylose and L-arabinose, as these sugars cannot be used by natural S. cerevisiae strains. In this study, we describe the first engineered S. cerevisiae strain (strain IMS0003) capable of fermenting mixtures of glucose, xylose, and arabinose with a high ethanol yield (0.43 g g ؊1 of total sugar) without formation of the side products xylitol and arabinitol. The kinetics of anaerobic fermentation of glucose-xylose-arabinose mixtures were greatly improved by using a novel evolutionary engineering strategy. This strategy included a regimen consisting of repeated batch cultivation with repeated cycles of consecutive growth in three media with different compositions (glucose, xylose, and arabinose; xylose and arabinose; and only arabinose) and allowed rapid selection of an evolved strain (IMS0010) exhibiting improved specific rates of consumption of xylose and arabinose. This evolution strategy resulted in a 40% reduction in the time required to completely ferment a mixture containing 30 g liter ؊1 glucose, 15 g liter ؊1 xylose, and 15 g liter ؊1arabinose.In recent years, the need for biotechnological manufacturing based on lignocellulosic feedstocks has become evident (6, 10). In contrast to the readily fermentable, mainly starch-or sucrose-containing feedstocks used in current biotechnological production processes, lignocellulosic biomass requires intensive pretreatment and hydrolysis, which yield complex mixtures of sugars (3,7,14,27). For cost-effective and efficient industrial processes, complete and fast conversion of all sugars present in lignocellulosic hydrolysates is a prerequisite. The major hurdles encountered in implementing these production processes are the conversion of substrates that cannot be utilized by the organism of choice and, even more importantly, the subsequent improvement of sugar conversion rates and product yields.The use of evolutionary engineering has proven to be very valuable for obtaining phenotypes of (industrial) microorganisms with improved properties, such as an expanded substrate range, increased stress tolerance, and efficient substrate utilization (16,17). Also, for the yeast Saccharomyces cerevisiae, the preferred organism for large-scale ethanol production for the past few decades, evolutionary engineering has been extensively used to select for industrially relevant phenotypes. For ethanol production from lignocellulose by S. cerevisiae, one of the main challenges is efficient conversion of the pentoses ...

Unravelling evolutionary strategies of yeast for improving galactose utilization through integrated systems level analysis

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Sequential Targeting of Xylose and Glucose Conversion in Fed-Batch Simultaneous Saccharification and Co-fermentation of Steam-Pretreated Wheat Straw for Improved Xylose Conversion to Ethanol

“…To attenuate the effect of inhibitors and improve co-fermentation efficiency, strategies that improve the natural tolerance of the microorganism have been developed. Improved tolerance to inhibitors and ability to convert xylose have been achieved by e.g., evolutionary engineering [12,13] and short-term adaptation during propagation [14]. However, xylose is still generally fermented by S. cerevisiae to ethanol at lower rates [15] and lower yields than glucose [15,16].…”

Catalysis, Structures, and Applications of Carbohydrate Epimerases

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