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(-1) arabinose.

FIG. 1.

Product formation and sugar consumption (A, B, and C) and specific consumption rates (D, E, and F) during anaerobic batch cultivation of strains IMS0003 (A and D), IMS0007 (B and E), and IMS0010 (C and F) in MY containing a mixture of 30 g liter⁻¹ glucose, 15 g liter⁻¹ d-xylose, and 15 g liter⁻¹ l-arabinose. The data are data from single batch cultivations and are representative of duplicate experiments. To correct for small differences in the initial biomass, the profiles were aligned using the beginning of the glucose consumption peak. Ethanol concentrations were derived from the cumulative CO₂ production. (A, B, and C) Symbols: ▾, concentration of glucose; •, concentration of xylose; ○, concentration of arabinose; ⧫, concentration of ethanol; □, dry weight of cells. (D, E, and F) Symbols: •, specific consumption rate for xylose; ○, specific consumption rate for arabinose; ▪, sum of the specific consumption rates for the two pentoses. DW, dry weight of cells.

FIG. 2.

Residual sugar concentrations, dry weights of cells, and specific consumption rates during selective anaerobic chemostat cultivation of S. cerevisiae strain IMS0003 in MY with medium reservoir concentrations of xylose and arabinose of 20 and 20 g liter⁻¹, respectively. The dotted vertical line at 21 days indicates the shift of the dilution rate from 0.03 to 0.05 h⁻¹. Symbols: •, concentration of xylose; ○, concentration of arabinose; ⧫, dry weight of cells; ▪, specific consumption rate for xylose; □, specific consumption rate for arabinose. D, dilution rate; DW, dry weight of cells; q, specific consumption rate.

FIG. 3.

(A and B) CO₂ production profiles (A) and estimated μ_max derived from CO₂ production (B) during repeated anaerobic batch cultivation in MY-XA. Gray line, batch 2; dotted line, batch 7; dashed line, batch 12; solid black line, batch 16. (C) CO₂ production profiles during anaerobic batch cultivation in MY containing 30 g liter⁻¹ glucose, 15 g liter⁻¹ d-xylose, and 15 g liter⁻¹ l-arabinose inoculated with a 100-ml sample taken from the repeated batch cultivation (solid black line) compared to strain IMS0003 (solid gray line) and single-colony isolate IMS0007 (dashed line). To correct for small differences in the initial biomass, the profiles were aligned using the beginning of the glucose consumption peak.

FIG. 4.

CO₂ production profiles (A) and estimated μ_max derived from the CO₂ production profiles (B) during anaerobic SBR cultivation with repeated cycles consisting of consecutive cultivation in MY-GXA, MY-XA, and MY-A. (A) Gray line, cycle 1; dotted line, cycle 7; dashed line, cycle 13; solid black line, cycle 20. (B) Symbols: •, MY-GXA; ▪, MY-XA; ▴, MY-A.