Evolutionary engineered Candida intermedia exhibits improved xylose utilization and robustness to lignocellulose-derived inhibitors and ethanol

Abstract

The development of robust microorganisms that can efficiently ferment both glucose and xylose represents one of the major challenges in achieving a cost-effective lignocellulosic bioethanol production. Candida intermedia is a non-conventional, xylose-utilizing yeast species with a high-capacity xylose transport system. The natural ability of C. intermedia to produce ethanol from xylose makes it attractive as a non-GMO alternative for lignocellulosic biomass conversion in biorefineries. We have evaluated the fermentation capacity and the tolerance to lignocellulose-derived inhibitors and the end product, ethanol, of the C. intermedia strain CBS 141442 isolated from steam-exploded wheat straw hydrolysate. In a mixed sugar fermentation medium, C. intermedia CBS 141442 co-fermented glucose and xylose, although with a preference for glucose over xylose. The strain was clearly more sensitive to inhibitors and ethanol when consuming xylose than glucose. C. intermedia CBS 141442 was also subjected to evolutionary engineering with the aim of increasing its tolerance to inhibitors and ethanol, and thus improving its fermentation capacity under harsh conditions. The resulting evolved population was able to ferment a 50% (v/v) steam-exploded wheat straw hydrolysate (which was completely inhibitory to the parental strain), improving the sugar consumption and the final ethanol concentration. The evolved population also exhibited a better tolerance to ethanol when growing in a xylose medium supplemented with 35.5 g/L ethanol. These results highlight the potential of C. intermedia CBS 141442 to become a robust yeast for the conversion of lignocellulose to ethanol.

Keywords: Lignocellulosic bioethanol; Microbial robustness; Non-conventional yeast; Xylose fermentation.

Fig. 1

Schematic representation of the evolutionary engineering of C. intermedia CBS 141442. The evolved population EVO 1 was obtained after 2 cycles of random mutagenesis with UV light and short-term adaptation in the presence of lignocellulose-derived inhibitors (5–30% (v/v) wheat straw hydrolysate). This intermediate population was subsequently subjected to short-term adaptation in the presence of 31.6 g/L ethanol, resulting in the final evolved population EVO 2

Fig. 2

Fermentation of mineral media with an initial xylose concentration of 20 g/L and a glucose concentration of a 10 g/L (MM10G20X) or b 40 g/L (MM40G20X) by the parent C. intermedia strain CBS 141442

Fig. 3

Effect of ethanol on the growth of C. intermedia strain CBS 141442 in rich media with glucose (YPD) or xylose (YPX). a Biomass formation in terms of OD_600nm after 48 h of cultivation. b Inhibition mechanisms according to the ethanol tolerance index (α). c Estimation of P_max and α in the glucose-containing medium. d Estimation of P_max and α in the xylose-containing medium

Fig. 4

Fermentation of 50% (v/v) hydrolysate by the final evolved population C. intermedia EVO 2

Fig. 5

Biomass formation expressed as OD_600nm for the parental C. intermedia CBS 141442, and the evolved populations EVO 1 and EVO 2 after 48 h of cultivation in rich medium with xylose containing 31.6 g/L, 35.5 g/L, or 39.5 g/L ethanol