D-xylose accelerated death of pentose metabolizing Saccharomyces cerevisiae

Abstract

Rapid and effective consumption of D-xylose by Saccharomyces cerevisiae is essential for cost-efficient cellulosic bioethanol production. Hence, heterologous D-xylose metabolic pathways have been introduced into S. cerevisiae. An effective solution is based on a xylose isomerase in combination with the overexpression of the xylulose kinase (Xks1) and all genes of the non-oxidative branch of the pentose phosphate pathway. Although this strain is capable of consuming D-xylose, growth inhibition occurs at higher D-xylose concentrations, even abolishing growth completely at 8% D-xylose. The decreased growth rates are accompanied by significantly decreased ATP levels. A key ATP-utilizing step in D-xylose metabolism is the phosphorylation of D-xylulose by Xks1. Replacement of the constitutive promoter of XKS1 by the galactose tunable promoter Pgal10 allowed the controlled expression of this gene over a broad range. By decreasing the expression levels of XKS1, growth at high D-xylose concentrations could be restored concomitantly with increased ATP levels and high rates of xylose metabolism. These data show that in fermentations with high D-xylose concentrations, too high levels of Xks1 cause a major drain on the cellular ATP levels thereby reducing the growth rate, ultimately causing substrate accelerated death. Hence, expression levels of XKS1 in S. cerevisiae needs to be tailored for the specific growth conditions and robust D-xylose metabolism.

Keywords: ATP; Bioethanol; D-xylose consumption; Xks1 expression; Yeast.

Fig. 1

Aerobic growth of the IMX730-▲H strain in mineral medium containing 0.5% d-xylose (●), 1.0% d-xylose (■), 2.0% d-xylose (▲), 4.0% d-xylose (◆) and 8.0% d-xylose (○) complemented with l-histidine. Error bars were obtained from biological duplicates

Fig. 2

Intracellular ATP analysis after adding (T0) 0.5% d-xylose (●), 1.0% d-xylose (■), 2.0% d-xylose (▲), 4.0% d-xylose (◆) and 8.0% d-xylose (○) in the IMX730-△H strain. The IMX730-△H pre-culture was grown aerobically for 16 h in mineral medium supplemented with 0.5% d-xylose and l-histidine. Error bars were obtained from biological triplicates

Fig. 3

A Transcript fold change levels of XKS1 in IMX730-△H (grey bars) and IMX730-pGAL::XKS1 (white bars) at various galactose concentrations ranging from 0 to 1%. WT-0 represented IMX730-△H was incubated in the absence of galactose, while WT-1 represented IMX730-△H incubated in the presence of 1% galactose. Cells were incubated aerobically in MM containing 0.5% d-xylose and 2 h after the addition of the galactose RNA was isolated. B Xks1 activity in IMX730-pGAL::XKS1 (white bars) at 0, 0.025 and 1% galactose and in IMX730-△H (grey bar, 0% galactose). All error bars were obtained from biological duplicates

Fig. 4

Aerobic growth in 96 wells micro-titer plates of IMX730-△H (A) and IMX730-pGAL::XKS1 (B) in mineral medium containing 0.5% d-xylose (●), 1.0% d-xylose (■), 2.0% d-xylose (▲), 4.0% d-xylose (◆) and 8.0% d-xylose (○) complemented with 0.0125% galactose. Error bars were obtained from biological triplicates

Fig. 5

Intracellular ATP analysis in the IMX730-pGAL::XKS1 strain which was pre-incubated for 2 h with 0.003125% galactose (open symbols) or 1.0% galactose (closed symbols). 0.5% d-xylose (squares) or 8% d-xylose (circles) was added to the cultures at T0 and ATP levels were analyzed after 1, 10, 30 and 60 min. Error bars were obtained from biological duplicates