Production of fuels and chemicals from xylose by engineered Saccharomyces cerevisiae: a review and perspective

Abstract

Efficient xylose utilization is one of the most important pre-requisites for developing an economic microbial conversion process of terrestrial lignocellulosic biomass into biofuels and biochemicals. A robust ethanol producing yeast Saccharomyces cerevisiae has been engineered with heterologous xylose assimilation pathways. A two-step oxidoreductase pathway consisting of NAD(P)H-linked xylose reductase and NAD⁺-linked xylitol dehydrogenase, and one-step isomerase pathway using xylose isomerase have been employed to enable xylose assimilation in engineered S. cerevisiae. However, the resulting engineered yeast exhibited inefficient and slow xylose fermentation. In order to improve the yield and productivity of xylose fermentation, expression levels of xylose assimilation pathway enzymes and their kinetic properties have been optimized, and additional optimizations of endogenous or heterologous metabolisms have been achieved. These efforts have led to the development of engineered yeast strains ready for the commercialization of cellulosic bioethanol. Interestingly, xylose metabolism by engineered yeast was preferably respiratory rather than fermentative as in glucose metabolism, suggesting that xylose can serve as a desirable carbon source capable of bypassing metabolic barriers exerted by glucose repression. Accordingly, engineered yeasts showed superior production of valuable metabolites derived from cytosolic acetyl-CoA and pyruvate, such as 1-hexadecanol and lactic acid, when the xylose assimilation pathway and target synthetic pathways were optimized in an adequate manner. While xylose has been regarded as a sugar to be utilized because it is present in cellulosic hydrolysates, potential benefits of using xylose instead of glucose for yeast-based biotechnological processes need to be realized.

Keywords: Metabolic engineering; Saccharomyces cerevisiae; Xylose.

Fig. 1

Overview of xylose assimilation pathways in yeasts. Red items indicate heterologous metabolic pathways which have been introduced into S. cerevisiae. 6-PGL 6-phosphoglucono-1,5-lactone, 6-PG 6-phosphogluconate, GA-3-P glyceraldehyde-3-phosphate

Fig. 2

Control of redox balance by manipulating endogenous metabolism (a, b) and furnishing heterologous electron sink reactions (c, d). Endogenous NADPH regenerating enzymes of the oxidative pentose phosphate pathway (a) and the ammonia utilization pathway were deleted to reduce XR activity and accordingly develop a lower XR/XDH activity ratio. Surplus NADH was alleviated via NADH oxidase reaction (a) or utilized as a driving force of acetate reduction pathway consisting of acetyl-CoA synthase (ACS) and acetylating acetaldehyde dehydrogenase (AADH) (b). Gene nomenclature: ZWF1 glucose-6-phsophate dehydrogenase, GND1 6-phosphogluconate dehydrogenase, GDH1 NADPH-dependent glutamate dehydrogenase, GDH2 NADH-dependent glutamate dehydrogenase, noxE NADH oxidase from L. lactis, adhE AADH from Escherichia coli

Fig. 3

Schematics of the non-oxidative pentose phosphate pathway. While other intermediates can be shunted to glycolysis, sedoheptulose-7-phosphate is converted into a dead-end metabolite sedoheptulose by promiscuous phosphatase activity of Pho13p. The expression of TAL1 is indirectly regulated by PHO13. Gene nomenclature: RKI1 ribose-5-phosphate isomerase, RPE1 ribulose-5-phosphate epimerase, TKL1 transketolase, TAL1 transaldolase

Fig. 4

Comparison of a synthetic PK-PTA-AADH pathway (left, blue) and native metabolism of xylulose-5-phosphate (right) via pentose phosphate pathway and glycolysis. The synthetic pathway is more carbon-conserving in ethanol production, compared to native metabolism, due to its absence of carbon losing enzymatic reactions, such as pyruvate decarboxylase. The dotted arrow indicates promiscuous acetyl phosphate phosphatase activities of S. cerevisiae

Fig. 5

Schematic comparison between glucose (orange) and xylose (blue) metabolisms in engineered S. cerevisiae. Colored distinction of arrows indicates which sugar induce each metabolic pathway to be predominant. Xylose-derived weaker glycolysis metabolic activity and dysregulation of glucose repressions on cytosolic acetyl-coA synthetic pathway and mitochondrial development (green) allowed S. cerevisiae to more efficiently produce derivatives of pyruvate and cytosolic acetyl-CoA