A Modified Saccharomyces cerevisiae Strain That Consumes l -Arabinose and Produces Ethanol

Abstract

Metabolic engineering is a powerful method to improve, redirect, or generate new metabolic reactions or whole pathways in microorganisms. Here we describe the engineering of a Saccharomyces cerevisiae strain able to utilize the pentose sugar L-arabinose for growth and to ferment it to ethanol. Expanding the substrate fermentation range of S. cerevisiae to include pentoses is important for the utilization of this yeast in economically feasible biomass-to-ethanol fermentation processes. After overexpression of a bacterial L-arabinose utilization pathway consisting of Bacillus subtilis AraA and Escherichia coli AraB and AraD and simultaneous overexpression of the L-arabinose-transporting yeast galactose permease, we were able to select an L-arabinose-utilizing yeast strain by sequential transfer in L-arabinose media. Molecular analysis of this strain, including DNA microarrays, revealed that the crucial prerequisite for efficient utilization of L-arabinose is a lowered activity of L-ribulokinase. Moreover, high L-arabinose uptake rates and enhanced transaldolase activities favor utilization of L-arabinose. With a doubling time of about 7.9 h in a medium with L-arabinose as the sole carbon source, an ethanol production rate of 0.06 to 0.08 g of ethanol per g (dry weight) ⅐ h ؊1 under oxygen-limiting conditions, and high ethanol yields, this yeast strain should be useful for efficient fermentation of hexoses and pentoses in cellulosic biomass hydrolysates.

Alcoholic fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae: current status

“…As demonstrated above, promising strategies are available for a further expansion of the substrate range of ethanol-producing S. cerevisiae strains, probably starting with the full optimization of the bacterial L-arabinose pathway (Becker and Boles 2003;Sedlak and Ho 2001). We are confident that a concerted, multidisciplinary effort in yeast metabolic engineering will result in rapid progress in the areas discussed in this review.…”

“…In our opinion, Becker and Boles (2003) have convincingly demonstrated that the overexpression of the bacterial L-arabinose pathway is the most promising basis for constructing L-arabinose-fermenting S. cerevisiae strains. This approach circumvents the intrinsic redox imbalances associated with expression of the fungal pathway.…”

“…An enhanced transaldolase (TAL1) activity was also reported to enhance L-arabinose fermentation. Overexpression of GAL2 was found not to be essential for growth on L-arabinose, suggesting that other yeast sugar transporters can also transport Larabinose (Becker and Boles 2003).…”

“…Although activities of all three enzymes were shown to be present in a yeast strain carrying the heterologous genes, the engineered strain only accumulated L-arabinitol and did not produce ethanol from L-arabinose (Sedlak and Ho 2001). Becker and Boles (2003), who followed essentially the same strategy, were more successful. In their work, the complete bacterial Larabinose pathway-consisting of E. coli araB and araD and Bacillus subtilis araA-was introduced into S. cerevisiae, simultaneous with the overexpression of the yeast galactose permease gene (GAL2).…”

“…Gal2p is known to transport Larabinose (Kou et al 1970). Although overexpression of the complete arabinose pathway did not result in immediate growth on L-arabinose as the sole carbon source, the growth rate of the transformants increased progressively after 4-5 days of incubation (Becker and Boles 2003). Eventually, an L-arabinose-utilizing strain was selected after several sequential transfers in Larabinose medium.…”