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). 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.

FIG. 1.

Schematic presentation of the l-arabinose utilization pathway engineered in S. cerevisiae. l-Arabinose is transported into the cell by the galactose permease Gal2 and subsequently converted to d-xylulose-5-P by three enzymatic steps. d-Xylulose-5-P is an intermediate of the pentose phosphate pathway and is directed to glycolysis by transketolase (TKL) and transaldolase (TAL). ZWF, glucose-6-P dehydrogenase; PGL, phosphogluconolactonase; GND, gluconate-6-P dehydrogenase; RPI, ribose-5-P isomerase; RPE, ribulose-5-P 3-epimerase.

FIG. 2.

Selection of yeast transformants for growth on l-arabinose medium by sequential transfer. Yeast transformants (JBY24-4P) were pregrown in synthetic liquid medium with 2% glucose, washed twice, and inoculated in synthetic medium with 2% l-arabinose, 0.1% yeast extract, and 0.2% peptone at an initial OD₆₀₀ of about 0.2. Cells were grown aerobically at 30°C in shaker flasks on a rotary shaker. Growth was monitored by measuring the OD₆₀₀s of the cultures. Whenever the transformants reached an OD₆₀₀ of up to 10, they were inoculated in fresh medium at an OD₆₀₀ of about 0.3 and grown further.

FIG. 3.

Growth of different yeast transformants in l-arabinose medium. Yeast transformants were pregrown in synthetic medium with 2% glucose, washed twice, and inoculated in synthetic medium with 2% l-arabinose at an initial OD₆₀₀ of about 0.2. Cells were grown aerobically at 30°C in shaker flasks on a rotary shaker. Growth was monitored by measuring the OD₆₀₀s of the cultures. All growth tests were performed two to four times, with similar results. Representative results are shown here. The strains are JBY25-4M (▪) (A and B); JBY24-4V (♦), JBY24-4P (∗), and JBY25-4P (▴) (A); and JBY25-3M (♦), JBY24-3T (▴), and JBY24-4M (∗) (B). The doubling time of JBY25-4M in the exponential growth phase was 7.9 h. The various plasmid compositions of the transformants are indicated on the right side of the growth curves. wt, wild-type background (JBY24); mut, mutant background (JBY25).

FIG. 4.

Ethanol production from l-arabinose and glucose. The yeast transformants JBY25-4M and JBY24-4V were pregrown in synthetic medium with 2% glucose or 2% l-arabinose until they reached an OD₆₀₀ of about 2, washed twice, and incubated at 30°C in synthetic medium with 2% glucose or 2% l-arabinose under oxygen-limiting conditions at an OD₆₀₀ of about 15 to 20. Ethanol concentrations in the culture supernatants were determined at various time points. Representative results of at least two independent experiments for each strain are shown. •, JBY24-4V with glucose; ♦, JBY24-4V with l-arabinose; ▴, JBY25-4M with glucose; ▪, JBY25-4M with l-arabinose. EtOH, ethanol.