Evolutionary engineering of mixed-sugar utilization by a xylose-fermenting strain

Abstract

We have recently reported about a Saccharomyces cerevisiae strain that, in addition to the Piromyces XylA xylose isomerase gene, overexpresses the native genes for the conversion of xylulose to glycolytic intermediates. This engineered strain (RWB 217) exhibited unprecedentedly high specific growth rates and ethanol production rates under anaerobic conditions with xylose as the sole carbon source. However, when RWB 217 was grown on glucose-xylose mixtures, a diauxic growth pattern was observed with a relatively slow consumption of xylose in the second growth phase. After prolonged cultivation in an anaerobic, xylose-limited chemostat, a culture with improved xylose uptake kinetics was obtained. This culture also exhibited improved xylose consumption in glucose-xylose mixtures. A further improvement in mixed-sugar utilization was obtained by prolonged anaerobic cultivation in automated sequencing-batch reactors on glucose-xylose mixtures. A final single-strain isolate (RWB 218) rapidly consumed glucose-xylose mixtures anaerobically, in synthetic medium, with a specific rate of xylose consumption exceeding 0.9 gg(-1)h(-1). When the kinetics of zero trans-influx of glucose and xylose of RWB 218 were compared to that of the initial strain, a twofold higher capacity (V(max)) as well as an improved K(m) for xylose was apparent in the selected strain. It is concluded that the kinetics of xylose fermentation are no longer a bottleneck in the industrial production of bioethanol with yeast.

Dynamic flux balance modeling of microbial co‐cultures for efficient batch fermentation of glucose and xylose mixtures

“…The error was 65.7% for the biomass concentration, 35.9% for the glucose concentration and Kuyper et al (2005) 95.2% for the xylose concentration. Diauxic growth was evident with significant xylose consumption occurring only after glucose was completely exhausted.…”

“…Furthermore, xylose is transported into recombinant S. cerevisiae cells by the same family of hexose transporters (Hxts) that are used for glucose uptake (Hamacher et al, 2002). Because these transporters have over a magnitude greater affinity for glucose than xylose, these recombinant yeast utilize xylose only after depletion of glucose in a pattern of diauxic growth (Kuyper et al, 2005). Escherichia coli can natively metabolize xylose but this bacterium does not naturally exhibit high ethanol yields due to the synthesis of mixed fermentation products.…”

“…Rather than attempting to engineer cells to deviate from their natural metabolic behavior, these consortia take advantage of the native capabilities of different species to metabolize different sugars and direct available carbon to targeted biofuels. This strategy allows metabolic engineering efforts to be focused on enhancing natural behavior rather than introducing foreign genes whose effects are often counteracted (Kuyper et al, 2005). For example, pentose fermenting microbes have been co-cultured with hexose fermenting yeasts such as S. cerevisiae to maintain low hexose sugar concentrations and avoid catabolite repression (Beck et al, 1990;De Bara et al, 2004;Leschine and CanaleParola, 1984;Qian et al, 2006;Taniguichi et al, 1997a,b).…”

Biofuel alternatives to ethanol: pumping the microbial well

“…Therefore, catabolism of monosaccharides has been an important target in the development of an optimized microbial platform. Pathways for xylose and arabinose use have recently been engineered into Saccharomyces cerevisiae [8][9][10] and Zymomonas mobilis [11]. Although developments in sugar catabolism were made in the context of homo-ethanol production, the advances are applicable to the production of a variety of fuel molecules through multiple biosynthetic pathways (discussed below).…”

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

“…Even with this suboptimal xylose consumption the metabolite profiles of RWB 217 compared very well to those on xylose alone and the wild type on glucose. To further improve the xylose consumption characteristics in mixed-substrate batch cultures, RWB 217 was subjected to evolutionary engineering (Kuyper et al 2005b). In a first stage, xylose-uptake kinetics were improved by cultivation for 85 generations in a xylose-limited chemostat.…”