Functional expression of a bacterial xylose isomerase in Saccharomyces cerevisiae

Abstract

In industrial fermentation processes, the yeast Saccharomyces cerevisiae is commonly used for ethanol production. However, it lacks the ability to ferment pentose sugars like d-xylose and l-arabinose. Heterologous expression of a xylose isomerase (XI) would enable yeast cells to metabolize xylose. However, many attempts to express a prokaryotic XI with high activity in S. cerevisiae have failed so far. We have screened nucleic acid databases for sequences encoding putative XIs and finally were able to clone and successfully express a highly active new kind of XI from the anaerobic bacterium Clostridium phytofermentans in S. cerevisiae. Heterologous expression of this enzyme confers on the yeast cells the ability to metabolize d-xylose and to use it as the sole carbon and energy source. The new enzyme has low sequence similarities to the XIs from Piromyces sp. strain E2 and Thermus thermophilus, which were the only two XIs previously functionally expressed in S. cerevisiae. The activity and kinetic parameters of the new enzyme are comparable to those of the Piromyces XI. Importantly, the new enzyme is far less inhibited by xylitol, which accrues as a side product during xylose fermentation. Furthermore, expression of the gene could be improved by adapting its codon usage to that of the highly expressed glycolytic genes of S. cerevisiae. Expression of the bacterial XI in an industrially employed yeast strain enabled it to grow on xylose and to ferment xylose to ethanol. Thus, our findings provide an excellent starting point for further improvement of xylose fermentation in industrial yeast strains.

FIG. 1.

Phylogenetic tree of the amino acid sequences of the tested XIs reported in the GenBank database.

FIG. 2.

Inhibition of XI from C. phytofermentans (A) and Piromyces sp. strain E2 (B) by xylitol. Strains carrying the gene for XI from C. phytofermentans or Piromyces sp. strain E2, respectively, on a multicopy vector were grown as shake-flask cultures at 30°C into the exponential growth phase in synthetic medium with 20 g liter⁻¹ glucose and without uracil. Crude extracts were prepared, and quantitative enzyme activity tests were performed. Symbols: •, 0 mM xylitol; ▪, 10 mM xylitol; ▴, 30 mM xylitol; ▾, 50 mM xylitol.

FIG. 3.

Growth of recombinant S. cerevisiae strains expressing different XIs. SC medium (without uracil) contained 20 g liter⁻¹ d-xylose as the sole carbon source. Yeast strains were grown aerobically as shake-flask cultures at 30°C. Yeast strain MKY9 contained the different XI genes. Symbols: ▪, opt-XI-Clos; ▴, opt-XI-Piro; ⧫, XI-Clos; □, empty vector. Shown are results of a typical experiment.

FIG. 4.

Aerobic growth of industrial S. cerevisiae strain expressing the codon-optimized XI from C. phytofermentans. SC medium contained 20 g liter⁻¹ d-xylose as the sole carbon source. Yeast strains were grown aerobically as shake-flask cultures at 30°C. Symbols: ⧫, BWY10Xyl; ▪, BarraGrande (wild type).

FIG. 5.

Anaerobic batch fermentation by recombinant S. cerevisiae. Shown is a graph of anaerobic batch fermentation of strain BWY10Xyl. The strain was grown in mineral medium supplemented with amino acids and with 30 g liter⁻¹ d-xylose as the sole carbon source. The strain was pregrown in the fermentor under aerobic conditions until about 5 g liter⁻¹ d-xylose was consumed and then shifted to anaerobic conditions (indicated by the arrow). Symbols: ⧫, d-xylose; ▪, ethanol; □, xylitol; ▴, biomass. Shown are results of a typical experiment.