Construction and application of multi-host integrative vector system for xylose-fermenting yeast

Abstract

The xylose-fermenting yeasts (CTG clade yeasts, e.g. Scheffersomyces stipitis, Spathaspora passalidarum, Candida amazonensis and Candida jeffriesii) have the potential to be superior platforms for the conversion of lignocellulosic hydrolysate into fuel-grade ethanol and other chemical products. Here, a genetic expression system compatible with the genetic coding characteristics of CTG clade yeasts was constructed for use in xylose-fermenting yeasts. The pRACTH-gfpm plasmid based on an 18S rDNA shuttle vector was capable of stable integration into the genomes of a wide range of heterologous hosts. Green fluorescent protein was transformed and functionally expressed in S. stipitis, S. passalidarum, C. jeffriesii, C. amazonensis and Saccharomyces cerevisiae under control of the SpADH1 promoter and SpCYC1 terminator. Finally, the expression system was useful in multiple yeast hosts for construction of the plasmid pRACTH-ldh. Scheffersomyces stipitis, S. passalidarum, C. jeffriesii, C. amazonensis and S. cerevisiae were enabled to produce lactate from glucose or xylose by pRACTH-based expression of a heterologous lactate dehydrogenase. Among them, C. amazonensis (pRACTH-ldh) exhibited the highest lactate fermentation capacity, which reached a maximum of 44 g L-1 of lactate with a yield of 0.85 g lactate/g xylose.

Keywords: 18S rDNA; L-lactate; general CTG clade yeast vector; heterologous gene expression; xylose-fermenting yeast.