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.
Subject(s)
Fermentation , Gene Expression , Genetic Vectors , Xylose/metabolism , Yeasts/genetics , Yeasts/metabolism , Genes, Reporter , Glucose/metabolism , Green Fluorescent Proteins/analysis , Green Fluorescent Proteins/genetics , L-Lactate Dehydrogenase/analysis , L-Lactate Dehydrogenase/genetics , Plasmids , Promoter Regions, Genetic , Transcription Termination, GeneticABSTRACT
OBJECTIVE: It is of great significance to improve the utilization of lignocellulosic material, the most abundant renewable resource on earth. METHODS: We studied the stress tolerance (temperature, ethanol and osmotic tolerance) of five xylose utilizing yeasts, Scheffersomyces stipitis, Candida tenuis, Spathaspora passalidarum, Candida amazonensis and Candida jeffriesii. We also tested their utilization ability of multiple carbon and nitrogen sources. RESULTS: S. passalidarum could tolerate at 44 degrees C and utilize various carbon and nitrogen sources effectively. S. passalidarum could metabolism xylose rapidly to produce ethanol, with an ethanol yield of 0.43 g/g under oxygen limiting condition. C. amazonensis could also torelate at 42 degrees C. Moreover, C. amazonensis could converse xylose to xylitol with ethanol as the main by-product. CONCLUSION: S. passalidarum is a potentially valuable workhorse in industrial utilization of lignocellulosic for its excellent characteristics. In addition, C. amazonensis may be a promising xylitol producer.
