N -Acetylglucosamine Utilization by Saccharomyces cerevisiae Based on Expression of Candida albicans NAG Genes

Abstract

ABSTRACT Synthesis of chitin de novo from glucose involves a linear pathway in Saccharomyces cerevisiae . Several of the pathway genes, including GNA1 , are essential. Genes for chitin catabolism are absent in S. cerevisiae . Therefore, S. cerevisiae cannot use chitin as a carbon source. Chitin is the second most abundant polysaccharide after cellulose and consists of N -acetylglucosamine (GlcNAc) moieties. Here, we have generated S. cerevisiae strains that are able to use GlcNAc as a carbon source by expressing four Candida albicans genes ( NAG3 or its NAG4 paralog, NAG5 , NAG2 , and NAG1 ) encoding a GlcNAc permease, a GlcNAc kinase, a GlcNAc-6-phosphate deacetylase, and a glucosamine-6-phosphate deaminase, respectively. Expression of NAG3 and NAG5 or NAG4 and NAG5 in S. cerevisiae resulted in strains in which the otherwise-essential Sc GNA1 could be deleted. These strains required the presence of GlcNAc in the medium, indicating that uptake of GlcNAc and its phosphorylation were achieved. Expression of all four NAG genes produced strains that could use GlcNAc as the sole carbon source for growth. Utilization of a GlcNAc catabolic pathway for bioethanol production using these strains was tested. However, fermentation was slow and yielded only minor amounts of ethanol (approximately 3.0 g/liter), suggesting that fructose-6-phosphate produced from GlcNAc under these conditions is largely consumed to maintain cellular functions and promote growth. Our results present the first step toward tapping a novel, renewable carbon source for biofuel production.