Ribose and ribulose production from mixed sugar with genetically engineered Candida tropicalis

Abstract

L-Arabinose is the second most abundant sugar in the xylose mother liquor. L-Arabinose is converted to L-ribulose using L-arabinose isomerase. L-Ribulose is utilized as starting material for synthesizing L-ribose, a promising L-rare sugar to produce antiviral and anticancer L-nucleosides. As L-nucleoside analogues have less toxicity, greater metabolic stability due to their own stereochemistry and similar or even greater antiviral activity than their D-counterparts, L-ribose gets more importance. In this study, we constructed genetically modified Candida tropicalis to convert L-arabinose into L-ribulose and L-ribose from glucose/L-arabinose mixture. For the uptake of L-arabinose as substrates, we tested two heterologous transporters GAL2 from Saccharomyces cerevisiae and STP2 from Arabidopsis thaliana. Endogenous xylose reductase harboring C. tropicalis L10 strain was transformed to constitutively translate codon-optimized GAL2 and STP2. The resulting strains, L10 CoGAL2 and L10 CoSTP2, were grown in fermentation medium containing L-arabinose. L10 CoSTP2 showed the highest L-arabitol production (40 g/L) from 50 g/L L-arabinose whereas L10 and L10 CoGAL2 produced 18 g/L L-arabinose during culture. The codon-optimized STP2 was rationale choice for efficient L-arabinose uptake in C. tropicalis. We constitutively expressed catalytically efficient L-arabinose isomerase from Bacillus licheniformis in C. tropicalis and an increased conversion yield achieved with STP2. Additionally, with the help of STP2, various L-arabinose isomerases originated from Lactobacillus species were tested to increase conversion yield. As a result, L-arabinose isomerase from L. sakei 23K exhibited the highest conversion yield (30%, w/w). Finally, L-ribose isomerase gene from Acinetobacter sp. strain DL-28 was optimized for construction of L-ribose-producing C. tropicalis strain. The resulting strains, K1 CoSTP2 23K AraA CoRI, produced 5 g/L of L-ribose and 3 g/L of L-ribulose from 30 g/L of L-arabinose. This constructed strain can be used for L-ribose production by fermentation with mixed sugars. Moreover, two stage culture was applied for consumption of residual sugars.