Functional screening for novel D-xylose isomerases from the gut of a wood feeding beetle reveals efficient expression in Saccharomyces cerevisiae

Abstract

Renewable sugar rich feedstocks such as lignocellulosic hydrolysates remain one of the primary sources of potentially renewable fuel and bulk chemicals. The pentose sugar D-xylose is often present in significant amounts along with hexoses such as glucose and galactose. The yeast Saccharomyces cerevisiae can acquire the ability to metabolize D-xylose through expression of heterologous D-xylose isomerase (XI). This enzyme is notoriously difficult to express in S. cerevisiae and only fourteen genes have been reported to be active. We cloned a new XI from microorganisms in the gut of the wood feeding beetle Odontotaenius disjunctus. The new enzyme, 8454_2 XI, was functionally screened from a pool of enzymes with potential XI activity based on its sequence similarity to the XI from Piromyces sp. strain E2. A phylogenetic analysis revealed that the enzyme 8454_2 XI shares high identity with XIs from Bacteroidia class of the Bacteroidetes phylum, and all XIs from Bacteroidia screened in yeast so far have exhibited high activity. Cells carrying the new XI in D-xylose containing media as the sole carbon source showed higher growth and D-xylose consumption rates to those of XI of Piromyces. Remarkably, the 8454_2 XI also exhibited 2.6 times higher Vmax and 37 % higher affinity, and retained substantially higher relative activity at 30 ºC. The new XI is a useful addition to the molecular toolbox for genetic modification of S. cerevisiae for the metabolism of second-generation substrates.