Functional Characterization of Corynebacterium alkanolyticum β-Xylosidase and Xyloside ABC Transporter in Corynebacterium glutamicum

TheCorynebacterium alkanolyticum xylEFGD gene cluster comprises thexylD gene that encodes an intracellular β-xylosidase next to thexylEFG operon encoding a substrate-binding protein and two membrane permease proteins of a xyloside ABC transporter. Cloning of the cluster revealed a recombinant β-xylosidase of moderately high activity (turnover forp -nitrophenyl-β-d -xylopyranoside of 111 ± 4 s−1 ), weak α-l -arabinofuranosidase activity (turnover forp -nitrophenyl-α-l -arabinofuranoside of 5 ± 1 s−1 ), and high tolerance to product inhibition (Ki for xylose of 67.6 ± 2.6 mM). Heterologous expression of the entire cluster under the control of the strong constitutivetac promoter in theCorynebacterium glutamicum xylose-fermenting strain X1 enabled the resultant strain X1EFGD to rapidly utilize not only xylooligosaccharides but also arabino-xylooligosaccharides. The ability to utilize arabino-xylooligosaccharides depended oncgR _2369 , a gene encoding a multitask ATP-binding protein. Heterologous expression of the contiguousxylD gene in strain X1 led to strain X1D with 10-fold greater β-xylosidase activity than strain X1EFGD, albeit with a total loss of arabino-xylooligosaccharide utilization ability and only half the ability to utilize xylooligosaccharides. The findings suggest some inherent ability ofC. glutamicum to take up xylooligosaccharides, an ability that is enhanced by in the presence of a functionalxylEFG -encoded xyloside ABC transporter. The finding thatxylEFG imparts nonnative ability to take up arabino-xylooligosaccharides should be useful in constructing industrial strains with efficient fermentation of arabinoxylan, a major component of lignocellulosic biomass hydrolysates.