Characterization of Xylan Utilization and Discovery of a New Endoxylanase in Thermoanaerobacterium saccharolyticum through Targeted Gene Deletions
ABSTRACTThe economical production of fuels and commodity chemicals from lignocellulose requires the utilization of both the cellulose and hemicellulose fractions. Xylanase enzymes allow greater utilization of hemicellulose while also increasing cellulose hydrolysis. Recent metabolic engineering efforts have resulted in a strain ofThermoanaerobacterium saccharolyticumthat can convert C5and C6sugars, as well as insoluble xylan, into ethanol at high yield. To better understand the process of xylan solubilization in this organism, a series of targeted deletions were constructed in the homoethanologenicT. saccharolyticumstrain M0355 to characterize xylan hydrolysis and xylose utilization in this organism. While the deletion of β-xylosidasexylDslowed the growth ofT. saccharolyticumon birchwood xylan and led to an accumulation of short-chain xylo-oligomers, no other single deletion, including the deletion of the previously characterized endoxylanase XynA, had a phenotype distinct from that of the wild type. This result indicates a multiplicity of xylanase enzymes which facilitate xylan degradation inT. saccharolyticum. Growth on xylan was prevented only when a previously uncharacterized endoxylanase encoded byxynCwas also deleted in conjunction withxynA. Sequence analysis ofxynCindicates that this enzyme, a low-molecular-weight endoxylanase with homology to glycoside hydrolase family 11 enzymes, is secreted yet untethered to the cell wall. Together, these observations expand our understanding of the enzymatic basis of xylan hydrolysis byT. saccharolyticum.