Development of Bacillus subtilis strains for conversion of xylans to xylooligosaccharides

Methylglucuronoxylans (MeGX_n) and methylglucuronoarabinoxylans (MeGAX_n) are the predominant polysaccharides in hemicelluloses and sources of pentoses for production of biofuels and chemicals, as well neutral (XOS) and acidic (U-XOS) xyloligosaccharides that have applications in nutrition and medicine. B. subtilis 168 secretes endoxylanases, GH11 XynA and GH30 XynC,  and GH43  xylanoarabinofuranosyl hydrolase (XynD). Deletion of the xynA gene provided B. subtilis MR44 in which XynC and XynD are the only secreted xylanolytic enzymes and which quantitatively converts MeGX_n and MeGAX_n to U-XOS. The genes xynD and xynC comprise an operon into which a promoterless chloramphenicol acetyltransferase gene (cat) was ligated downstream of xynC. This B. subtilis strain MR53 (MR44, xynDC::cat),  continuously transferred into media with higher concentrations of chloramphenicol (Cm), evolved strain HR1 which was tolerant in medium containing 350 µg mL^-1 Cm. HR1 contained 7 copies of xynDC::cat in the chromosome as determined by qPCR and  produced total reducing sugar from MeGAX_n at a rate 3-times faster than MR44. The increased levels of expression of the xynCD::cat operon result in an increase in levels of GH43 XynD and GH30 XynC, increasing the release arabinose as a preferred growth substrate and MeGX_n as a preferred substrate for GH30 XynC.  The new HR1 strain from B.subtilis MR44 produces acidic xylooligosaccharides rapidly, with MeGAX_n as a preferred source of U-XOS for applications in human and veterinary medicine.