Characterization of solute-binding protein XynE of the xylooligosaccharide transporter from Bacillus sp. N16-5

Abstract:

Abstract: The alkaliphilichemicellulolytic bacterium Bacillus sp．N16-5 was isolated from Lake Wudunao in Inner Mongolia．It has a broad substrate spectrum and exhibits a capacity to utilize complex carbohydrates such as galactomannan，xylan and pectin．Previous transcriptional analysis of differential carbohydrate utilization by Bacillus sp．N16-5 has identified a putative gene cluster related to xylan utilization．It contains a putative xylo-oligosaccharide ATPbinding cassette (ABC) transporter encoded by xynEFG gene cluster．［Objective］xynE gene is predicted to encode an extracellular solute-binding protein of the ABC transporter．Here，the physiological roles of xynE on the xylan utilization was investigated by gene deletion．［Methods］We obtained the xynE deficient strain N16-5(ΔxynE) through homologous recombination using a temperature sensitive shuttle vector pNNB194． The effects of xynE on xylan utilization by N16-5 were detected by comparing the growth profiles on xylan of the wild type and mutant strains as well as the variation of reducing sugars concentration in the medium during cultivation．We further verified the phenotype by constructing the complementary strain．Moreover，the substrate specificity of XynE was illustrated by the HPLC analysis results of the xylan medium components，which was supplemented with the growth profiles of the wild type strain and N16-5 (ΔxynE) strain on xylose．［Results］ Compared with the wild type strain，strain N16-5 (ΔxynE ) had a delayed exponential phase， obtained a lower maximum optical intensity OD600 value，and presented the accumulation and depletion of reducing sugars during cultivation．The complementary strain retrieved the phenotype of wild type strain，and grown slightly better than it．HPLC analysis showed that N16-5 ( ΔxynE ) strain degraded the xylan substrates more slowly than wild type，xylooligosaccharides like xylotetraose，xylotriose and xylobiose began to accumulate after 16 h cultivation．Moreover it still maitained a large number of the degradation product xylobiose in the medium after 60 h． When cultured in xylose medium，strain N16-5(ΔxynE) performed similar growth profile with the wild type strain． ［Conclusion］XynE played an important role in rapidly and effectively utilizing xylan in Bacillus sp．N16-5 and specifically related with xylo-oligosaccharide uptake．