嗜碱芽孢杆菌N16-5木寡糖结合蛋白XynE的功能表征

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

摘要:

摘要:嗜碱芽孢杆菌(Bacillus sp.)N16-5是本实验室从内蒙古乌都淖湖沉积物中分离的嗜碱菌，含有丰富的多糖水解酶，能够利用广泛的单糖和多糖。实验室前期转录组研究发现其基因组上存在一个21 kb大小的木聚糖利用相关基因簇，其中包括xynEFG基因簇编码的ABC转运蛋白。【目的】生物信息学分析预测xynE编码转运蛋白的底物结合蛋白，通过敲除xynE基因研究它对菌株N16-5利用木聚糖的影响。【方法】利用温敏型载体pNNB194介导的同源交换重组的方法构建了xynE基因敲除菌株N16-5(ΔxynE)，并通过基 因回补对敲除菌株表型进行验证。通过检测菌株在木聚糖培养基中的生长情况及培养基中还原糖含量的变化来分析xynE基因对菌株利用木聚糖的影响;通过HPLC检测分析不同培养时间点木聚糖培养基的组分，结合缺失菌株和野生型菌株在以木糖为唯一碳源的培养基中的生长情况来分析XynE所属ABC转运蛋白的底物特异性。【结果】相比野生型菌株，缺失型菌株N16-5(ΔxynE)在木聚糖培养基的生长曲线对数期明显延迟，最大生物量略低，且培养过程中出现了明显的还原糖的累积与消耗过程;回补菌株恢复了野生型表型， 且最大生物量比野生型略高。HPLC检测分析显示，相比野生型菌株，缺失菌株培养过程底物消耗速度较慢，且16 h后出现明显的木四糖、木三糖和木二糖的累积，直至60 h 后仍有较大量木二糖的存在;在木糖培养基中培养时，缺失型菌株和野生型菌株的生长趋势较一致。【结论】XynE蛋白特异性结合木寡糖，其所属ABC转运蛋白在嗜碱芽孢杆菌N16-5降解利用木聚糖过程中发挥着重要作用。

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．