细菌纤维素合成菌纳塔葡糖酸醋杆菌遗传操作方法的建立

谢文平　鲍素敏　刘权　张鸿

摘要 [目的]建立纳塔葡糖酸醋杆菌遗传操作系统，为采用基因工程方法改造该类菌株，提高纤维素产量，提升纤维素品质提供参考依据。[方法]通过优化培养基确定菌株电转化感受态制备条件，采用质粒或者线性化片段在不同电压下进行转化，优化最佳转化条件。[结果]采用添加了1%纤维素酶的C2培养基进行1次摇瓶转接，可有效制备纳塔葡糖酸醋杆菌电转化感受态;采用携带同源臂及Kan抗性基因的环状质粒或者线性化片段均实现了对纳塔葡糖酸醋杆菌葡萄糖脱氢酶基因（GDH）的同源双交换基因敲除;GDH基因的敲除降低了工程菌株发酵产纤维素膜过程的葡萄糖酸产生，提高了细菌纤维素的最终产量及品质。[结论]成功建立了纳塔葡糖酸醋杆菌遗传转化方法和基因敲除方法。

关键词 细菌纤维素;纳塔葡糖酸醋杆菌;电转化;基因敲除

中图分类号 S-3 文献标识码 A

文章编号 0517-6611（2020）11-0001-06

Abstract [Objective]Establishing a genetic operating method for Gluconacetobacter nataicola can provide reference for modifing the strains by genetic engineering，improving the yield and the quality of bacterial cellulose.[Method]The conditions for the preparation of competent cells were determined by the growth ability of the strain in different media，and the optimal transformation conditions were determined by positive clone obtained by transforming the plasmid or the linearized fragment at different electrotransformation voltages.[Result]The C2 medium supplemented with 1% cellulase was used for one time bottom transferring，which can effectively prepare the electroporation of G.nataicola.The homologous doubleexchange knockout of the glucose dehydrogenase gene （GDH） was achieved by transferring the circle plasmid or the linearized fragment carrying homologous arm of GDH and Kan resistance gene.Knockout of GDH gene reduced the production of gluconic acid in the process of fermentation，and improved the final yield and quality of bacterial cellulose membrane.[Conclusion]The genetic transformation method and gene knockout method of G.nataicola were successfully established.

Key words Bacterial cellulose;Gluconacetobacter nataicola;Electroporation;Gene knockout

細菌纤维素（bacterial cellulose，BC）是细菌利用葡萄糖合成的由β-1，4-糖苷键连接而成的纤维素。因其具有超高的纯度、高结晶性、高杨氏模量、优良的生物可降解性、高持水量和良好的生物相容性等特点，已作为新型生物可降解材料用于食品、化学工业和医学领域[1-2]。目前，细菌纤维素已经成功应用于烧伤敷料、退热贴、高端面膜等产品的基材而在各国实现了规模化生产[3-5]。

根据已有的报道，具有细菌纤维素合成功能的微生物包括葡糖酸醋杆菌属（Gluconacetobacter）、土壤杆菌属（Agrobacterium）、醋酸杆菌属（Acetobacter）、无色杆菌属（Achromobacter）、肠杆菌属（Enterobacter）等[3，6-7]。……