A food-grade expression system for d-psicose 3-epimerase production in Bacillus subtilis using an alanine racemase-encoding selection marker.
A food-grade expression system for d-psicose 3-epimerase production in Bacillus subtilis using an alanine racemase-encoding selection marker.
复制标题
使用丙氨酸种族友编码选择标记物在枯草芽孢杆菌中生产D-Psicose 3-二聚酶的食品级表达系统。
DOI：
10.1186/s40643-017-0139-7
复制
发表时间：
2017
影响因子：
4.6
通讯作者：
Zhang D
中科院分区：
文献类型：
--
作者：
Chen J;Jin Z;Gai Y;Sun J;Zhang D
食品级表达系统要求合成的菌株只能包含来自食品安全微生物的材料，并且不能使用抗生素耐药性标记。利用枯草芽孢杆菌丙氨酸外消旋酶编码基因作为选择标记，建立d-多聚糖3-差向异构酶的食品级表达系统。本研究利用Cre/lox系统从枯草芽孢杆菌1A751的染色体上删除了编码d-丙氨酸消旋酶的基因dal，以产生食品级宿主。随后，在食品级寄主中补充了质粒编码的选择标记Dal，从而在Dal缺失菌株中成功地表达了RDPE，而不添加d-丙氨酸。选育条件严格，在培养过程中保持了稳定的表达。培养72 h时，RDPE活力最高，达到46U/ml，与以卡那霉素为基础的发酵体系中RDPE产量相当。最后对食品级枯草杆菌1A751D2R在7.5℃L发酵罐中进行了补料分批发酵。编码丙氨酸消旋酶的基因可以作为选择标记，该食品级表达系统适合于枯草芽孢杆菌生产外源蛋白。本文的在线版本(doi：10.1186/s40643-0170139-7)包含补充材料，授权用户可以使用。
Food-grade expression systems require that the resultant strains should only contain materials from food-safe microorganisms, and no antibiotic resistance marker can be utilized. To develop a food-grade expression system for d-psicose 3-epimerase production, we use an alanine racemase-encoding gene as selection marker in Bacillus subtilis. In this study, the d-alanine racemase-encoding gene dal was deleted from the chromosome of B. subtilis 1A751 using Cre/lox system to generate the food-grade host. Subsequently, the plasmid-coded selection marker dal was complemented in the food-grade host, and RDPE was thus successfully expressed in dal deletion strain without addition of d-alanine. The selection appeared highly stringent, and the plasmid was stably maintained during culturing. The highest RDPE activity in medium reached 46 U/ml at 72 h which was comparable to RDPE production in kanamycin-based system. Finally, the capacity of the food-grade B. subtilis 1A751D2R was evaluated in a 7.5 l fermentor with a fed-batch fermentation. The alanine racemase-encoding gene can be used as a selection marker, and the food-grade expression system was suitable for heterologous proteins production in B. subtilis. The online version of this article (doi:10.1186/s40643-017-0139-7) contains supplementary material, which is available to authorized users.