Background

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.

Results

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.

Conclusion

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.

中文翻译：

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食品级表达系统，用于在枯草芽孢杆菌中使用丙氨酸消旋酶编码选择标记生产d-庚糖3-表异构酶。

背景

食品级表达系统要求所得菌株应仅包含来自食品安全微生物的材料，并且不能使用抗生素抗性标记。为了开发食品级表达系统以生产d-聚蔗糖3-表异构酶，我们使用丙氨酸消旋酶编码基因作为枯草芽孢杆菌中的选择标记。

结果

在这项研究中，使用Cre / lox系统从枯草芽孢杆菌1A751的染色体中删除了d-丙氨酸消旋酶编码基因dal，以生成食品级宿主。随后，在食品级宿主中对质粒编码的选择标记dal进行了互补，从而在dal缺失菌株中成功表达了RDPE，而无需添加d-丙氨酸。选择看起来非常严格，并且在培养过程中稳定地维持了质粒。培养基中最高的RDPE活性在72 h达到46 U / ml，这与基于卡那霉素的系统中RDPE的产生相当。最后，食品级枯草芽孢杆菌的能力 1A751D2R在7.5升发酵罐中进行分批补料发酵。

结论

可以将丙氨酸消旋酶编码基因用作选择标记，并且食品级表达系统适用于枯草芽孢杆菌中的异源蛋白质生产。