A food-grade expression system for d-psicose 3-epimerase production in Bacillus subtilis using an alanine racemase-encoding selection marker

Abstract

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.

Keywords

Bacillus subtilis / Cre/lox system / d-Psicose 3-epimerase')">d-Psicose 3-epimerase / Fed-batch fermentation / Food-grade system

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Funding

National Nature Science Foundation of China(31370089)

State Key Development 973 Program for Basic Research of China(2013CB733601)

Nature Science Foundation of Tianjin City(CN)(16JCYBJC23500)

the Key Projects in the Tianjin Science and Technology Pillar Program(11ZCZDSY08400)

Natural Science Foundation of Liaoning Province of China(2014026012)