Rational design and medium optimization for shikimate production in recombinant Bacillus licheniformis strains

Abstract

Shikimate is a key intermediate for the synthesis of an anti-influenza drug sold under the trade name Tamiflu®. The metabolic pathway of shikimate has been extensively explored in Escherichia coli. This paper presents a rational strategy via cumulative disruption and overexpression of rate-limiting genes in Bacillus licheniformis, which is a GRAS (Generally Recognized as Safe) strain with high potential for the production of shikimate. The enhanced production of shikimate was achieved by the deletion of shikimate kinase (AroK) and pyruvate kinase (Pyk) as well as overexpression of transketolase (Tkt), 3-deoxy-d-arabinoheptulosonate-7-phosphate (DAHP) synthase (AroA), and shikimate dehydrogenase (AroD) respectively and the amount of byproducts was reduced significantly. Furthermore, the medium was optimized by an orthogonal test, and the titer of shikimate (21.8 g/L) produced by the strain WX-02ΔaroKΔpyk/pHY300-aroD increased by 56.8% compared to the original medium. This work revealed a new GRAS platform for the production of shikimate. These findings also suggest that B. licheniformis has great potential for the production of aromatic compounds of the extended shikimate pathway.