Modification Of The Metabolic Pathway For High Yield Shikimate And Exploring The Synthesis Pathway Of Melanin In Bacillus Licheniformis

Shikimate is a crucial 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.Firstly,the Bacillus licheniformis strain WX-02 began to accumulate shikimate by the deletion of shikimate kinase(Aro K),which blocks the downstream metabolic pathway of shikimate.The enhanced production of shikimate was achieved by the deletion of pyruvate kinase(Pyk),the inactivation of phosphotransferase system(PTS)glucose-specific enzyme IICBA component(Pts G)and overexpression of hexose transporter Glc U,as well as overexpression of transketolase(Tkt),3-deoxy-D-arabinoheptulosonate-7-phosphate(DAHP)synthase(Aro A),shikimate dehydrogenase(Aro D)and glucose-6-phosphate 1-dehydrogenase(Zwf)respectively and the number 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-02Δaro KΔpyk/p HY300-aro D increased by 56.8%compared to the original medium.During the fermentation of engineered strain which produced shikimate in this study,the fermentative liquid would gradually turn into black,and the black substance was initially speculated to be melanin.Melanin is an oxidized polymer composed of various phenolic or indole monomers.So far,melanin produced by microorganisms has not been studied and reported in detail.In this study,we found that WX-02 strain produced black substances only after the knockout of shikimate kinase(Aro K),indicating that the melanin synthesis pathway in this study is closely related to aro K gene.After purification of melanin by acid-base extraction method,the ultraviolet full-wavelength scanning of melanin was carried out and the result was consistent with those reported in the literature.After the determination of intracellular amino acid content and feeding experiments,it was found that the consumption of homogentisic acid and aromatic amino acids,in which phenylalanine played an important role,were related to the synthesis of melanin.After comparing the transcriptome data of WX-02Δpyk and WX-02ΔarokΔpyk,the strains WX-02ΔarokΔpykΔtyr A and WX-02ΔarokΔpykΔphe A were constructed.It was found that the production of melanin increased by 25.8%to 6.82 g/L compared with WX-02ΔarokΔpyk(5.42 g/L)after the knockout of tyr A gene.After the knockout of phe A gene,the production of melanin reduced by 63.5%to 1.98 g/L.According to the previous results,we concluded that the melanin produced by B.licheniformis is pyomelanin in this study and predicted the synthesis pathway using homogentisic acid and phenylalanine as precursors.This work revealed a new GRAS platform for the production of shikimate.These findings also suggest that B.licheniformis has excellent potential for the production of aromatic compounds of the extended shikimate pathway.