Cofactor Self-Sufficient Whole-Cell Biocatalysts for the Relay-Race Synthesis of Shikimic Acid

Abstract

Shikimic acid (SA) is a key intermediate in the aromatic amino-acid biosynthetic pathway, as well as an important precursor for synthesizing many valuable antiviral drugs. The asymmetric reduction of 3-dehydroshikimic acid (DHS) to SA is catalyzed by shikimate dehydrogenase (AroE) using NADPH as the cofactor; however, the intracellular NADPH supply limits the biosynthetic capability of SA. Glucose dehydrogenase (GDH) is an efficient enzyme which is typically used for NAD(P)H regeneration in biocatalytic processes. In this study, a series of NADPH self-sufficient whole-cell biocatalysts were constructed, and the biocatalyst co-expressing Bmgdh–aroE showed the highest conversion rate for the reduction of DHS to SA. Then, the preparation of whole-cell biocatalysts by fed-batch fermentation without supplementing antibiotics was developed on the basis of the growth-coupled l-serine auxotroph. After optimizing the whole-cell biocatalytic conditions, a titer of 81.6 g/L SA was obtained from the supernatant of fermentative broth in 98.4% yield (mol/mol) from DHS with a productivity of 40.8 g/L/h, and cofactor NADP+ or NADPH was not exogenously supplemented during the whole biocatalytic process. The efficient relay-race synthesis of SA from glucose by coupling microbial fermentation with a biocatalytic process was finally achieved. This work provides an effective strategy for the biosynthesis of fine chemicals that are difficult to obtain through de novo biosynthesis from renewable feedstocks, as well as for biocatalytic studies that strictly rely on NAD(P)H regeneration.