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.

Whole-Cell Bioconversion of Renewable Biomasses-Related Aromatics to cis,cis-Muconic Acid

“…Taking into account that the Ado enzyme belongs to the carotenoid cleavage oxygenase family containing iron as a prosthetic group, the effect of Fe 2+ on the oxygenase reaction was investigated: adding 1 mM FeSO 4 in the medium (see the Materials and Methods section) did not affect the reaction rate and the bioconversion yield of the overall biotransformation of ferulic acid into vanillin. Moreover, the simultaneous addition of IPTG and 10 mM benzyl alcohol, to induce chaperone co-expression, did not improve the bioconversion yield, and the supplement of 10 g/L glucose in the reaction mixture, to enhance the NAD­( P )H cofactor regeneration, significantly inhibited the vanillin production: a 30.2 ± 1.4% bioconversion yield was observed after 24 h of incubation.…”

Multi-step biosynthesis of the biodegradable polyester monomer 2-pyrone-4,6-dicarboxylic acid from glucose

“…For the preparation of DHS fermentative broth, fed-batch fermentation of the WJ060 strain was conducted as previously described [ 29 ]. After fermentation, the cells were removed by centrifugation, and the supernatant with a high concentration of DHS was used as the substrate for whole-cell bioconversion.…”

Design of a cofactor self‐sufficient whole‐cell biocatalyst for enzymatic asymmetric reduction via engineered metabolic pathways and multi‐enzyme cascade

“…Therefore, improving the efficiency of whole-cell biocatalysis through regulating intracellular NADP(H) pools based on redox balance remains a challenging issue. [7,9,10] L-Phosphinothricin (L-PPT) is a broad-spectrum and highly efficient chiral herbicide. [37] The reductive amination of PPO to produce L-PPT by NADPH-dependent glufosinate dehydrogenases (GluDH) is a green method.…”

“…Many strategies focused on the supply and regeneration of NAD(P)H cofactor have been developed in biocatalysis and biotransformation by researchers. [ 5–10 ] For example, cheaper artificial analogs of nicotinamide cofactors have emerged as alternatives to more expensive natural nicotinamide cofactors because of their similar chemical structure [ 11 ] ; however, using artificial cofactors resulted in the enzymatic efficiencies being substantially decreased compared with the same amount of stoichiometric use of natural cofactors. Besides, NAD(P)H was recycled to regenerate under a sacrificial substrate as the electron donor by introducing a heterologous NAD(P)+ dependent enzyme coupled with NAD(P)H dependent enzyme.…”

“…Therefore, improving the efficiency of whole‐cell biocatalysis through regulating intracellular NADP(H) pools based on redox balance remains a challenging issue. [ 7,9,10 ]…”