A baculovirus–insect cell whole-cell biocatalysis platform for NeuNAc biosynthesis

Industrial biocatalytic applications are frequently constrained by poor recombinant enzyme solubility and limited functional recovery in conventional microbial expression systems. To address this challenge, we developed a baculovirus–insect cell whole-cell biocatalysis platform and evaluated its feasibility using N-acetylneuraminic acid (NeuNAc) biosynthesis as a model system. Comparative analysis of three heterologous expression hosts— Escherichia coli , Pichia pastoris , and baculovirus-infected insect cells—showed that, under the baseline expression conditions evaluated in this study, insect cells provided higher soluble enzyme recovery and process-level bioconversion performance for GlcNAc 2-epimerase (2ep) and NeuNAc aldolase (NanA). Using baculovirus-infected Sf9 cells, both purified-enzyme and whole-cell configurations were established for sequential and one-pot biocatalysis. The whole-cell system enabled direct conversion without protein purification and achieved approximately 23% conversion to a product assigned as NeuNAc based on agreement of its HPLC retention time with that of an authentic standard under laboratory-scale one-pot conditions. Process optimization identified suitable conditions for suspension culture, infection, substrate concentrations, and catalyst loading, and the platform was further evaluated using cells produced in a 3-L bioreactor. In addition to simplifying catalyst preparation, the whole-cell strategy reduced the functional catalyst preparation requirements associated with purified-enzyme workflows. Although a comprehensive techno-economic assessment was beyond the scope of this work, a preliminary laboratory-scale cost estimation suggested lower raw material expenditure for the whole-cell workflow. Overall, this platform provides a proof-of-concept complementary strategy for biocatalytic applications in which enzyme solubility, folding, or functional expression limits the performance of conventional microbial hosts.