Design and modularized optimization of one-step production of N-acetylneuraminic acid from chitin in Serratia marcescens.

Abstract

Chitin is an abundant, biorenewable, nitrogen-rich biomass feedstock that can be potentially developed for biochemical production; however, efficient bioprocesses have yet to be established. Here, we demonstrate an engineered bioprocess to produce N-acetylneuraminic acid (Neu5Ac) directly from chitin using the chitinolytic bacterium, Serratia marcescens by selecting and characterizing promoters, characterization of heterologous enzyme activity, and optimization of pathway fluxes. By generating RNASeq data for S. marcescens growth in different carbon-limited conditions (glucose, N-acetylglucosamine, and glycerol), 12 promoters with varying strength were identified and characterized to implement for transcriptional control. Neu5Ac production was initially engineered into S. marcescens through heterologous expression of N-acetylglucosamine 2-epimerase (slr1975) and N-acetylneuraminic acid aldolase (nanA). The activity of both genes was characterized in vitro for kinetics and in vivo expression using promoters identified in this study. Optimization of Neu5Ac production was accomplished by balancing pathways fluxes through promoter swapping and replacing the reversible nanA with the irreversible gene neuB. The optimized recombinant strain P T5 -slr1975-P rplJ -neuB was able to produce 0.48 g/L Neu5Ac from 20 g/L N-acetylglucosamine, and 0.30 g/L Neu5Ac from 5 g/L crystal chitin. These results represent the first demonstration of direct conversion of crystal chitin to N-acetylneuraminic acid.