Virginia Commonwealth University, USA
Title: Modularization strategies in metabolic engineering Serratia marcescens for N-acetylneuraminic acid production
Biography: Qiang Yan
Annually, large amounts of waste material are generated from seafood (crab, shrimp and lobster shells) that typically go to landfills with high expense (e.g. $150/ton in Australia). The biopolymer chitin makes up ~40% total weight of such waste and it can potentially be used as a sustainable feedstock for value-added products. In this study, we developed a consolidated bioprocess to produce N-acetylneuraminic acid (NeuNAc) directly from chitin using the chitinolytic organism Serratia marcescens by developing and implementing genetic engineering tools to modify S. marcescens. Starting from RNA sequencing data, 10 native promoters of varying strength were identified and characterized using a fluorescent reporter gene (sfGFP). Two heterologous genes (N-acetylglucosamine isomerase and NeuNAc lyase) were introduced in S. marcescens, and expression was transcriptionally controlled using three different strength promoters (high-high, medium-medium, and low-low). An initial 34.8 mg/L NeuNAc was produced under a high-high promoter combination. Improvements to NeuNAc were conducted by changing expression strength of the two heterologous genes to balance metabolic flux. Using a high strength promoter for the isomerase and a medium strength promoter for the lyase improved both NeuNAc production (1.42-fold) and growth (2.57-fold). Further improvements were tested by characterizing the kinetics of each heterologous enzyme and implementing alternative genes. Swapping N-acetylglucosamine isomerase gene did not show any improvement in production; however, swapping the second-step reversible NeuNAc lyase (nanA) to an irreversible NeuNAc synthase (neuB) improved NeuNAc production (3.25-fold). Overall, we identified and characterized promoter sequences that can be used for genetic engineering of S. marcescens and have implemented the tools to demonstrate and improve production of NeuNAc.