Abstract
N-acetylglucosamine (GlcNAc) is an amino sugar that has been widely used in the nutraceutical and pharmaceutical industries. Recently, microbial production of GlcNAc has been developed. One major challenge for efficient biosynthesis of GlcNAc is to achieve appropriate carbon flux distribution between growth and production. Here, a synergistic substrate co-utilization strategy was used to address this challenge. Specifically, glycerol was utilized to support cell growth and generate glutamine and acetyl-CoA, which are amino and acetyl donors, respectively, for GlcNAc biosynthesis, while glucose was retained for GlcNAc production. Thanks to deletion of the 6-phosphofructokinase (PfkA and PfkB) and glucose-6-phosphate dehydrogenase (ZWF) genes, the main glucose catabolism pathways of Escherichia coli were blocked. The resultant mutant showed a severe defect in glucose consumption. Then, the GlcNAc production module containing glucosamine-6-phosphate synthase (GlmS*), glucosamine-6-phosphate N-acetyltransferase (GNA1*) and GlcNAc-6-phosphate phosphatase (YqaB) expression cassettes was introduced into the mutant, to drive the carbon flux from glucose to GlcNAc. Furthermore, co-utilization of glucose and glycerol was achieved by overexpression of glycerol kinase (GlpK) gene. Using the optimized fermentation medium, the final strain produced GlcNAc with a high stoichiometric yield of 0.64 mol/mol glucose. This study offers a promising strategy to address the challenge of distributing carbon flux in GlcNAc production.
Highlights
N-acetylglucosamine (GlcNAc) is the monomer unit of chitin, which is the second most abundant polysaccharide on Earth and can be commonly found in crustaceans, fungi and insects [1]
To achieve a high yield of GlcNAc from glucose, carbon flux distribution at principal branch points (glucose-6-phosphate (G-6-P) and F-6-P) in the central metabolic network of E. coli must be significantly modified from that observed during balanced growth, so that the GlcNAc precursor F-6-P can be synthesized in the optimal stoichiometric ratio
For E. coli strains, G-6-P could be driven toward phosphate pathway (PPP) through ZWF, while F-6-P was mainly broken down to pyruvate in Embden-MeyerhofParnas pathway (EMP) via PFK encoded by the pfkA and pfkB genes (Figure 1)
Summary
N-acetylglucosamine (GlcNAc) is the monomer unit of chitin, which is the second most abundant polysaccharide on Earth and can be commonly found in crustaceans, fungi and insects [1]. It is a basic component of various heterologous biopolymers, such as hyaluronic acid and chondroitin sulfate, which play important roles in cartilage and joint health [2,3]. The biosynthesis pathway of GlcNAc from the precursor fructose-6-phosphate (F-6-P) involves three crucial enzymes, glucosamine-6phosphate synthase (GlmS), glucosamine-6-phosphate N-acetyltransferase (GNA1) and GlcNAc-6-phosphate phosphatase (Figure 1). There are relatively few studies focusing on this strategy, which may limit the further improvement of microbial production of GlcNAc
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