Synergetic Fermentation of Glucose and Glycerol for High-Yield N-Acetylglucosamine Production in Escherichia coli

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.

Metabolically Engineered Escherichia coli for Conversion of D-Fructose to D-Allulose via Phosphorylation-Dephosphorylation

“…In E. coli cells, fructose-6-phosphate can also be phosphorylated into fructose-1, 6-bisphosphate by 6-phosphofructokinases ( pfkA , pfkB ) and utilized as a carbon source through the EMP pathway ( Wang et al, 2022 ). Therefore, blocking the phosphorylation of fructose-6-phosphate may favor the synthesis of D-allulose.…”

“…As shown in Figure 4C, buffering LB medium with 100 mM potassium phosphate was able to limit this acidification to pH 5.7 after 72 h, and the D-allulose titer increased to 0.51 g/L, with depletion of 4.03 g/L D-fructose. In E. coli cells, fructose-6-phosphate can also be phosphorylated into fructose-1, 6-bisphosphate by 6phosphofructokinases (pfkA, pfkB) and utilized as a carbon source through the EMP pathway (Wang et al, 2022). Therefore, blocking the phosphorylation of fructose-6phosphate may favor the synthesis of D-allulose.…”

Reprogramming the Metabolic Network in Kluyveromyces lactis with a Transcriptional Switch for De Novo Lacto-N-biose Synthesis

“…The addition of GlcNAc in the salvage pathway increased the cost in LNB production, and it has been reported that GlcNAc can be generated via microbial fermentation (Table S5). − Thus, the de novo pathway should be developed in K. lactis for synthesizing LNB using lactose as the sole substrate (without the addition of GlcNAc).…”

Metabolic Engineering of Escherichia coli for High-Level Production of Salicin

“…E. coli can utilize glucose and glycerol as carbon sources to produce various chemicals. Significant production enhancement could be realized through synergetic utilization of glucose and glycerol, which has been successfully applied to many valuable chemical biosynthesis processes such as myo-inositol, 38 lycopene, 39 N-acetylglucosamine, 40 and so forth. To achieve higher salicin production, the optimization of medium was conducted by varying the carbon sources in modified M9Y medium.…”