Metabolic Engineering of Corynebacterium glutamicum for Production of UDP-N-Acetylglucosamine

Abstract

Uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) is an acetylated amino sugar nucleotide that naturally serves as precursor in bacterial cell wall synthesis and is involved in prokaryotic and eukaryotic glycosylation reactions. UDP-GlcNAc finds application in various fields including the production of oligosaccharides and glycoproteins with therapeutic benefits. At present, nucleotide sugars are produced either chemically or <i>in vitro</i> by enzyme cascades. However, chemical synthesis is complex and non-economical, and <i>in vitro</i> synthesis requires costly substrates and often purified enzymes. A promising alternative is the microbial production of nucleotide sugars from cheap substrates. In this study, we aimed to engineer the non-pathogenic, Gram-positive soil bacterium <i>Corynebacterium glutamicum</i> as a host for UDP-GlcNAc production. The native <i>glmS</i>, <i>glmU</i>, and <i>glmM</i> genes and <i>glmM</i> of <i>Escherichia coli</i>, encoding the enzymes for UDP-GlcNAc synthesis from fructose-6-phosphate, were over-expressed in different combinations and from different plasmids in <i>C. glutamicum</i> GRS43, which lacks the glucosamine-6-phosphate deaminase gene (<i>nagB</i>) for glucosamine degradation. Over-expression of <i>glmS</i>, <i>glmU</i> and <i>glmM,</i> encoding glucosamine-6-phosphate synthase, the bifunctional glucosamine-1-phosphate acetyltransferase/N-acetyl glucosamine-1-phosphate uridyltransferase and phosphoglucosamine mutase, respectively, was confirmed using activity assays or immunoblot analysis. While the reference strain <i>C. glutamicum</i> GlcNCg1 with an empty plasmid in the exponential growth phase contained intracellularly only about 0.25 mM UDP-GlcNAc, the best engineered strain GlcNCg4 accumulated about 14 mM UDP-GlcNAc. The extracellular UDP-GlcNAc concentrations in the exponential growth phase did not exceed 2 mg/L. In the stationary phase, about 60 mg UDP-GlcNAc/L was observed extracellularly with strain GlcNCg4, indicating the potential of <i>C. glutamicum</i> to produce and to release the activated sugar into the culture medium. To our knowledge, the observed UDP-GlcNAc levels are the highest obtained with microbial hosts, emphasizing the potential of <i>C. glutamicum</i> as a suitable platform for activated sugar production.