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 in vitro by enzyme cascades. However, chemical synthesis is complex and non-economical, and in vitro 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 Corynebacterium glutamicum as a host for UDP-GlcNAc production. The native glmS, glmU, and glmM genes and glmM of Escherichia coli, encoding the enzymes for UDP-GlcNAc synthesis from fructose-6-phosphate, were over-expressed in different combinations and from different plasmids in C. glutamicum GRS43, which lacks the glucosamine-6-phosphate deaminase gene (nagB) for glucosamine degradation. Over-expression of glmS, glmU and glmM, 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 C. glutamicum 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 C. glutamicum 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 C. glutamicum as a suitable platform for activated sugar production.

Keywords: Corynebacterium glutamicum; UDP-N-acetylglucosamine; activated amino sugars; metabolic engineering; sugar nucleotide.

FIGURE 1

Schematic overview of the metabolic pathway responsible for UDP-GlcNAc biosynthesis in C. glutamicum and metabolic engineering strategies implemented to overproduce UDP-GlcNAc. Abbreviations are given in the box. The desired product UDP-GlcNAc is boxed. Product of genes that are knocked out or over-produced are shown in red or green text, respectively.

FIGURE 2

Specific GlmU (A) and GlmS (B) activities in cell extracts of C. glutamicum GRS43 and the recombinant strains C. glutamicum GlcNCg1 to GlcNCg7. For strain description, Table 1. Data represent mean values of triplicate assays from at least three individual cultivations. Error bars represent standard deviations (SD).

FIGURE 3

Western blot analysis of heterologous C-terminally His-tagged GlmM in cell extracts of recombinant C. glutamicum strains GRS43 and GlcNCg4 to GlcNCg9 (for strain description Table 1). The GlmM _Eco protein (calculated mass ∼49 kDa) is highlighted with a red rectangle.

FIGURE 4

Extracellular accumulation of UDP-GlcNAc in C. glutamicum GlcNCg1, GlcNCg3 and GlcNCg4. For strain description Table 1. The cells were cultured in CgXII minimal medium with 2% glucose supplemented with inducers (1 mM IPTG, 250 ng/ml ATc). Data represents mean values of triplicate (exponential phase) or duplicate (stationary phase) assays from individual cultivations and error bars represent deviations.