Metabolic engineering of Lactobacillus casei for production of UDP-N-acetylglucosamine.

ABSTRACT

UDP-sugars are used as glycosyl donors in many enzymatic glycosylation processes. In bacteria UDP-N-acetylglucosamine (UDP-GlcNAc) is synthesized from fructose-6-phosphate by four successive reactions catalyzed by three enzymes Glucosamine-6-phosphate synthase (GlmS), phosphoglucosamine mutase (GlmM), and the bi-functional enzyme glucosamine-1-phosphate acetyltransferase/N-acetylglucosamine-1-phosphate uridyltransferase (GlmU). In this work several metabolic engineering strategies, aimed to increment UDP-GlcNAc biosynthesis, were applied in the probiotic bacterium Lactobacillus casei strain BL23. This strain does not produce exopolysaccharides, therefore it could be a suitable host for the production of oligosaccharides. The genes glmS, glmM, and glmU coding for GlmS, GlmM, and GlmU activities in L. casei BL23, respectively, were identified, cloned and shown to be functional by homologous over-expression. The recombinant L. casei strain over-expressing simultaneously the genes glmM and glmS showed a 3.47 times increase in GlmS activity and 6.43 times increase in GlmM activity with respect to the control strain. Remarkably, these incremented activities resulted in about fourfold increase of the UDP-GlcNAc pool. In L. casei BL23 wild type strain transcriptional analyses showed that glmM and glmU are constitutively transcribed. By contrast, glmS transcription is down-regulated with a 21-fold decrease of glmS mRNA in cells cultured with N-acetylglucosamine as the sole carbon source compared to cells cultured with glucose. Our results revealed for the first time that GlmS, GlmM, and GlmU are responsible for UDP-GlcNAc biosynthesis in lactobacilli.