Combinatorial pathway engineering of Bacillus subtilis for production of structurally defined and homogeneous chitooligosaccharides.

Chitooligosaccharides (COSs) have a widespread range of biological functions and an incredible potential for various pharmaceutical and agricultural applications. Although several physical, chemical, and biological techniques have been reported for COSs production, it is still a challenge to obtain structurally defined COSs with defined polymerization (DP) and acetylation patterns, which hampers the specific characterization and application of COSs. Herein, we achieved the de novo production of structurally defined COSs using combinatorial pathway engineering in Bacillus subtilis. Specifically, the COSs synthase NodC from Azorhizobium caulinodans was overexpressed in B. subtilis, leading to 30 ± 0.86 mg/L of chitin oligosaccharides (CTOSs), the homo-oligomers of N-acetylglucosamine (GlcNAc) with a well-defined DP lower than 6. Then introduction of a GlcNAc synthesis module to promote the supply of the sugar acceptor GlcNAc, reduced CTOSs production, which suggested that the activity of COSs synthase NodC and the supply of sugar donor UDP-GlcNAc may be the limiting steps for CTOSs synthesis. Therefore, 6 exogenous COSs synthase candidates were examined, and the nodCM from Mesorhizobium loti yielded the highest CTOSs titer of 560 ± 16 mg/L. Finally, both the de novo pathway and the salvage pathway of UDP-GlcNAc were engineered to further promote the biosynthesis of CTOSs. The titer of CTOSs in 3-L fed-batch bioreactor reached 4.82 ± 0.11 g/L (85.6% CTOS5, 7.5% CTOS4, 5.3% CTOS3 and 1.6% CTOS2), which was the highest ever reported. This is the first report proving the feasibility of the de novo production of structurally defined CTOSs by synthetic biology, and provides a good starting point for further engineering to achieve the commercial production.

Metabolic engineering for the production of chitooligosaccharides: advances and perspectives.

Chitin oligosaccharides (CTOs) and its related compounds chitosan oligosaccharides (CSOs), collectively known as chitooligosaccharides (COs), exhibit numerous biological activities in applications in the nutraceutical, cosmetics, agriculture, and pharmaceutical industries. COs are currently produced by acid hydrolysis of chitin or chitosan, or enzymatic techniques with uncontrollable polymerization. Microbial fermentation by recombinant Escherichia coli, as an alternative method for the production of COs, shows new potential because it can produce a well-defined COs mixture and is an environmentally friendly process. In addition, Bacillus subtilis, a nonpathogenic, endotoxin-free, GRAS status bacterium, presents a new opportunity as a platform to produce COs. Here, we review the applications of COs and differences between CTOs and CSOs, summarize the current preparation approaches of COs, and discuss the future research potentials and challenges in the production of well-defined COs in B. subtilis by metabolic engineering.

Combinatorial promoter engineering of glucokinase and phosphoglucoisomerase for improved N-acetylglucosamine production in Bacillus subtilis.

In previous work, a recombinant Bacillus subtilis strain was successfully constructed for microbial production of N-acetylglucosamine (GlcNAc). In this study, GlcNAc titer was further improved by combinatorial promoter engineering of key genes glck encoding glucokinase and pgi encoding phosphoglucoisomerase. First, three promoters including constitutive promoter P43, xylose inducible promoter PxylA, and isopropyl-ß-d-thiogalactoside inducible Pgrac were used to replace the native promoters of glcK and pgi, yielding 12 recombinant strains. It was found that when glcK and pgi were both under the control of promoter PxylA, the highest GlcNAc titer in 3-L fed-batch bioreactor reached 35.12g/L, which was 52.6% higher than that of the initial host. Next, the transcriptional levels of the related genes in glycolysis, GlcNAc synthesis pathway, peptidoglycan synthesis pathway, and pentose phosphate pathway were investigated by quantitative real-time PCR analysis. Fine-tuning upper GlcNAc synthesis pathway by combinatorial promoter substitution significantly enhanced GlcNAc production in engineered B. subtilis.