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Systems Metabolic Engineering Of Bacillus Subtilis For Efficient N-acetyl Glucosamine Production | 33970
Journal of Biotechnology & Biomaterials
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N-acetyl glucosamine (GlcNAc) is a pharmaceutically and nutraceutically important compound with wide applications
and now is mainly produced by hydrolysis from crab and shrimp shells which can cause severe environmental pollution
and has potential risk of allergic reactions. In this work, we achieved the over-production of GlcNAc by systems metabolic
engineering of Bacillus subtilis, a generally regarded as safe strain. Specifically, GlcNAc synthesis pathway was strengthened
by co-over expression of Glucosamine-6-phosphate (GlmS) synthase and GlcNAc-6-phosphate N-acetyl transferase (GNA1)
which realized GlcNAc production (240 mg/L). Next, GlcNAc uptake pathway and intracellular degradation pathway were
entirely blocked by knockout of all the encoding genes in GlcNAc catabolic pathway to facilitate GlcNAc accumulation.
Then, to balance and strengthen GlcNAc synthetic pathway, DNA-guided scaffold system was introduced and increased
GlcNAc titer from 1.83 g/L to 4.55 g/L. Synthetic small regulatory RNAs were then employed to optimize expression level
of key enzymes in the nodes of GlcNAc-related network including 6-phosphofructokinase (Pfk) and phospho glucosamine
mutase (GlmM). GlcNAc titer was improved to 8.30 g/L by modular regulation of the activities of GlcNAc-related modules.
In fed-batch fermentation, the GlcNAc titer was further increased to 31.65 g/L which was 3.8-fold that in the shake flask.
Finally, to understand kinetics of metabolite changes in GlcNAc synthesis pathway and glycolysis, targeted metabolomics and
dynamic labeling were implemented. Inefficient GlcNAc6P dephosphorylation and undesired GlcNAc phosphorylation were
identified as rate-limiting step for GlcNAc synthesis which pin-pointed future direction for further pathway optimization. The
used systems metabolic engineering strategies may be useful for the construction of versatile B. subtilis cell factories for the
production of the other industrially important chemicals.
Long Liu is currently a life-time professor at School of Biotechnology, Jiangnan University, Wuxi, China. He has been working in the area of bioprocess engineering and metabolic engineering with special reference to bioprocess optimization and control. He has authored 2 books, 3 book chapters, 7 review papers, 20 patents, 37 research papers in SCI journals, and 10 conference papers. He has been a recipient of First prize of Science and Technology progress, Jiangsu, China (2010), First prize of Science and Technology Progress, China Petroleum and Chemical Industry Federation (2011), the Technological Invention Award of China National Light Industry Council (2013), and Excellent Young Teacher of Jiangsu, China (2014).