Glucosamine and its derivatives have recently been used in various applications, including dietary supplements, cosmetics, pharmaceuticals and the like, and thus the market demand therefor has increased. To meet this increased demand, metabolic engineering studies on the production of glucosamine and is derivatives have been actively conducted. One recent study has shown that glucosamine and its derivatives can relieve arthritis, suggesting that the application thereof will be expanded to clinical treatment of arthritis.
However, glucosamine and its derivatives, which are currently produced, are mainly extracted from crustacean shell waste, and thus can cause side effects such as allergic reactions in the human body. Accordingly, there have been attempts to produce glucosamine and its derivatives in strains recognized as safe strains. For example, the production of glucosamine by use of Bacillus subtilis has been reported (Liu Y, Zhu Y, Li J, Shin H D, Chen R R, Du G, Liu L, Chen J. 2014. Modular pathway engineering of Bacillus subtilis for improved N-acetylglucosamine production. Metab Eng 23:42-52).
Methods of effectively producing metabolites such as glucosamine in microorganisms include a method that redirects flux from competing pathways toward a desired target metabolite. One example may be a method of controlling central carbon metabolism to reduce glycolytic flux and increase the yield of the target metabolite. As a method for controlling glycolytic flux, a study has been reported indicating that glycolytic flux is controlled by modulating irreversible enzymes, such as hexokinase, phosphofructokinase and pyruvate kinase, in bacteria.
However, the regulation of glycolysis in eukaryotic cells considerably differs from the regulation of glycolysis in prokaryotic cells. Namely, in eukaryotic cells, additional allosteric effects play an important role in the regulation of glycolysis, and thus glycolysis is regulated by a mechanism which is much more complex than that in prokaryotic cells. As a result, studies on the use of eukaryotic cells have not yet shown worthy results. S. cerevisiae can be considerably advantageous for the production of glucosamine and its derivatives, due to its advantage of having excellent resistance to various chemical substances. However, studies on S. cerevisiae merely include a recent report indicating that the glycolytic flux was regulated by modulating hexokinase activity in S. cerevisiae to enhance the production of gluconate (Tan S Z, Manchester S, Prather K L. 2015. Controlling Central Carbon Metabolism for Improved Pathway Yields in Saccharomyces cerevisiae. ACS Synth Biol). However, hexokinase, an enzyme catalyzing the first step of glycolysis in S. cerevisiae, has a problem in that regulation of the activity of hexokinase is not suitable for producing metabolites, such as glucosamine and its derivatives, in the downstream steps of glycolysis.
Accordingly, the present inventors have made extensive efforts to develop a method for regulation of glycolytic flux, which is suitable for the production of N-acetylglucosamine which is highly useful as an intermediate metabolite of glycolysis. As a result, the present inventors have found that, when the gene encoding phosphofructokinase-2 (PFK-2) is disrupted or deleted in a microorganism having glycolysis and N-acetylglucosamine biosynthesis pathways, N-acetylglucosamine production can be effectively increased, thereby completing the present invention.
USPTO Backgrounds
USPTO Backgrounds