Heterologous expression and characterization of the key enzymes involved in sugar donor synthesis of polysaccharide in Ganoderma lucidum

Abstract:

[Background] Ganoderma lucidum polysaccharide (GLP), as a macromolecular substance with various biological activities, has attracted a lot of attention in recent years. Some researchers have improved the yield of GLP through optimization of fermentation regulation or strain improvement for G. lucidum. However, it is a fact that the biosynthesis pathway of GLP is still unclear completely, and the understanding about characteristics of key enzymes involved in GLP synthesis are insufficient. Thus, there is a bottleneck on substantially improving the yield of GLP. [Objective] Through heterologous expression of the genes of phosphoglucomutase (PGM), UDP-glucose pyrophosphorylase (UGPG), and phosphomannose isomerase (PMI) cloned from G. lucidum in E. coli, decent amounts of the purified enzymes were obtained. We characterized their enzymatic properties and compared them with other sources, to provide insights into the characteristic information of them, and build an efficient strategy for the synthetic of GLP on fermentation. [Methods] Genes of gl-pgm, gl-ugpg and gl-pmi were cloned from G. lucidum strain CGMCC 5.26 and expressed in E. coli, the recombinases were purified with Co-NTA resin respectively. Then, the enzymatic properties of the purified enzymes were studied. [Results] GLpgm, GLugpg and GLpmi, were successfully expressed in E. coli. The optimum reaction pH of GLpgm was 8.5, and it was 7.5 for GLugpg and GLpmi. The optimum reaction temperature of GLpgm, GLugpg and GLpmi were 35, 40 and 30 °C, respectively. For them, 1 mmol/L of Ag+ and Cu2+ had strong inhibitory effects, while Mn2+ and Mg2+ had activation effects on GLpgm and GLpmi, especially the use of Mn2+ could increase the GLpgm activity by 2.7 times. The kcat/Km values of GLpgm, GLugpg and GLpmi were 196.08, 818.60 and 1 105.22 mmol/(L·s), respectively. [Conclusion] GLpgm, GLugpg and GLpmi were similar to the sources of plant and other fungi in terms of optimum pH, temperature and metal ions action, while the catalytic efficiency was relatively higher than them. Their properties could provide more informantion for the regulation of GLP based on it’s pathway.

Key words:Phosphoglucosemutase, UDP-glucose pyrophosphorylase, Phosphomannose isomerase, Heterologous expression, Enzymatic characterization