Abstract
The natural product pneumocandin B0 is the precursor of the antifungal drug caspofungin. We found that replacing glucose in the initial fermentation medium with 20 g/L fructose is more conducive to pneumocandin B0 production and biomass accumulation. In order to explore the mechanism of the different metabolic responses to fructose and glucose, we used each as the sole carbon source, and the results showed that fructose increased the total pneumocandin B0 yield and biomass by 54.76 and 13.71%, respectively. Furthermore, we analyzed the differences of gene expression and metabolic pathways between the two different carbon sources by transcriptomic analysis. When fructose was used as the carbon source, genes related to the pentose phosphate pathway (PPP), glycolysis and branched-chain amino acid metabolism were significantly upregulated, resulting in increased intracellular pools of NADPH and acetyl-CoA in Glarea lozoyensis for cell growth and pneumocandin B0 product synthesis. Interestingly, the pneumocandin B0 biosynthetic gene cluster and the genes of the TCA cycle were significantly downregulated, while the FAS genes were significantly upregulated, indicating that more acetyl-CoA was used for fatty acid synthesis. In particular, we found that excessive synthesis of fatty acids caused lipid accumulation, and lipid droplets can sequester lipophilic secondary metabolites such as pneumocandin B0 to reduce cell damage, which may also be an important reason for the observed increase of pneumocandin B0 yield. These results provide new insights into the relationship between pneumocandin B0 biosynthesis and carbon sources in G. lozoyensis. At the same time, this study provides important genomic information for improving pneumocandin B0 production through metabolic engineering strategies in the future.
Highlights
In nature, fungi are challenged by a variety of biotic and abiotic stressors, ranging from attack by other microorganisms to nutrient deficiencies or extremes of pH and temperature
Li et al (2015) found that when Actinosynnema mirum was cultured with fructose as the sole carbon source, the yield of Ansamitocin P-3 was increased fourfold compared to glucose. These results indicated that fructose could replace glucose as a carbon source for G. lozoyensis fermentation, but the effects of fructose on the metabolism of G. lozoyensis remain to be elucidated
At 6 days of fermentation, the change of Pneumocandin B0 (PB0) yield and biomass was the most significant, with increases of 53.75 and 42.85%, respectively. This indicated that fructose as a carbon source is more conducive to the production of PB0 and the accumulation of biomass than glucose
Summary
Fungi are challenged by a variety of biotic and abiotic stressors, ranging from attack by other microorganisms to nutrient deficiencies or extremes of pH and temperature. They produce a large number of secondary metabolites (SMs), including antibiotics, cytochromes, and other active substances, some of which are applied as antitumor of cholesterol-lowering drugs. Antibiotics are arguably the most important secondary metabolites, and are the focus of a large body of research (Li and Tan, 2017). Tkacz et al (1993) found that using 100 g/L mannitol as the sole carbon source improved the yield of PB0 to 800 mg/L. Connors et al (2000) found that by using a high concentration of 125 g/L fructose as the sole carbon source, the fermentation time of PB0 can be effectively reduced to 375 h, while reducing the concentration of by-products and obtaining a final PB0 yield of approximately 400 mg/L. In our previous study (Song et al, 2018b), 20 g/L glucose and 80 g/L mannitol were used as carbon source, and an improved PB0 yield of 2768 mg/L was obtained
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