Abstract
BackgroundAlternaria sp. MG1, an endophytic fungus isolated from grape, is a native producer of resveratrol, which has important application potential. However, the metabolic characteristics and physiological behavior of MG1 still remains mostly unraveled. In addition, the resveratrol production of the strain is low. Thus, the whole-genome sequencing is highly required for elucidating the resveratrol biosynthesis pathway. Furthermore, the metabolic network model of MG1 was constructed to provide a computational guided approach for improving the yield of resveratrol.ResultsFirstly, a draft genomic sequence of MG1 was generated with a size of 34.7 Mbp and a GC content of 50.96%. Genome annotation indicated that MG1 possessed complete biosynthesis pathways for stilbenoids, flavonoids, and lignins. Eight secondary metabolites involved in these pathways were detected by GC–MS analysis, confirming the metabolic diversity of MG1. Furthermore, the first genome-scale metabolic network of Alternaria sp. MG1 (named iYL1539) was reconstructed, accounting for 1539 genes, 2231 metabolites, and 2255 reactions. The model was validated qualitatively and quantitatively by comparing the in silico simulation with experimental data, and the results showed a high consistency. In iYL1539, 56 genes were identified as growth essential in rich medium. According to constraint-based analysis, the importance of cofactors for the resveratrol biosynthesis was successfully demonstrated. Ethanol addition was predicted in silico to be an effective method to improve resveratrol production by strengthening acetyl-CoA synthesis and pentose phosphate pathway, and was verified experimentally with a 26.31% increase of resveratrol. Finally, 6 genes were identified as potential targets for resveratrol over-production by the recently developed methodology. The target-genes were validated using salicylic acid as elicitor, leading to an increase of resveratrol yield by 33.32% and the expression of gene 4CL and CHS by 1.8- and 1.6-fold, respectively.ConclusionsThis study details the diverse capability and key genes of Alternaria sp. MG1 to produce multiple secondary metabolites. The first model of the species Alternaria was constructed, providing an overall understanding of the physiological behavior and metabolic characteristics of MG1. The model is a highly useful tool for enhancing productivity by rational design of the metabolic pathway for resveratrol and other secondary metabolites.
Highlights
The roles of cofactors in the regulation of resveratrol biosynthesis were investigated, and both biochemical and genetic strategies were proposed for the improvement of resveratrol production
Genome sequencing and characteristics To obtain a comprehensive understanding of the functional genes, especially those response for the biosynthesis of resveratrol and other functional metabolites in Alternaria sp
A genome scale metabolic network model (GSMM) for the species Alternaria was constructed, providing an overall understanding of the physiological behavior and metabolic characteristics of the tested strain. Both biochemical and genetic strategies were proposed for the improvement of resveratrol production in silico and verified with wet experiments
Summary
MG1, an endophytic fungus isolated from grape, is a native producer of resveratrol, which has important application potential. Under the current market demand, endophytic fungi exhibit significant potential for producing plant-original drugs and functional compounds, such as taxol, which is a renowned antitumor agent that was originally isolated from the bark of the Pacific Yew, Taxus brevifolia [4]. Endophytes offer the advantages of accumulating higher concentrations of functional compounds that would be toxic to genetically modified Escherichia coli and yeast since they possess higher resistance to their self-produced metabolites [5]. Compared to the complex procedures and toxic organic solvents needed for its chemical synthesis [8, 9], genomic instability for plant cell culture [10], and the time-consumption and product inhibition for genetically modified E. coli and yeast [11], Alternaria sp. MG1, since it has not been verified in any microorganisms at the gene level
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