Improvement and Prediction of Secondary Metabolites Biosynthesis Under Yeast Extract Elicitation of Neem Cell Suspension Culture Using Response Surface Methodology

Abstract

Neem is the medicinal plant used as antimalarial, antibacterial, antiviral, insecticide and antimicrobial. The aim of this study was investigate effect of yeast extract and sampling time on cell growth, secondary metabolites synthesis, SQS1 and MOF1 genes expression and prediction of secondary metabolites synthesis by response surface methodology. Highest fresh and dry cell weight were 580.25 g/L and 21.01 g/L obtained 6 days after addition 100 mg/L yeast extract. Highest azadirachtin accumulation and production were 16.08 mg/g DW and 219.78 mg/L obtained at 25 mg/L yeast extract for 2 and 4 days. Maximum mevalonic acid accumulation and production were 1.746 mg/g DW and 23.77 mg/L observed 2 days after addition 50 mg/L yeast extract addition. Highest amount of squalene accumulation (0.215 mg/g DW) and production (4.526 mg/L) were achieved 4 days after application of 50 mg/L yeast extract. Prediction results showed that highest azadirachtin accumulation (13.607 mg/g DW) and production (190.50 mg/L), mevalonic acid accumulation (0.500 mg/g DW) and production (5.57 mg/L) and squalene accumulation (0.301 mg/g DW) obtained by using 245 mg/L yeast extract for 2 days, 71 mg/L yeast extract for 2 days, 200 mg/L yeast extract for 4.96 days, whitout yeast extract for 6.54 days and 4 days, respectively and predicted that highest squalene production is achieved by long-term exposure to high concentrations of yeast extract. qRT-PCR analysis showed the maximum relative gene expression of SQS1 and MOF1 by addition 150 mg/L yeast extract for 4 days and 25 mg/L yeast extract for 2 days, respectively.