Improvement and prediction of secondary metabolites production under yeast extract elicitation of Azadirachta indica cell suspension culture using response surface methodology

Abstract Neem is the medicinal plant used as antimalarial, antibacterial, antiviral, insecticide, and antimicrobial. This study aimed to investigate and prediction the effect of yeast extract and sampling time on cell growth, secondary metabolites synthesis, SQS1 and MOF1 genes expression by response surface methodology. The highest fresh and dry cell weights were 580.25 g/L and 21.01 g/L obtained 6 days after using 100 mg/L yeast extract. The highest azadirachtin accumulation and production were 16.08 mg/g DW and 219.78 mg/L obtained 2 and 4 days after using 25 mg/L yeast extract. Maximum mevalonic acid accumulation and production were 1.75 mg/g DW and 23.77 mg/L observed 2 days after application of 50 mg/L yeast extract. The highest amount of squalene accumulation (0.22 mg/g DW) and production (4.53 mg/L) were achieved 4 days after using 50 mg/L yeast extract. Prediction results showed that the highest azadirachtin accumulation (13.61 mg/g DW) and production (190.50 mg/L), mevalonic acid accumulation (0.50 mg/g DW) and production (5.57 mg/L), and squalene accumulation (0.30 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, without yeast extract for 6.54 days and 4 days, respectively. Also, predicted that the highest squalene production is achieved by long-term exposure to high concentrations of yeast extract. The qRT-PCR analysis showed the maximum relative gene expression of SQS1 and MOF1 obtained by using 150 mg/L yeast extract for 4 days and 25 mg/L yeast extract for 2 days, respectively.