Construction And Optimization Of Saccharomyces Cerevisiae For Synthesizing Forskolin

Forskolin,a class of labdane-type diterpenoid in the cork tissue of the root of Coleus forskohlii,has significant medicinal value in anti-cancer,antiasthmatic,antihypertensive,strengthen heart and weight loss.Natural forskolin is mainly obtained by plant extraction.However,plant extraction is faced with many problems.In recent years,the construction of microbial cell factories to produce medicinal natural products by methods of synthetic biology is an effective way to solve the current problems.Saccharomyces cerevisiae is a model eukaryotic cell,the complete organelle structure and mature expression system ensure the activity of heterologous enzymes and facilitate the expression of heterologous proteins.The diterpene precursor GGPP high-yield strain 3HP was used as the parent strain and the t Cf TPS2 and t Cf TPS3 genes encoding 13R-manoyl oxide synthase were overexpressed to construct a Saccharomyces cerevisiae strain capable of synthesizing13R-manoyl oxide（13R-MO）.The key genes t HMG1 and BTS1～ERG20^F96C in the MVA pathway were further overexpressed to obtain the strain LJ4,which can produce13R-MO 145.8 mg/L.On the basis of the 13R-MO high-yield strain LJ4,the cytochrome P450 enzyme genes Cf CYP76AH15,Cf CYP76AH11,Cf CYP76AH16 and the acetyltransferase gene Cf ACT1-8 were overexpressed to construct a Saccharomyces cerevisiae strain capable of synthesizing forskolin.Two different sources of cytochrome P450 reductase CPR were selected for adaptation,and it was found that the Cf CPR from Coleus forskohlii,which was homologous to P450s,was the best.Using the self-sufficient fusion protein body construction idea,the Cf CYP76AH15～t66Cf CPR fusion reduced the production of 11-OXO-MO,while the production of forskolin increased by 1.03 times.Aa CYB5from Artemisia annua is the most conducive to product forskolin in exploring the role of three CYB5 in the production of forskolin.Fusion of t30Aa CYB5 with Cf CYP76AH15～t66Cf CPR showed that the production of 11-OXO-MO decreased even more,and the production of forskolin accumulated to 583.24μg/L.In order to further increase the yield,the constructed strain LJ24 can produce 5.78 mg/L forskolin by using a step-by-step multi-copy integration strategy.Modification of the endoplasmic reticulum of LJ24 and the encoding activating phospholipid synthesis gene INO2 was overexpressed,resulted in 15.1 mg/L forskolin,which was 2.61 times of strains LJ24.In order to speed up the use of ethanol by the strain and the supply of important cofactors,we overexpressed the ACSse^L641P,ALD6,ZWF1,and GND1 genes to obtain the strain LJ26 with a forskolin production of 21.47 mg/L.This study optimized the carbon source and fermentation conditions at the shake flask level.The high forskolin-producing strain LJ26 was subjected to batch and fed-batch fermentation in a 5 L bioreactor,with ethanol as a supplementary carbon source,and the concentration was controlled at 1-6 g/L.Finally the forskolin production reached 79.33 mg/L.