Construction Of Saccharomyces Cerevisiae Cell Factory For Efficient Accumulation Of (+)-Nootkatone

(+)-Nootkatone is a high value-added sesquiterpene compound found in grapefruit peel and heartwood of yellow cedar,which can be used as an edible flavor for blending citrus fragrance and widely applied in daily fragrance and perfume.In our previous study,it was found that the catalytic efficiency of the membrane-anchored cytochrome oxidase P450/reductase CPR（HPO/At CPR）in the（+）-nootkatone metabolic pathway is low,which limits the efficient accumulation of（+）-nootkatone to some degree.In this thesis,a high-throughput screening platform was developed and applied to HPO/At CPR activity screening to obtain its optimal expression ratios using Saccharomyces cerevisiae as the original strain.Further,an efficient yeast cell for producing（+）-nootkatone was constructed by global regulatory factor interference,precursor supply enhancement,branched circuit blocking and enzyme stability enhancement.In this study,the significant effect of enzyme/coenzyme expression levels on the catalytic efficiency was investigated by regulating the various expression modes of HPO/At CPR including the change of linkers and the binding modes to endoplasmic reticulum.The yield ofβ-nootkatol from substrate（+）-valencene was enhanced from 9.6 mg/L to 35.2mg/L with resting-cell assay.Based on the component characteristics of yeast cell wall,an efficient and versatile high-throughput screening platform was developed in this study,which achieving uniform and efficient release of yeast cell contents with only 5 U/m L Zymolyase for 2 h for the detection of intracellular material.The platform was used to screen the HXT7 promoter mutation library,and the strengths of promoter library was in the range of 0.07 to 3.5.The mutants with varying strengths were used to control expression of HPO and At CPR,respectively,thus regulating the expression ratios of HPO/At CPR more finely.The optimal strain yielded 66.2mg/Lβ-nootkatol from substrate（+）-valencene.According to the above findings,de novo biosynthetic pathway of（+）-nootkatone was constructed successfully by increasing the flux of precursor（+）-valencene supply through overexpression of valencene synthase gene Cn VS and t HMG1 which is a key enzyme gene of MVA pathway,relieving the global inhibition to MVA pathway with knockout of ROX1 gene,weakening the branched pathway of the cell membrane component ergosterol using the knockdown of ERG9 gene,enhancing the stability of the endoplasmic reticulum membrane protein At CPR through overexpression of ICE2 gene,and coupling of ADH3 which is a key dehydrogenase for oxidation ofβ-nootkatol to（+）-nootkatone.Through the optimization of fermentation mode and the verification of 3 L fermenter scale-up,the engineered strain PK2m RI-At C/H_m6A reached a cell density of 113 g/L CDW after 198 h fed-batch fermentation.A total terpene yield of 4.89 g/L was achieved,including 1.02 g/L（+）-nootkatone,which are the highest yields reported so far.This study provides a simple and feasible technical strategy for the fine-tuning of P450and its coenzyme CPR for efficient biotransformation.And the efficient biosynthesis of（+）-nootkatone in situ also provides an excellent example for the construction of efficient cell factories in yeast cell.