Prodution Of Monoterpene Geraniol By Reconstitution Of The Mevalonate Pathway In Saccharomyces Cerevisiae

Monoterpenes are C10 compounds with significant applications in medicine,industry,cosmetics,fuels,and anticancer,etc.To date,monoterpenes are mainly prepared by plant extracts and chemical synthesis,which have disadvantages such as difficulties in isolation and purification,or high consumption pollution.In this dissertation,Saccharomyces cerevisiae strain CEN.PK2-1D was employed to express geraniol synthase encoding gene GES from Ocimum basilicum.To further improve the production of geraniol,we introduced isopentenyl phosphate kinase encoding gene IPK from Methanothermobacter thermautotrophicus into S.cerevisiae and constructed an isopentenyl phosphate reflux utilization pathway(IRUP)that can efficiently utilize isopentenyl phosphate(IP)as the precursor for isopentenyl diphosphate(IPP)synthesis.Subsequently,through protein fusion,overexpression of key rate-limiting enzymes of the mevalonate pathway and medium optimization,the yield of geraniol was increased from 0.84 mg/L to 18.09mg/L.The main results are described as follows:1.The gene encoding geraniol synthase(GES)was heterologous expressed in S.cerevisiae,allowing S.cerevisiae acquire the ability to accumulate geraniol at the yield of 0.84 mg/L.Subsequently,to increase the utilization of the metabolic flux of the mevalonate pathway,the gene encoding isopentenyl phosphate kinase(IPK)from M.thermautotrophicus was expressed to phosphorylated isopentenyl phosphate(IP),generated by the promiscuous activity of diphosphomevalonate decarboxylase(PMD),to isopentenyl diphosphate(IPP).The introduction of IPK further increased geraniol to 2.74 mg/L,an increase of 224.9%compared with the star strain.These results suggest that expression of IPK in S.cerevisiae can enhance the yield of geraniol;2.In order to optimize the ratio of IPP/DMAPP and reduce the spatial block of adjacent enzymatic reactions to improve the efficiency of monoterpene geraniol synthesis in S.cerevisiae,the IPK was fusion with the mevalonate pathway rate-limiting enzyme encoding gene ERG20^F96WN127W or IDI1(IPK-IDI1,IDI1-IPK,IPK-ERG20^WW and ERG20^WW-IPK fusion proteins)and control strains were constructed to co-express IPK with ERG20^ww or IDI1.Compared to the controls,IPK-IDI1 and IDI1-IPK fusion proteins expression increased geraniol yield by 57.1%and 24.9%,respectively,while IPK-ERG20^WW and ERG20^WW-IPK fusion expression decreased geraniol yield by 132.8%and 97.9%,respectively.The above results confirmed that the rational regulation of the ratio of IPP and DMAPP by fusion IPK with IDI1 could effectively improve the geraniol yield,but the fusion of ERG20^WW with IPK had a negative effect on the geraniol yield.Finally,overexpression of ERG20^WW based on the fusion protein IPK-IDI1 expressing strain increased the accumulation of geraniol by 133.1%to7.81 mg/L;3.To reduce the cost of fermentation production,two strains SG9E and SG12,which had higher geraniol production among the above strains,were used to investigate the production of geraniol with molasses as the cheap carbon source.To remove hazardous components in molasses,we pretreated the molasses using four different pretreatment methods.Both SG9E and SG12 strains showed the improvement in geraniol production when molasses was used as the fermentation carbon source after microwave sulfuric acid treatment,increasing 68.8%and8.1%,respectively.Subsequently,by optimizing the addition time and amount of dodecane,the nitrogen source of the medium and the concentration of molasses,the accumulation of geraniol reached 16.64 mg/L and 18.09 mg/L,respectively,which increased by 160.8%and 106.0%,respectively compared to the untreated molasses medium.These results indicate that the use of molasses as a cheap carbon source can achieve efficient accumulation of geraniol in S.cerevisiae.