Modular Metabolic Engineering To Improve Geraniol Production In Saccharomyces Cerevisiae

Terpenoids constitute a large family of metabolites,and have been widely applied in energy,medical,food and chemical industry.Traditionally,terpenoids are extracted from natural sources(generally plants),or are chemically synthesized.However,these production processes often result in low yield and high cost,and waste plants sources seriously.The development and deployment of metabolic engineering and synthetic biology provides a new environment-friendly approach for the high-value chemicals production through engineering microbial cell factories.In nature,two independent biosynthetic pathways have evolved for the terpenoid synthesis.One is the mevalonate(MVA)pathway that exists in plants,most eukaryotes and some bacteria;the other is the methylerythritol phosphate(MEP)pathway that exists in plants,most bacteria and green algae.Saccharomyces cerevisiae is an attractive microbial cell factory due to its inherent MVA pathway,clear genetic background,easy genetic manipulation,excellent fermenting property and easy expression of eukaryotic enzymes.Most of previous studies have been focusing on sesquiterpenes synthesis in S.cerevisiae,such as artemisinic acid.However,compared with sesquiterpenes,the study of engineering S.cerevisiae for monoterpenes production was lagging behind.Many researches demonstrated that the low production of monoterpenes was attributed to the low enzyme activity of monoterpene synthase from plants,the shortage of precursor geranyl disphosphate(GPP)supply,and low tolerance for monoterpene in S.cerevisiae.Geraniol is an acyclic monoterpene alcohol that is widely used in the food and cosmetic industries,and has also been developed as an anti-cancer drug,antimicrobial reagent and advanced biofuels.The modular metabolic engineering was applied in this study to improve geraniol production.Specifically,the geraniol biosynthesis pathway was divided into four modules,including geraniol synthesis and MVA pathway module,precursor GPP synthesis module,geraniol downstream metabolism module,and acetyl-CoA synthesis module.The production of geraniol was significantly improved to 1.69 g/L through the the regulation of each metabolism module and fermentation condition optimization,which is the highest titer reported in engineered yeast strain.The main results of this study are showed as follows:(1)It is essential to express geraniol synthase with high activity for geraniol production in yeast strain.In this study,three geraniol synthases from diverse plants were evaluated in S.cerevisiae.Geraniol synthase from Valeriana officinalis(VoGES)showed the highest activity.Engineered S.cerevisiae strain containing VoGES could produce 0.82 mg/L geraniol,and those containing geraniol synthase from Ocimum basilicum(ObGES)and Lippia dulcis(LdGES)could produce 0.5 mg/L and 0.2 mg/L geraniol,respectively.Since terpenoid synthases from plants generally harbor a plastid-targeting peptide,we compared the truncated version of geraniol synthases without plastid-targeting peptide,and found out that the overexpression of tVoGES(a truncated version of VoGES)improved geraniol production by three folds,up to 2.5 mg/L.Other two GESs without plastid-targeting peptide also improved geraniol production.To further increase geraniol production,two rate-limiting enzymes,isopentenyl diphosphate isomerase encoded by ID11 and the catalytic domain of HMG-CoA reductase encoded by tHMG1,were overexpressed to enhance the flux of MVA pathway in strain overexpressing GESs.The results showed that 7.98 mg/L of geraniol was obtained through tVoGES overexpression and the enhanced MVA pathway.Therefore,tVoGES with the highest activity was selected for further engineering of geraniol-producing yeast strain.(2)The shortage of precursor GPP supply limits monoter:pene production due to the lack of a specific GPP synthase in wide-type S.cerevisiae.To increase intracellular GPP accumulation,we expressed three specific GPP synthase genes that are derived from different plants,including Abies grandis GPPS2,Picea abies GPPS2,and Catharanthus roseus GPPS.However,engineered strains with these GPP synthases failed to improve geraniol production.We then expressed endogenous ERG20 and its two mutants ERG20K197G and ERG2F096W-N127W,which showed more GPP synthase activity than FPP synthase activity in engineered strain(tVoGES-,IDI1-and tHMG1-expressing strain).The results showed that the geraniol production in engineered strains containing ERG20K197G and ERG20F96W-N127W were significantly improved,reaching to 16.9 mg/L and 27.3 mg/L,respectively.In addition,overexpression of a global transcription factor of MVA pathway,further enhanced geraniol production to 39.3 mg/L.To better utilize GPP,we constructed an tVoGES-ERG20F96W-N127W fusion protein,which helps geraniol production increase to 66.5 mg/L after optimizing the amino acid linker and the order of these two proteins.The expression of ERG20,a key branch point regulating GPP flux,was controlled by promoters with different strength to redistribute GPP and FPP synthesis.The result indicated that direct down-regulation of ERG20 expression did not increase geraniol production.ERG20 expression can be controlled by HXT1 promoter dynamically in response to glucose concentration change to redistribute the flux of the precursor GPP and geraniol production was significantly improved through regulating the precursor GPP metabolism module.The geraniol production can achieve 867.7 mg/L after carbon source feeding was optimized.(3)Our result also indicated that geraniol can be converted into other monoterpenoids in yeast.Geraniol production was dramatically decreased at the end of fermentation process,while by-products such as citronellol appeared.Since NADPH-oxidoreductase encoded by OYE2 and alcohol acetyltransferase encoded by ATF1 were the primary enzymes involved in geraniol downstream metabolism in S.cerevisiae.Geraniol production was further improved through the deletion of OYE2 and ATF1 to block geraniol downstream metabolism.The geraniol synthesis module,precursor GPP synthesis module and geraniol downstream metabolism modules were then assembled in S.cerevisiae,and the final engineered strain was able to produce 984.6 mg/L geraniol,and the geraniol yield reached 66.1 mg/g DCW in the fed-batch fermentation.,We the further constructed LEU2 prototrophic geraniol-producing strain,and LEU2 complementation enhanced geraniol production up to 1.69 g/L,which is the highest reported production in engineered yeast.(4)Acetyl-CoA is the starting compound of MVA pathway,and the sufficient supply of acetyl-CoA is important to enhance terpenoid biosynthesis in microbial cell factories.Citrate is catalyzed to generate acetyl-CoA and oxaloacetate by citrate lyase(ACL).However,the introduction of heterologous ACL pathway gene from Yarrowia lipolytica(YlACL)or Mus muculus(MmACL)into tVoGES-ERG20F96W-N127W expressing strain,does not improve geraniol production.In order to increase citrate accumulation,intracellular citrate metabolism pathway was further modified.Among the modified pathways,deletion of ICL1 improved geraniol production from 17.0 mg/L to 18.8 mg/L in strains expressing YIACL;Further deletion of IDH1 improved geraniol production to 22.1 mg/L,which is about 30%increase over that in the control strain.Acetyl-CoA synthesis was also further optimized to increase acetyl-CoA supply for geraniol production by balancing metabolism of related intermediate metabolite.In summary,the strategy of modular metabolic engineering was applied in this work to improve geraniol production,and the geraniol production in S.cerevisiae were significantly improved through the regulation of four geraniol biosynthesis pathway modules and fermentation condition optimization,which could provide a useful platform and paradigm to synthesize other high-value monoterpenes and their derivatives,such as monoterpene indole alkaloids derived from geraniol.