ProductionofMiltiradienebyMetabolically
EngineeredSaccharomycescerevisiae
ZhuboDai,1YiLiu,1,2LuqiHuang,3XueliZhang1
1KeyLaboratoryofSystemsMicrobialBiotechnology,TianjinInstituteofIndustrial
Biotechnology,ChineseAcademyofSciences,32XiQiDao,TianjinAirportEconomicPark,
Tianjin,300308,China;telephone:86-22-84861983;fax:86-22-84861983;
e-mail:zhang_xl@
2CollegeofBiotechnology,TianjinUniversityofScience&Technology,Tianjin,China
3InstituteofChineseMateriaMedica,ChineseAcademyofChineseMedicalScience,
No.16Nanxiaojie,DongzhimenneiAve,Beijing,100700,China;
telephone:þ86-10-64014411;fax:þ86-10-64013996;e-mail:huangluqi@
ABSTRACT:Metabolicengineeringofmicroorganismsisanalternativeandattractiverouteforproductionofvaluableterpenoidsthatareusuallyextractedfromplantsources.TanshinonesarethebioactivecomponentsofSalviamiltiorrhizhaBunge,whichisawell-knowntraditionalChinesemedicinewidelyusedfortreatmentofmanycardiovasculardiseases.Asasteptowardmicrobialproduc-tionoftanshinones,copalyldiphosphate(CPP)synthase,andnormalCPPkaurenesynthase-likegenes,whichconverttheuniversalditerpenoidprecursorgeranylgeranyldiphos-phate(GGPP)tomiltiradiene(animportantintermediateofthetanshinonessyntheticpathway),wasintroducedintoSaccharomycescerevisiae,resultinginproductionof4.2mg/Lmiltiradiene.Improvingsuppliesofisoprenoidprecursorswastheninvestigatedforincreasingmiltiradieneproduc-tion.Althoughover-expressionofatruncated3-hydroxyl-3-methylglutaryl-CoAreductase(tHMGR)andamutatedglobalregulatoryfactor(upc2.1)genedidimprovesupplyoffarnesyldiphosphate(FPP),productionofmiltiradienewasnotincreasedwhilelargeamountsofsqualene(78mg/L)wereaccumulated.Incontrast,miltiradieneproductionincreasedto8.8mg/LbyimprovingsupplyofGGPPthroughover-expressionofafusiongeneofFPPsynthase(ERG20)andendogenousGGPPsynthase(BTS1)togetherwithaheterologousGGPPsynthasefromSulfolobusacidocaldarius(SaGGPS).Auxotrophicmarkersintheepisomalplasmidswerethenreplacedbyantibioticmarkers,sothatengineeredyeaststrainscoulduserichmediumtoobtainbettercellgrowthwhilekeepingplasmidstabilities.Over-expressingERG20-BTS1andSaGGPSgenesincreasedmiltiradieneproductionfrom5.4to28.2mg/binatorialover-expressionoftHMGR-upc2.1andERG20-BTS1-SaGGPSgeneshadasynergeticeffectsonmiltiradieneproduction,increasingtiterto61.8mg/L.Finally,fed-batchfermentationwasperformed,and488mg/Lmiltiradienewasproduced.Theyeaststrainsengineeredinthisworkprovideabasisforcreatinganalternativewayforproductionoftanshinonesinplaceofextractionfromplantsources.