Employment In Engineering Terpenoid Backbone Biosynthesis Pathway In Saccharomyces Cerevisiae For Squalene Overproduction

Metabolic engineering is employed to construct genetically customized and robust microbial cell factories that can overproduce value-added chemicals.The overproduction of biofuels and pharmaceuticals through microbial engineering has rendered great socio-economic impact on the world.Isoprenoids are a broad class of natural products,though,produced in small quantities but harbor myriad pharmaceutical and industrial applications.However,isoprenoid production through traditional methods(extraction from natural sources)and chemical synthesis suffer various economic and environmental constraints.Thus it would be advantageous to devise robust and environment friendly alternative for overproduction of isoprenoids.Saccharomyces cerevisiae has an inbuilt potential to overproduce high-value compounds such as squalene,ergosterol and oleic acid.Herein,tandem terpenoid backbone biosynthesis pathway(TBBP)was overexpressed under newly characterized yeast promoters for overproduction of squalene in S.cerevisiae.A total of24 constitutive and 2 inducible promoters were characterized.4 hybrid promoters were engineered via ligating the transcriptional binding sites of HHF2 p and TEF1 p upstream of ERG20 p,HHF2p and TEF1 p.Strength of promoters of TBBP was also determined and knowledge of their used to select the comparatively stronger promoters for overexpression of tandem TBBP.TBBP pathway was integrated in the genome of S.cerevisiae as module-1(mevalonate pathway)and module-2(IDI1,ERG20,and 2 X PHO8?60).Upon integration and overexpression of TBBP in engineered strain(FOH-2),it turned into pink color and produced 3 fold higher biomass than wild type strain.Analysis and quantification of metabolites revealed an enhanced production of ergosterol 3.37 fold(34 mg/L)and oleic acid11.258 fold(213.907 mg/L)by engineered strain than wild type strain.Engineered strain overexpressing FDP dephosphorylating enzyme(PHO8?60)instead of farnesol produced 37mg/L longifolene and unlike wild type strain it did not even accumulate trace amount of squalene.Hence,it was presumed that overexpression of TBBP proteins bearing acidic isoelectric point and together release of phosphate during their enzymatic reactions lowered the intracellular pH of the customized strain.Therefore acidic intracellular environment might have converted the acid labile compounds farnesol and squalene into longifolene and oleic acid.To prove our hypothesis,farnesol and squalene were treated separately with methanolic-HCl,which resulted in longifolene and oleic acid production from farnesol and oleic acid from squalene.As aconsequence,acidic intracellular environment was found to be responsible for longifolene and oleic acid overproduction in engineered strain(FOH-2).Ergosterol and oleic acid play complementary role to maintain the rigidity and fluidity of yeast cell membrane.Therefore,it was anticipated that ergosterol overproduction could also enhance oleic acid production.Thus,a strain(HMG)was engineered by overexpressing ERG genes(erg2,erg3,erg4 and erg6)which consequently increased the transcription of Ole1 gene(oleic acid synthase)and aggrandized 1.52 fold oleic acid biosynthesis.Finally,the acidic intracellular environment and enhanced ergosterol biosynthesis together increased the oleic acid content of the engineered strain.The overexpression of TBBP reduced 3.618 fold ethanol production by engineered strain than the wild type strain.Inhibition of squalene monooxygenase with terbinafine turned white the engineered strain and accumulated highest 304.492 mg/L squalene,a 6.680 fold higher than wild type strain under same conditions.Inhibition of 14-alpha-demethylase with ketoconazole intensified the pink color of the engineered strain without accumulation of lanosterol and squalene epoxide.However squalene epoxide is putatively modified under acidic intracellular environment then converted to lycopene like novel compound.Successful overexpression of tandem TBBP drained the flux of acetyl CoA in isoprenoid biosynthesis to overproduce longifolene,squalene,and ergosterol,and alongside it also enhanced oleic biosynthesis.