Biosynthesis Of Geranylgeraniol And ?-Ionon In Saccharomves Cerevisiae

Geranylgeraniol(GGOH)is an important intermediate for the production of spices,drugs,vitamin A and E,with(E,E,E)-GGOH as the bioactive enantiomer.The initial strain constructed by expressing fusion proteins of geranylgeranyl diphosphate synthase-diacylglycerol diphosphate phosphatase(BST1-DPP1)and geranylgeranyl diphosphate synthase-famesyl diphosphate synthetase-famesyl diphosphate synthetase(BST1-ERG20)in Saccharomyces cerevisiae syn thesized 34.85 mg/L of(E,E,E)-GGOH.In this work,metabolic regulation was further explored to redirect the metabolic flow at the farnesyl pyrophosphate(FPP)node.Overexpression of a GGPP synthase mutant(CrtE03M)increased the yield of GGOH by 1.6 times,reaching 56.04 mg/L,whereas down-regulation of the competitive squalene synthetic branch by replacing the native PERG9 with a glucose-inducible weak promoter PHXT1 increased GGOH production by 4.5 times,reaching 156 mg/L.Meanwhile,a glucose-responsive GAL system was constructed by GAL80 deletion and used to control the synthetic genes of GGOH.The glucose-repressive PGAL-driven GGOH synthetic pathway and the glucose-inducible PHXT1-driven squalene pathway constituted a sequential expression system,which allowed for balanced cell growth and product accumulation.Finally,a GGOH production of 183.07 mg/L was obtained after 72 h in shake flask by biphasic fermentation using dodecane as the upper layer,which represents a 5-fold improvement over the initial strain.Therefore,YWWP11-12DEE03(+)was selected for high density fermentation to achieve high GGOH production.In the end,the OD600 reached 310 and 1.86 g/L GGOH was producted after 120 h of fermwntation.These results demonstrated the power of metabolic flux redirection at the FPP node,and may provide reference for biosynthesis of other natural production sharing FPP as a precursor.?-ionon was chosen as another target terpenoid for biosynthesis in S.cerevisiae.?-ionon is highly appreciated in the flavoring industry due to its characteristic aromatic notes.At present,?-ionon is mainly synthesized from citral.The increasing concerns in healthy living leads to growing market demand of natural spices,including those produced by microbial fermentation.In this experiment,the ?-carotene producing strain YXPW74 previously constructed was used as the starting strain for construction of P-ionon synthesizing S.cerevisiae.After expressing the exogenous gene RdCCDl from rose,about 4.2 mg/L ?-ionon was detected.However,further overexpression of RdCCDl did not obviously improve the production of ?-ionon(4.9 mg/L).Subsequently,another member of the CCD protein family,AtCCD7 was cloned from Arabidopsis thaliana and expressed to improve the production of?-ionon.In order to examine the synergy between RdCCDl and AtCCD7 in conversion of?-carotene to ?-ionone,two strains were constructed,respectively by expressing AtCCD7 alone and by co-expressing RdCCD1 and AtCCD7.Expression of AtCCD7 alone yielded only 0.6 mg/L ?-ionone,and further overexpression of this protein did not improve the production.In contrast,co-expression of RdCCD1 and AtCCD7 produced 6.1 mg/L ?-ionone,representing an obvious improvement over the RdCCD1-expressing strain,indicating a synergistic effect between CCD1 and CCD7 in Saccharomyces cerevisiae.Considering the unsatisfactory catalytic activity of both enzymes,directed evolution was conducted using a color-based high-throughput screening method.However,no positive mutants were obtained.The synergy caused by co-expressing RdCCD1 and AtCCD7 provides reference for synthetic biology in artificially designed metabolic pathways.