Rational Design Of Oxidative Cleavage Enzyme CCD2 And The Construction Of Crocetin Biosynthesis Pathway

The carotenoid biosynthesis pathway has drawn much attention in recent years,and the carotenoid products lycopene,β-carotene,and zeaxanthin were successfully synthesized in Saccharomyces cerevisiae by our research group.However,when crocetin,the oxidative cleavage product of zeaxanthin,was synthesized,the yield was only at the microgram level.Previous studies have shown that carotenoid cleavage dioxygenase 2(CsCCD2)is the rate-limiting enzyme in the crocetin biosynthesis pathway.Therefore,in this study,the structural domains of CsCCD2 were resolved and redesigned to facilitate the conversion of the carotenoid precursors to crocetin.The engineered CsCCD2 was used for the synthesis of crocetin in S.cerevisiae and enzymecatalyzed “one-pot” in vitro synthesis.The active center of CsCCD2 is located inside the protein.The tunnels connecting the internal active center and the external solvent are responsible for the transport of substrate,cofactor and solvent,and therefore also determine the substrate recognition specificity and catalytic site specificity of CsCCD2.In this paper,a“hybrid-tunnel”strategy was proposed.Through integrating the traits of CCD1 and CCD4 access tunnels to the CsCCD2 access tunnel,the CsCCD2 with “hybrid-tunnel” would obtain a broad substrate profile while retaining its precise cleavage mode for catalyzing the synthesis of crocetin from diverse carotenoid precursors.Guided by the “hybrid-tunnel” strategy,the S323 A mutant,which could catalyze a new substrate β-carotene for the synthesis of crocetin,was obtained,reshaping the synthesis pathway of crocetin in S.cerevisiae.Meanwhile,the crocetin titer in yeastbased shake fermentation was increased 5-fold to 6 mg/L.Besides,through optimizing the genetic stability of the strain and the fed-batch fermentation process in a 5 L fermenter,the yield of crocetin in S.cerevisiae was further increased to 107 mg/L.Based on the excretion characteristic of crocetin during fermentation,the ratio of organic phase to medium during the two-phase fermentation was optimized at 1:1,and thus 99% of crocetin in yeast cells was excreted and stored in the extracellular organic phase,while the carotenoid precursors were completely retained in the cell.The spatial segregation of the carotenoid precursors and the product crocetin creates conditions for rapid screening of crocetin high-producing yeast strains.A CsCCD2 and aldehyde dehydrogenase(SynALD)tandem-catalyzed “two enzyme one-pot” method was developed for the in vitro synthesis of crocetin using zeaxanthin as substrate.The two enzymes were added to the reaction system simultaneously in equimolar ratio,and the conversion of zeaxanthin exceeded 95%.However,the competitive catalysis of SynALD also resulted in a by-product proportion of 17% in the reaction system.In view of the difference in catalytic activity of CsCCD2 and SynALD,a dual enzyme catalytic balance was achieved by adjusting the addition ratio of CsCCD2 to SynALD at 3:1.Finally,the proportion of the by-product in the reaction system decreased to 5%,while the proportion of crocetin was increased to 88%,which laid the foundation for the industrial application of crocetin.