Engineered Yeast Cell For Producing Crocetin

For heterologous synthesize natural carotenoid crocetin in microorganism,a ?-carotene producing Saccharomyces cerevisiae was chozen as the host cell to construct engineered yeast with synthetic biology method.The key enzymes ?-carotene hydroxylase encoding gene CrtZ were integrated in the chromosomal,the zeaxanthin cleavage dioxygenase encoding gene CCD and the aldehyde dehydrogenase encoding gene ALD were also introduced in the form of plasmids.Since yeast endogenous containing aldehyde dehydrogenase,so co-expression of eight sources CrtZ and four sources CCD at frist.As the result,the strain caring CrtZ from Erwinia uredovora and CCD from Crocus sativus L.achieved the highest titer of crocetin(231?g/L).And then the crocetin yeild increased 1.88-fold(from 231?g/L to 434?g/L)through introducing the plasmid containing ALD from Synechocystis sp.PCC6803.In order to further improve the yield of crocetin,the precursor zeaxanthin yield was improved through knocking out competition pathway gene and increasing the CrtZ gene expression level.The conversion from farnesyl pyrophosphate(an important precursor for terpenoid natural products)to farnesol was blocked by knocking out gene Lpp1 and Dpp1,providing more precursors for zeaxanthin synthesis.The zeaxanthin yield increased 1.27-fold accordingly.Furthermore,a titer of zeaxanthin was achieved as 96.2 mg/L in shake-flask through increasing the CrtZ gene copy number and regulating the its promoter's activity,then the crocetin yeild increased to 1.17mg/L by introducing the multicopy plasmid caring CCD from Crocus sativus L.,which is the highest reported titer in heterologous synthesis crocetin.