The Construction And Optimization Of High Astaxanthin Producing Saccharomyces Cerevisiae

Astaxanthin(3,3'-dihydroxy-4,4'-keto-?,??-carotene)is a natural carotenoid pigment.Due to its powerful antioxidant activity and the ability of quenching singlet oxygen,astaxanthin can prolong life,keep skin healthy,boost the immune system,reduce the risk of cardiovascular disease,thus have the potential market value in the field of food,cosmetic and pharmaceutical industries.Therefore,the research of astaxanthin synthesis and production in engineering strain by using synthetic biology has important significance.The tendency of reaction process in astaxanthin biosynthesis catalyzed by hydroxylase(CrtZ)and ketolase(CrtW)decides the number and quantity of accumulated intermediate metabolites,thus the key to reduce the reticular metabolism network and intermediates accumulation of astaxanthin synthesis process is to solve metabolic balance between the hydroxylase and ketolase.In this study,?-carotene of astaxanthin direct precursor was obtained through ? site integration of ?-carotene synthesis gene,then the strain(SyBE_Sc118012)with highest titer was selected for integrating one copy of thmgr and fusion gene bts1-erg20 at endogenous ypl062 w gene site to increase the supply of precursors and the production of ?-carotene was improved by 1.65 fold,up to 162.1 mg/L in engineered Saccharomyces cerevisiae SyBE_Sc118030.On this basis,through attempting a combination of different sources of hydroxylase and ketolase and adjusting relative expression intensity between the hydroxylase and ketolase,the high astaxanthin producing Saccharomyces cerevisiae SyBE_Sc118076 was obtained with the titer of 28.25 mg/L(4.4 mg/g DCW).The production of astaxanthin was improved by 1.25 fold in 5L fermentor by the optimization of fed batch fermentation,up to 53.5 mg/L.Therefore,this study screened a combination of different sources of hydroxylase and ketolase gene suitable for chassis to increase the production of astaxanthin and reduce the accumulation of metabolic intermediates,has a certain reference value for the optimization of astaxanthin metabolic pathway.