Metabolic Engincering Of Saccharomyces Cerevisiae For Lutein Biosynthesis

Lutein,as a carotenoid with strong antioxidant capacity and an important component of macular pigment in the retina,has wide applications in pharmaceutical,food,feed and cosmetics industries.In this study,aiming to explore fermentative production of lutein,lutein-producing Saccharomyces cerevisiae strains were constructed by enzyme mining,protein engineering and dynamic regulation.First of ail,according to the natural biosynthetic pathway of lutein,codon-optimized lycopene ε-cyclase from Tagetes erecta and β-cyclase from Xanthophyllomyces dendrorhous were synthesized and expressed in a previously constructed lycopeneproducing S.cerevisiae strain.It turned out that the activity of β-cyclase(CrtYB)was far higher than ε-cyclase(OmLCYE).leading to failure to synthesize α-carotene as the key precursor for lutein.By introducing the temperature-sensitive regulatory protein Gal4M9 and placing CrtYB under the GAL promoter,the expression of CrtYB was controlled by temperature.Meanwhile,the other genes in the pathway were all constitutively expressed.In this way,OmLCYE and CrtYB were expressed in order,leading to successful biosyntheis of α-carotene.By subcellular recolization of OmLCYE via fusing with a cell membrane anchor and directed evolution of this enzyme towards improved catalytic performance adopting a color-based highthroughput screening method,the ratio of α-carotene/β-carotene was increased to 1:1.5,and the production of α-carotene in the resulting strain YTA08(PMSeV-C-OmLCYEF61N)reached 294.3μg/L,laying the foundation for lutein synthesis.In order to construct the complete lutein synthesis pathway in yeast,carotene hydroxylases were screened.By extending the heterologous synthetic pathway in strain YTA08 by introducing the carotene β-hydroxylase(CYP97A3)and ε-hydroxylase(LUT1)from Arabidopsis thaliana with high substrate specificity and catalytic activity,lutein was detected.During this process,the order of lutein synthesis in S.cerevisiae was determined as following:α-carotene was catalyzed by CYP97A3 to form zeinoxanthin,and then under the action of LUT1 to produce lutein.Through overexpression of the rate-limiting enzyme CYP97A3,the production of lutein was improved to 595.3 μg/L(438 μg/g DCW),and 538.8 μg/L of α-carotene was accumulated.These results indicated the metabolic flux was mainly shifted to the abranch.Due to the low activity of OmLCYE.it was expressed on a high-copy episomal plasmid,which leads to the issue of genetic stability.To address this issue,a luteinproducing strain with good stability was constructed by screening and genomic integration of lycopene ε-cyclase with higher catalytic activity(At-LCYE from Arabidopsis thaliana).Meanwhile,the reductases for the carotene hydroxylases were screened and optimized.When RFNR1 and FD3 were used as the reductases,the catalytic activity of the carotene hydroxylases was the highest.In order to further increase the production of lutein,balanced expression of lycopene ε-cyclase,caroteneβ-hydroxylase and its reductase was explored.The final strain YTL-AE-07 expressing two copies of At-LCYE,three copies of CYP97A3 and one copy of RFNR1 and FD3 produced 6.91 mg/L(1.88 mg/g DCW)of lutein in shake-flask clutures,and the yield reached 28.27 mg/L in high-density fermentation,which is the highest lutein yield by engineered strains.In strain YTL-AE-07.negative effect of early carotenoids accumulation on cell growth was observed.To solve the conflict between growth and intermediate accumulation,a dynamic regulation system using glucose concentration and temperature as dual signals was constructed by combining glucose-responsive promoter and temperature regulation system.After confirming the strict glucose response of PADH2,it was used to replace the constitutive promoters controlling the δ-carotene synthetic pathway in strain YTL-AE-07.In combination with the temperature-regulatedδ-carotene transformation,a multi-level regulated lutein-producing yeast strain was successfully constructed.Using this system,cell growth,accumulation of the key intermediate δ-carotene,and synthesis of the final product lutein could be divided into three stages.The lutein production of the resulting strain reached 19.92 mg/L(4.53 mg/g DCW)in shake-flask cultures,which was much higher than that of the temperature-regulated strain YTL-AE-07 and close to that of the algae naturally producing lutein.The three-stage dynamic control system simultaneously solved the two key issues including the conflict between cell growth and product accumulation.and intra-pathway competition for common precursor.This work would lay a foundation for the fermentative production of lutein.Meanwhile,the two-stage sequential control and three-stage dynamic control strategies developed in this study would provide reference for the regulation of heterologous synthesis of other natural products.