Metabolic Regulation Of Astaxanthin Biosynthesis In Saccharomvces Cerevisiae

As an important carotenoid with strong antioxidant activity,astaxanthin,in particular its 3S,3’S isomer,has wide applications in aquaculture,pharmaceutical,food and cosmetic industries.In this study,to improve the productivity of astaxanthin and to lower the production costs,a Saccharomyce cerevisiae cell factory for fermentative production of astaxanthin was developed by means of pathway construction,protein engineering and dynamic metabolic regulation.Firstly,the β-carotene ketolase and hydroxylase genes(bkt and crtZ)were cloned from H.pluvialis and their expression in S.cerevisiae was improved by codon optimization.Introduction of these two genes into a p-carotene-hyperproducing S.cerevisiae strain generated an astaxanthin producer.The astaxanthin configuration was confirmed to be optically pure(3S,3’S).Furthermore,the substrate selectivity ofβ-carotene hydroxylase and ketolase in astaxanthin biosynthesis was characterized.Subsequently,the copy number of Obkt and OcrtZ genes was adjusted.The strains YPP9 harboring two copies each of Obkt and OcrtZ produced 3.54 mg/g DCW of astaxanthin,while in the strain YPP13 containing three copies each of Obkt and OcrtZ,the production of astaxanthin was about 4.20 mg/g DCW.Elevation of precursor supply and accelerated conversion of P-carotene to astaxanthin are key issues for further enhancement of astaxanthin production.Firstly,a positive mutant of GGPP synthase(CrtE03M)from Xanthophyllomyces dendrorhous was overexpressed leading to 56%enhancement in precursor(β-carotene)accumulation of the resulting strain Ycarot-02,reaching about 16.37 mg/g DCW.Meanwhile,to accelerate the conversion of P-carotene to astaxanthin,the catalytic performance of OBKT was improved via directed evolution adopting a color-based high throughput screening method,generating a positive mutant ObktM.In comparison with those of the control strain harboring the nativeObkt gene,integration of the ObktM gene leading to 50%decrease of β-carotene accumulation and 2.4-fold increase in canthaxanthin production.By combining the expression of CrtE03M,OBKTM and other pathway rate-limiting enzymes with iron cofactor supplementation,the diploid strain YastD-03 produced 8.10 mg/g DCW(47.18 mg/L)astaxanthin.However,the stability of the diploid strain was not satisfactory.In addition,OCrtZ was identified as another key rate-limiting enzyme in this process.Therefore,the method of directed co-evolution of OCrtZ and OBKTM was established,generating mutants OCrtZM1 and OBKTM29,which were then introduced into Ycarot-02 strain.The resulting strain YPP27 accumulated about 5.70 mg/g DCW of astaxanthin,with 38.7%improvement as compared with the strain containing wild-type OCrtZ and OBKTM.Although the astaxanthin production was lower than that of YastD-03,this strain was genetically more stable.However,high-density fermentation failed with the above constructed strains,which could be possibly attributed to the early expression of pathway genes which posed adverse effect on cell growth.Therefore,in order to address the common issue of conflict between cell growth and product accumulation in biosynthesis,we developed a temperature-dependent dynamic control strategy based on GAL regulation system to facilitate decoupling of production from growth.After knocking out GAL80,the transcriptional activator Gal4 plays the sole controlling role in GAL regulation system.Firstly,a temperature-sensitive Gal4 mutant Gal4M9 was created by directed evolution based on a lycopene-indicated high-throughput screening method,and the induction temperature was subsequently optimized to 24℃.Moreover,the performance of this temperature-responsive system was evaluated by fluorescence measurement of the reporter EGFP and transcriptional analysis of the lycopene pathway genes.The sensitivity and stringency of this system was further demonstrated using lycopene as an example product.In high-density fermentation of the temperature-responsive Ylyc-TS03 strain,successful decoupling of growth and production was observed upon temperature shift and the lycopene titer at the end of fermentation was about 1.14 g/L.Finally,this temperature-responsive system was employed for regulation of astaxanthin biosynthesis.The astaxanthin of the resulting strain Yast-TS9 accumulated to about 4.0 mg/g DCW in flask-shaking fermentation.In addition,the biomass reached OD600= 250 and total carotenoids accumulated in a temperature-responsive manner to about 504 mglL after completion of fed-batch fermentation with temperature shift.In summary,by combining strategies of gene mining,gene expression optimization,protein engineering and dynamic metabolic regulation,an efficient astaxanthin-producing Saccharomyces cerevisiae strain was constructed.This study provides a basis for further improving biotechnological production of astaxanthin and provides methodological reference for biosynthesis of other value-added compouds.