Astaxanthin overproduction in yeast by strain engineering and new gene target uncovering

Background: Astaxanthin is a natural carotenoid pigment with tremendous antioxidant activity and great commercial value. Microbial production of astaxanthin via metabolic engineering has become a promising alternative. Although great efforts have been conducted by tuning the heterologous modules and precursor pools, the astaxanthin yields in these non-carotenogenic microorganisms were still unsatisfactory for commercialization, indicating that in addition to targeted tailoring limited targets guided by rationally metabolic design, combining more globe disturbances in astaxanthin biosynthesis system and uncovering new molecular mechanisms seem to be much more crucial for further development. Since combined metabolic engineering with mutagenesis by screening is a powerful tool to achieve more global variations and even uncover more molecular targets, this study would apply a comprehensive approach integrating heterologous module engineering and mutagenesis by atmospheric and room temperature plasma (ARTP) to promote astaxanthin production in Saccharomyces cerevisiae. Results: Here, compared to the strain with beta-carotene hydroxylase (CrtZ) from Alcaligenes sp. strain PC-1, involving new CrtZ from Agrobacterium aurantiacum enhanced astaxanthin yield to 1.78-fold and increased astaxanthin ratio to 88.7% (from 66.6%). Astaxanthin yield was further increased by 0.83-fold (to 10.1 mg/g DCW) via ARTP mutagenesis, which is the highest reported yield at shake-flask level in yeast so far. Three underlying molecular targets (CSS1, YBR012W-B and DAN4) associated with astaxanthin biosynthesis were first uncovered by comparative genomics analysis. To be noted, individual deletion of CSS1 can recover 75.6% improvement on astaxanthin yield achieved by ARTP mutagenesis, indicating CSS1 was a very promising molecular target for further development. Eventually, 217.9 mg/L astaxanthin (astaxanthin ratio was 89.4% and astaxanthin yield was up to 13.8 mg/g DCW) was obtained in 5-L fermenter without any addition of inducers. Conclusions: Through integrating rational engineering of pathway modules and random mutagenesis of hosts efficiently, our report stepwise promoted astaxanthin yield to achieve the highest reported one in yeast so far. This work not only breaks the upper ceiling of astaxanthin production in yeast, but also fulfills the underlying molecular targets pools with regard to isoprenoid microbial overproductions.