Enhanced astaxanthin production in S. cerevisiae by combinatorial engineering of gene targets outside the synthetic pathway

In recent years, microbial synthesis of astaxanthin with strong antioxidant activity has received widespread attention. In addition to the extensively engineered genes within the carotenogenic pathway, certain genes outside this pathway have also been found to influence carotenoid production. To investigate the combined effect of these gene targets, we first engineered a previously constructed β-carotene-producing S. cerevisiae strain using the tri-functional CRISPR system to simultaneously upregulate, downregulate or delete these targets, selecting out an optimal strain producing 53.71 mg/g DCW of β-carotene. With sufficient precursor supply, the astaxanthin synthetic pathway was then constructed by introducing and balancing the β-carotene hydroxylase and ketolase. Further deletion of the lipid droplet-associated gene HRD1 and conditional downregulation of OPI3, as well as overexpression of the pleiotropic drug resistance transcription factor Pdr3p, generated a final strain with high astaxanthin yield, reaching 12.26 mg/g DCW. This work underscores the significant, albeit not entirely elucidated, roles played by genes beyond the target synthetic pathway, along with their interactions in regulating carotenoid biosynthesis. The findings suggest that not only the genes in the pathway but also those outside the pathway should be considered targets during strain engineering.