The yeast Saccharomyces cerevisiae serves as a promising host for the production of a wide range of chemical compounds and fuels. Currently, simultaneous expression of several genes could be achieved via the use of 2A viral peptides, yet detailed characterizations to assess the discrepancy of different orders of genes linked by 2A peptides are rarely sufficient. In this study, we investigated the effects of the order of genes linked by porcine teschovirus-1 2A (P2A) peptide on the metabolic pathway in S. cerevisiae. A heterologous carotenoid biosynthetic system involving nine kinds of polycistronic expression of codon-optimized carotenogenic genes GGPPS, CARB and CARRP from Blakeslea trispora was introduced into S. cerevisiae. The order of genes in the polycistronic segment was exchanged; β-carotene production by engineered yeasts was significantly different. The highest β-carotene yield was achieved in transformants carrying the plasmid, with CARB as the first gene in the polycistronic construct regardless of the location of GGPPS, CARRP. In addition, we found that β-carotene production was coupled with the growth in engineered strain with the highest β-carotene content during the shake flask fermentation and fed-batch fermentation. A novel microbial heterologous carotenoid production system was established by optimizing the order of genes linked by P2A peptide sequences in a polycistronic expression construct. The observation of the importance of the order in a polycistronic construct may be used to increase yields in other P2A peptide-containing expression systems.
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