Figure 3.

“PCR & Go” system for the construction of yeast cell factories for β-carotene, zeaxanthin, and astaxanthin. (A) Overview of the carotenoid biosynthetic pathway. CrtE: geranylgeranyl diphosphate synthase; CrtYB: phytoene synthase/lycopene cyclase; CrtI: phytoene desaturase; CrtZ: β-carotene hydroxylase; CrtW: β-carotene ketolase. Multiple arrows indicate multiple reaction steps. (B) Integration efficiency of the three-gene, four-gene, and five-gene pathways using the “PCR & Go” system. 1.5 μg of the PCR amplified gene fragments with 40 bp homology arms together with the corresponding gRNA plasmid(s) were co-transformed into the expression chassis and the integration efficiency was calculated based on both genotypic (diagnostic PCR) and phenotypic (pigment formation) results. The integration efficiency of the β-carotene pathway was calculated as the percentage of pigmented colonies (number of orange colonies/total number of colonies on the agar plate). The integration efficiency of the astaxanthin pathway was calculated as the number of colonies with correct PCR patterns for five genes/total number of colonies on the agar plate. (C) Genotyping of the nine colonies in red on the astaxanthin agar plate. Diagnostic PCR results for the yellow colonies and white colony were provided in Supplementary Figure 2. 1, 2, 3, 4, 5 denoted CrtE, CrtI, CrtYB, CrtW, and CrtZ integrated into the expression chassis, respectively.