pESC-Trp vector

Beta-carotene is the precursor of vitamin A and itself has various medicinal functions. Compared with chemical synthesis, the production of β-carotene in microorganisms through metabolic engineering strategies is relatively cheap. Identification of genes that enhance β-carotene production in microorganisms is important for engineering strains that produce higher β-carotene production. Most previous efforts in identifying gene targets have focused on the isoprenoid pathway to which β-carotene biosynthesis belongs. However, seemingly unrelated genes outside the isoprenoid pathway may also influence β-carotene biosynthesis due to complex interactions between metabolic fluxes.

In this study, the laboratory yeast strain WAT11 was used as the host to construct a recombinant β-carotene production strain by integrating the carotenoid pathway genes derived from Xanthomonas dendriticum into its genome. The resulting β-carotene producing strain was then transformed with the Saccharomyces cerevisiae cDNA library. By screening colony color and then measuring β-carotene production, researchers here report several new amplification targets that significantly increase β-carotene production in Saccharomyces cerevisiae. Among these targets, class E proteins of the vacuolar protein sorting pathway (Did2) led to the highest improvement.

In this study, a yeast cDNA library was first screened by examining the colony color phenotype, and six candidate genes (CAR1, DID2, PDC5, BMH1, TIF5, and VOA1) were screened out. To examine whether the positive cDNA insert truly enhanced β-carotene production in S. cerevisiae, the ORF of the cDNA candidate was amplified and individually inserted into the yeast expression vector pESC-TRP under the control of a galactose-inducible promoter, yielding the constructs of pESC-TRP-BMH1, pESC-TRP-CAR1, pESC-TRP-DID2, pESC-TRP-PDC5, pESC-TRP-TIF5, and pESC-TRP-VOA1. To examine whether the above positive cDNA inserts truly enhance β-carotene production in S. cerevisiae, the ORFs of the cDNA candidates were amplified and individually inserted into the yeast expression vector pESC-TRP under the control of a galactose inducible promoter, yielding the constructs of pESC-TRP-BMH1, pESC-TRP-CAR1, pESC-TRP-DID2, pESC-TRP-PDC5, pESC-TRP-TIF5, and pESC-TRP-VOA1.

Figure 1. Production of β-carotene by the recombinant S. cerevisiae strains. WYIB, a WAT11 yeast strain integrating the β-carotene biosynthetic pathway. The WYIB strain was transformed with the empty vector pESC-TRP to obtain the control strain WYIB-TRP, and transformed with the target genes to generate recombinant yeast strains (WYIB-BMH1, WYIB-CAR1, WYIB-DID2, WYIB-PDC5, WYIB-TIF5 and WYIB- VOA1). (Li J, et al. 2017)