Abstract
A genetic screen to identify mutants that can increase flux in the isoprenoid pathway of yeast has been lacking. We describe a carotenoid-based visual screen built with the core carotenogenic enzymes from the red yeast Rhodosporidium toruloides. Enzymes from this yeast displayed the required, higher capacity in the carotenoid pathway. The development also included the identification of the metabolic bottlenecks, primarily phytoene dehydrogenase, that was subjected to a directed evolution strategy to yield more active mutants. To further limit phytoene pools, a less efficient version of GGPP synthase was employed. The screen was validated with a known flux increasing gene, tHMG1. New mutants in the TATA binding protein SPT15 were isolated using this screen that increased the yield of carotenoids, and an alternate isoprenoid, α-Farnesene confirming increase in overall flux. The findings indicate the presence of previously unknown links to the isoprenoid pathway that can be uncovered using this screen.
Highlights
Isoprenoids or terpenoids represent the largest class of natural products with more than 40,000 known structures (Bohlmann and Keeling, 2008)
Based on the recently released genome sequences of Rhodosporidium toruloides by multiple groups (Kumar et al, 2012; Zhu et al, 2012) we identified and carried out codon-optimised expression of the genes for the core biosynthetic carotenogenic enzymes upto β-carotene from R. toruloides into S. cerevisiae
The genes for Geranylgeranyl diphosphate (GGPP) synthase (RtGGPPS), Phytoene synthase (RtPSY1) and Phytoene dehydrogenase (RtCRTI) of R. toruloides were codon optimised by using EnCor Biotechnology Inc. software and custom synthesised by GenScript USA
Summary
Isoprenoids or terpenoids represent the largest class of natural products with more than 40,000 known structures (Bohlmann and Keeling, 2008) Many of these terpenoids are of immense commercial value. As carotenoids are coloured compounds, their production by yeast cells provides a good visual phenotype, and this has been extensively exploited in the past (Mitchell et al, 2015; Schmidt-Dannert et al, 2000; Wang et al, 2009; Xie et al, 2014). Surprisingly, despite their extensive use in a variety of different screens and assays, their development as a measure of isoprenoid flux has remained unsuccessful so far
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