Isoprenoids or terpenoids are naturally occurring, biologically significant diverse hydrocarbons derived from five-carbon isoprene units, many of which are of high value. However, the majority of the commercially important isoprenoids derive from plants [1]. As there are difficulties in obtaining high yields from plant sources, efforts are ongoing to develop microbial hosts reconstituted with these pathways and devise methods to increase the carbon flux into these pathways. Among the various hosts, Saccharomyces cerevisiae, an eukaryote, often allows heterologous enzymes to fold and function better in yeast compared to bacteria [2]. These organisms, however, do not natively produce high levels of isoprenoids and might not have evolved to cater to carrying a high flux of isoprenoids. One way of approaching this limitation is to develop carotenogenic or oleaginous yeasts that might have higher flux in these pathways as alternate hosts. However, these organisms are still challenging to manipulate, and the tools are not yet fully developed [3], [4]. An alternative way around the problem is to use enzymes from these organisms in S. cerevisiae and improve them.

The red yeast Rhodosporidium toruloides is an oleaginous yeast that is also the highest-known carotenoid producer among yeasts [3]. The possibility that the mevalonate pathway enzymes from this yeast might have been uniquely involved in carry a higher flux than the non-oleaginous, non-carotenoid-producing S. cerevisiae exists. With the genome sequence available from this yeast [5], [6], the possibility exists to investigate the mevalonate pathway enzymes of R. toruloides and examine if some of the key enzymes of this pathway might be better sources for synthetic biology applications.

Although sporadic reports have used enzymes from different carotenogenic yeasts [7], [8], [9], a systematic analysis and comparison have not yet been carried out. Therefore, in this study, we initiated a more systematic investigation of the mevalonate pathway genes of R. toruloides. The mevalonate (MVA) pathway genes were analyzed, and we custom synthesized some of the key enzymes in the pathway. During the initial evaluation, we observed two genes of R. toruloides, GGPPS and HMG-CoA reductase, as candidates that possibly merited further investigation. This study describes their characterization and our further efforts to improve these enzymes. GGPPS of R. toruloides was shown to have better catalytic activity than ScGGPPS, but additional mutants isolated did not show improved activity. In the case of RtHMG, we observed that the stability in S. cerevisiae could be enhanced by a specific mutant that could be advantageous for synthetic biology applications.