The potential of R. toruloides mevalonate pathway genes in increasing isoprenoid yields in S. cerevisiae: Evaluation of GGPPS and HMG-CoA reductase

The enzymes of the mevalonate pathway need to be improved to achieve high yields of isoprenoids in the yeast Saccharomyces cerevisiae. The red yeast Rhodosporidium toruloides produces high levels of carotenoids and may have evolved to carry a naturally high flux of isoprenoids. Enzymes from such yeasts are likely to be promising candidates for improvement. Towards this end, we have systematically investigated the various enzymes of the mevalonate pathway of R. toruloides and custom synthesized, expressed, and evaluated six key enzymes in S. cerevisiae. The two nodal enzymes geranyl pyrophosphate synthase (RtGGPPS) and truncated HMG-CoA reductase (RttHMG) of R. toruloides showed a significant advantage to the cells for isoprenoid production as seen by a visual carotenoid screen. These two were analyzed further, and attempts were also made at further improvement. RtGGPPS was confirmed to be superior to the S. cerevisiae enzyme, as seen from in vitro activity determinations and in vivo production of the heterologous diterpenoid sclareol. Four mutants were created through rational mutagenesis but were unable to improve the activity further. In the case of RttHMG, functional evaluation of the enzyme revealed that it was very unstable despite functioning very well in S. cerevisiae. We succeeded in stabilizing the enzyme through mutation of a conserved serine in the catalytic region, which did not alter the enzyme activity per se. In vivo evaluation of the mutant revealed that it could enable better sclareol yields. Therefore, these two enzymes from the red yeast are excellent candidates for heterologous isoprenoid production.