Engineering Saccharomyces Cerevisiae for Prostratin Production

The need for effective
anti-HIV drugs is urgent. Over 34 million people worldwide are infected with
HIV, many of whom live in countries where medicine is inaccessible due to cost.
Prostratin, a natural product derived from Homalanthus nutans, a small rainforest tree grown in Samoa,
is being actively developed as an adjuvant therapy to clear latent viral
reservoirs, the primary obstacle to eradication of HIV. Currently, prostratin
is produced via a semisynthetic route. However, such processes are inefficient
and costly; the development of a microbial production system promises to
significantly reduce the cost of prostratin. The
biosynthetic pathway of prostratin begins with the
non-mevalonate pathway, after which prenyltransferases and a diterpene
synthase produce casbene, a major precursor to prostratin. The biosynthetic steps in the prostratin pathway after casbene
are unknown, although several groups have postulated that P450s catalyze the
ring closures seen in prostratin. A Saccharomyces cerevisiae
strain was constructed with an upregulated isoprenoid pathway for production of the diterpene casbene at titers of up
to 500mg/L, a 16-fold increase from the highest previously reported titers. Using
coexpression analysis and comparative genomics on the
Euphorbiaceae species Homalanthus nutans, Jatropha curcas, Euphorbia resinifera, and Euphorbia fischeriana,
we found P450 oxidative enzymes that modify casbene. The
resulting P450s were integrated into the casbene
overproducing yeast strain using CRISPR-Cas genome
engineering, resulting in the production of oxidized casbanes,
which are important intermediates in the prostratin
pathway. This result is a significant step towards the development of an
industrial microbial system for the production of prostratin.