(235c) Biosynthesis of the Antimalarial Drug FR-900098 in E. Coli

Antibiotics containing C-P bonds, phosphonates and phosphinates, are a potent group of bioactive compounds. The phosphonic acid FR-900098 is a novel chemotherapeutic agent for the treatment of malaria and has been shown to be effective in humans and other animals. This compound inhibits 1-deoxy-D-xylulose 5-phosphate (DXP) reductoisomerase, the first committed enzyme in the nonmevalonate pathway for isoprenoid biosynthesis in many bacteria as well as Plasmodium falciparum, the parasite responsible for the most virulent form of the disease. The biosynthetic cluster for FR-900098 has recently been cloned from Streptomyces rubellomurinus and heterologously expressed in Streptomyces lividans and E. coli. Whole cell feeding experiments and in vitro assays with purified enzymes were used to verify each of the steps in the biosynthetic pathway. These studies revealed a route for production of a second phosphonate antibiotic, FR-33289, whose potency in malaria treatment was also investigated. Here we present metabolic engineering strategies towards improving the production of FR-900098 in E. coli to industrially desirable levels. These strategies include (1) using metabolic flux analysis to identify the accumulating intermediates which limit the overall yield of the compound, (2) increasing the precursor supply by gene knockouts and over-expression and (3) balancing gene expression levels by optimizing promoter strengths.