Jack D. Newman

High-level production of amorpha-4,11-diene, a precursor of the antimalarial agent artemisinin, in Escherichia coli.

Background Artemisinin derivatives are the key active ingredients in Artemisinin combination therapies (ACTs), the most effective therapies available for treatment of malaria. Because the raw material is extracted from plants with long growing seasons, artemisinin is often in short supply, and fermentation would be an attractive alternative production method to supplement the plant source. Previous work showed that high levels of amorpha-4,11-diene, an artemisinin precursor, can be made in Escherichia coli using a heterologous mevalonate pathway derived from yeast (Saccharomyces cerevisiae), though the reconstructed mevalonate pathway was limited at a particular enzymatic step. Methodology/ Principal Findings By combining improvements in the heterologous mevalonate pathway with a superior fermentation process, commercially relevant titers were achieved in fed-batch fermentations. Yeast genes for HMG-CoA synthase and HMG-CoA reductase (the second and third enzymes in the pathway) were replaced with equivalent genes from Staphylococcus aureus, more than doubling production. Amorpha-4,11-diene titers were further increased by optimizing nitrogen delivery in the fermentation process. Successful cultivation of the improved strain under carbon and nitrogen restriction consistently yielded 90 g/L dry cell weight and an average titer of 27.4 g/L amorpha-4,11-diene. Conclusions/ Significance Production of >25 g/L amorpha-4,11-diene by fermentation followed by chemical conversion to artemisinin may allow for development of a process to provide an alternative source of artemisinin to be incorporated into ACTs.

Optimization of the mevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene.

The introduction or creation of metabolic pathways in microbial hosts has allowed for the production of complex chemicals of therapeutic and industrial importance. However, these pathways rarely function optimally when first introduced into the host organism and can often deleteriously affect host growth, resulting in suboptimal yields of the desired product. Common methods used to improve production from engineered biosynthetic pathways include optimizing codon usage, enhancing production of rate-limiting enzymes, and eliminating the accumulation of toxic intermediates or byproducts to improve cell growth. We have employed these techniques to improve production of amorpha-4,11-diene (amorphadiene), a precursor to the anti-malarial compound artemisinin, by an engineered strain of Escherichia coli. First we developed a simple cloning system for expression of the amorphadiene biosynthetic pathway in E. coli, which enabled the identification of two rate-limiting enzymes (mevalonate kinase (MK) and amorphadiene synthase (ADS)). By optimizing promoter strength to balance expression of the encoding genes we alleviated two pathway bottlenecks and improved production five fold. When expression of these genes was further increased by modifying plasmid copy numbers, a seven-fold increase in amorphadiene production over that from the original strain was observed. The methods demonstrated here are applicable for identifying and eliminating rate-limiting steps in other constructed biosynthetic pathways.

Activity, structure, and stratification of membrane-attached methanotrophic biofilms cometabolically degrading trichloroethylene

A membrane-attached methanotrophic biofilm reactor was developed for the cometabolic degradation of trichloroethylene (TCE). In this reactor, CH4 and O2 are supplied to the interior of the biofilm through the membrane, while TCE-contaminated water is supplied to the exterior, creating a “counter-diffusional” effect that minimizes competitive inhibition between TCE and CH4. In addition, this novel design provides 100% CH4 and O2 transfer efficiencies, promotes the development of a thick biofilm, and minimizes the negative effects of TCE byproduct toxicity. The reactor sustained 80-90% TCE removals at TCE loading rates ranging from 100-320 μmol/m2/d. Chloride mass balances demonstrated that 60-80% of the TCE removed was mineralized. The maximum TCE transformation yield was 1.8 mmol of TCE removed per mole of CH4 utilized, although higher transformation yields are expected at higher TCE loading rates. The CH4 utilization rate was 0.20 mol/m2/d. Scanning electron microscopy (SEM) revealed a dense biofilm with a thickness of at least 400 μm. SEM and transmission electron microscopy (TEM) analyses indicated that the “holdfast” material associated with rosette formation in planktonic Methylosinus trichosporium OB3b ( M.t. OB3b) cells might also contribute to pure-culture biofilm development. In addition, fimbriae-like structures not commonly associated with methanotrophic bacteria were observed in pure-culture M.t. OB3b biofilms. Finally, fluorescent in situ hybridization (FISH) analyses showed the presence of discrete microcolonies of serine-pathway methanotrophs within mixed-culture biofilms.