Larry C. Anthony

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.

A Coiled-Coil from the RNA Polymerase β′ Subunit Allosterically Induces Selective Nontemplate Strand Binding by σ70

Abstract For transcription to initiate, RNA polymerase must recognize and melt promoters. Selective binding to the nontemplate strand of the −10 region of the promoter is central to this process. We show that a 48 amino acid (aa) coiled-coil from the β′ subunit (aa 262–309) induces σ 70 to perform this function almost as efficiently as core RNA polymerase itself. We provide evidence that interaction between the β′ coiled-coil and region 2.2 of σ 70 promotes an allosteric transition that allows σ 70 to selectively recognize the nontemplate strand. As the β′ 262–309 peptide can function with the previously crystallized portion of σ 70 , nontemplate recognition can be reconstituted with only 47 kDa, or 1/10 of holoenzyme.

Mutational Analysis of β′260–309, a ς70 Binding Site Located on Escherichia coliCore RNA Polymerase

In eubacteria, the ς subunit binds to the core RNA polymerase and directs transcription initiation from any of its cognate set of promoters. Previously, our laboratory defined a region of the β′ subunit that interacts with ς70 in vitro. This region of β′ contained heptad repeat motifs indicative of coiled coils. In this work, we used 10 single point mutations of the predicted coiled coils, located within residues 260–309 of β′, to look at disruption of the ς70-core interaction. Several of the mutants were defective for binding ς70 in vitro. Of these mutants, three (R275Q, E295K, and A302D) caused cells to be inviable in an in vivoassay in which the mutant β′ is the sole source of β′ subunit for the cell. All of the mutants were able to assemble into the core enzyme; however, R275Q, E295K, A302D were defective for Eς70 holoenzyme formation. Several of the mutants were also defective for holoenzyme assembly with various minor ς factors. In the recently published crystal structure of Thermus aquaticus core RNA polymerase (Zhang, G., Campbell, E. A., Minakhin, L., Richter, C., Severinov, K., and Darst, S. A. (1999)Cell 98, 811–824), the region homologous to β′260–309 of Escherichia coli forms a coiled coil. Modeling of our mutations onto that coiled coil places the most defective mutations on one face of the coiled coil.

Expression, Purification of, and Monoclonal Antibodies to σ Factors from Escherichia coli

Publisher Summary This chapter explores the escherichia coli DNA-dependent RNA polymerase that is the sole enzyme responsible for the synthesis of messenger, transfer, and ribosomal RNA. The E. Coli housekeeping σ factor, σ 70 , was the first prokaryotic σ factor to be purified and characterized. Since then, six additional σ factors have been found in E. Coli K12. All seven sigma factors have been categorized in two families by means of sequence similarity. In addition to unique spacing requirements within promoters, each sigma factor recognizes specific promoter sequences, allowing E. coli the means to regulate gene expression. The major sigma factors of E. coli can be purified using the basic principles of overexpression, isolation of inclusion bodies, denaturation and protein refolding, and purification over an ion-exchange column. Their significant negative charge at physiological pH allows the purification on anion-exchange resin such as PorosHQ or Mono Q resins. The chapter reviews protocols describing the purification of these sigma factors that have been published previously. Therefore, the purification protocols described in this chapter merely summarize, and refine the established protocols using the most current chromatography techniques.

Using Disulfide Bond Engineering To Study Conformational Changes in the β′260-309 Coiled-Coil Region of Escherichia coli RNA Polymerase during σ70 Binding

RNA polymerase of Escherichia coli is the sole enzyme responsible for mRNA synthesis in the cell. Upon binding of a sigma factor, the holoenzyme can direct transcription from specific promoter sequences. We have previously defined a region of the β′ subunit (β′260-309, amino acids 260 to 309) which adopts a coiled-coil conformation shown to interact with σ70 both in vitro and in vivo. However, it was not known if the coiled-coil conformation was maintained upon binding to σ70. In this work, we engineered a disulfide bond within β′240-309 that locks the β′ coiled-coil region in the coiled-coil conformation, and we show that this “locked” peptide is able to bind to σ70. We also show that the locked coiled-coil is capable of inducing a conformational change within σ70 that allows recognition of the −10 nontemplate strand of DNA. This suggests that the coiled-coil does not adopt a new conformation upon binding σ70 or upon recognition of the −10 nontemplate strand of DNA.