BackgroundArtemisinin 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 findingsBy 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/ significanceProduction 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.

The study of protein-protein interactions is becoming increasingly important for understanding the regulation of many cellular processes. The ability to quantify the strength with which two binding partners interact is desirable but the accurate determination of equilibrium binding constants is a difficult process. The use of Luminescence Resonance Energy Transfer (LRET) provides a homogeneous binding assay that can be used for the detection of protein-protein interactions. Previously, we developed an LRET assay to screen for small molecule inhibitors of the interaction of sigma 70 with the beta' coiled-coil fragment (amino acids 100-309). Here we describe an LRET binding assay used to monitor the interaction of E. coli sigma 70 and sigma 32 with core RNA polymerase along with the controls to verify the system. This approach generates fluorescently labeled proteins through the random labeling of lysine residues which enables the use of the LRET assay for proteins for which the creation of single cysteine mutants is not feasible. With the LRET binding assay, we are able to show that the interaction of sigma 70 with core RNAP is much more sensitive to NaCl than to potassium glutamate (KGlu), whereas the sigma 32 interaction with core RNAP is insensitive to both salts even at concentrations >500 mM. We also find that the interaction of sigma 32 with core RNAP is stronger than sigma 70 with core RNAP, under all conditions tested. This work establishes a consistent set of conditions for the comparison of the binding affinities of the E. coli sigma factors with core RNA polymerase. The examination of the importance of salt conditions in the binding of these proteins could have implications in both in vitro assay conditions and in vivo function.

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 series of low-copy expression vectors that permits the stable maintenance and regulated expression of highly toxic gene products has been developed. These vectors utilize the lactose promoter/operator system, and protect against read-through transcription from other promoters on the plasmid by placement of the rrnB T1T2 terminators upstream of the lactose promoter. For additional regulatory control, the vectors utilize low-copy origins of replication. Either the pMPP6 origin (pSC101-derived) is used for cloning into Escherichia coli or related species, or the broad-host-range RK2 origin of replication is utilized for cloning into the majority of Gram-negative bacteria. The resulting plasmids have no detectable leaky expression. To test these vectors, the genes for the bacteriocidal colicins D, E3, and E7 were cloned and stably maintained in the absence of their immunity genes. Upon induction with isopropyl-β-d-thiogalactopyranoside (IPTG), cell death was observed, indicating expression of each colicin. These low-copy expression vectors will be useful for the cloning and expression of toxic genes in bacterial systems.

A new Escherichia coli host/vector system has been engineered to allow tight and uniform modulation of gene expression and gamma origin (ori) plasmid copy number. Regulation of gamma ori plasmid copy number is achieved through arabinose-inducible expression of the necessary Rep protein, pi, whose gene was integrated into the chromosome of the host strain under control of the P(BAD) promoter. gamma ori replication can be uniformly modulated over 100-fold by changing the concentration of l-arabinose in the growth medium. This strain avoids the problem of all-or-nothing induction of P(BAD) because it is deficient in both arabinose uptake and degradation genes. Arabinose enters the cell by a mutant LacY transporter, LacYA177C, which is expressed from the host chromosome. Although this strain could be compatible with any gamma ori plasmid, we describe the utility of a gamma ori expression vector that allows especially tight regulation of gene expression. With this host/vector system, it is possible to independently modulate gene expression and gene dosage, facilitating the cloning and overproduction of toxic gene products. We describe the successful use of this system for cloning a highly potent toxin, Colicin E3, in the absence of its cognate immunity protein. This system could be useful for cloning genes encoding other potent toxins, screening libraries for potential toxins, and maintaining any gamma ori vector at precise copy levels in a cell.

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.

ABSTRACT 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 σ 70 both in vitro and in vivo. However, it was not known if the coiled-coil conformation was maintained upon binding to σ 70 . 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 σ 70 . We also show that the locked coiled-coil is capable of inducing a conformational change within σ 70 that allows recognition of the −10 nontemplate strand of DNA. This suggests that the coiled-coil does not adopt a new conformation upon binding σ 70 or upon recognition of the −10 nontemplate strand of DNA.

Prokaryotic RNA polymerase holoenzyme is composed of core subunits (alpha(2)betabeta'omega) plus a sigma factor that confers promoter specificity allowing for regulation of gene expression. Holoenzyme is known to undergo several conformational changes during the multiple steps of transcription initiation. However, the effects of these changes on the functions of specific regions have not been well characterized. In this work, we addressed the role of possible conformational change in region 2 of Escherichia coli sigma(70) by engineering disulfide bonds that "lock" region 2.1 with region 2.2 and region 2.2 with region 2.3. When these mutant holoenzymes were characterized for gross defects in multiple-round transcription, we found that insertion of either disulfide bond did not result in a fundamental block, indicating that the disulfide-containing holoenzymes are active. However, both disulfide-containing holoenzymes exhibited defects in formation and stability of the open complex. Our results suggest that conformational flexibility within sigma(70) region 2 facilitates open complex formation and transcription initiation.

Fluorescence labeling of proteins has become increasingly important since fluorescent techniques like FRET and fluorescence polarization are now commonly used in protein binding studies, proteomics, and for high-throughput screening in drug discovery. In our efforts to study the binding of the β′-subunit from Escherichia coli RNA polymerase (RNAP) to σ70, we synthesized a fluorescent-labeled β′-fragment (residues 100–309) in a very convenient way, that could be used as a general protocol for hexahistidine-tagged proteins. By performing all the following steps, purification, reduction, derivatization with IC5–maleimide, and free dye removal while the protein was bound to the column, we were able to reduce the procedure time significantly and at the same time achieve better labeling efficiency and quality. The β′-fragment with a N-terminal His6-tag was purified from inclusion bodies and could be refolded prior to or after binding to a Ni–NTA affinity column. Reduction prior to labeling was achieved with TCEP that does not interfere with Ni–NTA chemistry. The labeled β′-fragment was tested with σ70 that was labeled with an europium-based fluorophore for binding in a electrophoretic mobility-shift assay. The sigma-to-core protein interaction in bacterial RNA polymerase offers a potentially specific target for drug discovery, since it is highly conserved among the eubacteria, but differs significantly from eukaryotes.

It is clear that multiple sites of interaction exist between sigmas and core subunits, likely reflecting the changing pattern of interactions that occur sequentially during the complex process of holoenzyme formation, open promoter formation, and initiation of transcription. Recent studies have revealed that a major site of interaction of Escherichia coli sigma factors is the amino acid 260-309 coiled-coil region of the beta' subunit of core RNA polymerase. This region of beta' interacts with region 2.1-2.2 of sigma(70). Binding of this region of beta' to sigma(70) triggers a conformational change in sigma that allows it to bind to a -10 nontemplate promoter DNA strand oligonucleotide.

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 σ70 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 σ70 promotes an allosteric transition that allows σ70 to selectively recognize the nontemplate strand. As the β′ 262–309 peptide can function with the previously crystallized portion of σ70, nontemplate recognition can be reconstituted with only 47 kDa, or 1/10 of holoenzyme.

eubacteria, the a 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 beta' subunit that interacts with sigma(70) in vitro. This region of beta' 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 beta', to look at disruption of the sigma(70)-core interaction, Several of the mutants mere defective for binding sigma(70) in vitro. Of these mutants, three (R275Q, E295K, and A302D) caused cells to be inviable in an in vivo assay in which the mutant beta' is the sole source of beta' subunit for the cell. All of the mutants were able to assemble into the core enzyme; however, R275Q, E295K, A302D were defective for E sigma(70) holoenzyme formation. Several of the mutants were also defective for holoenzyme assembly with various minor a factors. In the recently published crystal structure of Thermus aquaticus core RNA polymerase (Zhang, G., Campbell, E. k, Minakhin, L,, Richter, C,, Severinov,,, and Darst, S, A. (1999) Cell 98, 811-824), the region homologous to beta'(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.

Bacillus subtilis core RNA polymerase, containing a His6-fusion to the C-terminus of the β′ subunit, was isolated by Ni–NTA, Superdex 200 gel filtration, and Mono Q anion-exchange chromatography. The purified core enzyme was shown to be free of the major sigma factor ςA and the transcription factors NusA and GreA. The purification procedure can be completed within 1 working day, is scalable, and yields highly purified and active core RNA polymerase.

In eubacteria, the final sigma 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 beta' subunit that interacts with final sigma(70) in vitro. This region of beta' 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 beta', to look at disruption of the final sigma(70)-core interaction. Several of the mutants were defective for binding final sigma(70) in vitro. Of these mutants, three (R275Q, E295K, and A302D) caused cells to be inviable in an in vivo assay in which the mutant beta' is the sole source of beta' subunit for the cell. All of the mutants were able to assemble into the core enzyme; however, R275Q, E295K, A302D were defective for Efinal sigma(70) holoenzyme formation. Several of the mutants were also defective for holoenzyme assembly with various minor final sigma 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 beta'(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.