Novel production of terpenoids in Escherichia coli and activities against breast cancer cell lines.

Metabolic engineering of heterologous pathways has allowed the production of therapeutically important compounds in microbial systems. Here, we report the engineering of a monoterpenoid biosynthetic pathway into Escherichia coli. Five genes encoding sequential enzymes for perillyl alcohol biosynthesis from the precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) were engineered into E. coli. Expression of these genes allowed the production of the intermediate limonene, but the downstream monoterpenoid, perillyl alcohol, was not detected. A new compound was detected but could not be identified based on the data obtained. Only 1.6 µg/ml of the compound was being produced from the engineered E. coli strain, but, when these cultures were fed limonene as a substrate, the production was nearly 250 µg/ml. This unknown compound inhibited the cell proliferation of MCF-7 and MDA-MB-231 breast cancer cells in 48-h treatment experiments. This compound may have potential benefits in breast cancer treatment. This is the first report showing the production of a monoterpenoid in engineered E. coli and its antiproliferative effects in breast cancer cells.

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 conserved structural module regulates transcriptional responses to diverse stress signals in bacteria.

A transcriptional response to singlet oxygen in Rhodobacter sphaeroides is controlled by the group IV sigma factor sigma(E) and its cognate anti-sigma ChrR. Crystal structures of the sigma(E)/ChrR complex reveal a modular, two-domain architecture for ChrR. The ChrR N-terminal anti-sigma domain (ASD) binds a Zn(2+) ion, contacts sigma(E), and is sufficient to inhibit sigma(E)-dependent transcription. The ChrR C-terminal domain adopts a cupin fold, can coordinate an additional Zn(2+), and is required for the transcriptional response to singlet oxygen. Structure-based sequence analyses predict that the ASD defines a common structural fold among predicted group IV anti-sigmas. These ASDs are fused to diverse C-terminal domains that are likely involved in responding to specific environmental signals that control the activity of their cognate sigma factor.

A transcriptional response to singlet oxygen, a toxic byproduct of photosynthesis.

The ability of phototrophs to convert light into biological energy is critical for life on Earth. However, there can be deleterious consequences associated with this bioenergetic conversion, including the production of toxic byproducts. For example, singlet oxygen (1O2) can be formed during photosynthesis by energy transfer from excited triplet-state chlorophyll pigments to O2. By monitoring gene expression and growth in the presence of 1O2, we show that the phototrophic bacterium Rhodobacter sphaeroides mounts a transcriptional response to this reactive oxygen species (ROS) that requires the alternative sigma factor, sigma(E). An increase in sigma(E) activity is seen when cells are exposed to 1O2 generated either by photochemistry within the photosynthetic apparatus or the photosensitizer, methylene blue. Wavelengths of light responsible for the generating triplet-state chlorophyll pigments in the photosynthetic apparatus are sufficient for a sustained increase in sigma(E) activity. Continued exposure to 1O2 is required to maintain this transcriptional response, and other ROS do not cause a similar increase in sigma(E)-dependent gene expression. When a sigma(E) mutant produces low levels of carotenoids, 1O2 is bacteriocidal, suggesting that this response is essential for protecting cells from this ROS. In addition, global gene expression analysis identified approximately 180 genes (approximately 60 operons) whose RNA levels increase > or = 3-fold in cells with increased sigma(E) activity. Gene products encoded by four newly identified sigma(E)-dependent operons are predicted to be involved in stress response, protecting cells from 1O2 damage, or the conservation of energy.

Interactions between the Rhodobacter sphaeroides ECF sigma factor, sigma(E), and its anti-sigma factor, ChrR.

Rhodobacter sphaeroides sigma(E) is a member of the extra cytoplasmic function sigma factor (ECF) family, whose members have been shown to regulate gene expression in response to a variety of signals. The functions of ECF family members are commonly regulated by a specific, reversible interaction with a cognate anti-sigma factor. In R.sphaeroides, sigma(E) activity is inhibited by ChrR, a member of a newly discovered family of zinc containing anti-sigma factors. We used gel filtration chromatography to gain insight into the mechanism by which ChrR inhibits sigma(E) activity. We found that formation of the sigma(E):ChrR complex inhibits the ability of sigma(E) to form a stable complex with core RNA polymerase. Since the sigma(E):ChrR complex inhibits the ability of the sigma factor to bind RNA polymerase, we sought to identify amino acid substitutions in sigma(E) that altered the sensitivity of this sigma factor to inhibition by ChrR. This analysis identified single amino acid changes in conserved region 2.1 of sigma(E) that either increased or decreased the sensitivity of sigma(E) for inhibition by ChrR. Many of the amino acid residues that alter the sensitivity of sigma(E) to ChrR are located within regions known to be important for interacting with core RNA polymerase in other members of the sigma(70) superfamily. Our results suggest a model where solvent-exposed residues with region 2.1 of sigma(E) interact with ChrR to sterically occlude this sigma factor from binding core RNA polymerase and to inhibit target gene expression.

Purification of Rhodobacter sphaeroides RNA polymerase and its sigma factors.

This article summarized methods to obtain RNA polymerase and sigma factors that can be used to analyze the in vitro control of gene expression by the facultative phototroph R. sphaeroides. While not a topic of this article, these purified components also allow one to analyze R. sphaeroides promoters that use activators to stimulate transcription. We expect that these approaches will be increasingly useful as investigators continue to dissect the number of unusual signal transduction pathways that control gene expression in this and other related species.