Analysis of heterologous taxadiene production in K- and B-derived Escherichia coli.

Taxa-4(5),11(12)-diene is the first dedicated intermediate in the metabolic pathway responsible for synthesizing the anticancer compound Taxol. In this study, the heterologous production of taxadiene was established in and analyzed between K- and B-derived Escherichia coli strains. First, recombinant parameters associated with precursor metabolism (the upstream methylerythritol phosphate (MEP) pathway) and taxadiene biosynthesis (the downstream pathway) were varied to probe the effect different promoters and cellular backgrounds have on taxadiene production. Specifically, upstream MEP pathway genes responsible for the taxadiene precursors, dimethylallyl diphosphate and isopentenyl diphosphate, were tested with an inducible T7 promoter system within K and B E. coli strains. Whereas, inducible T7, Trc, and T5 promoters were tested with the plasmid-borne geranylgeranyl diphosphate synthase and taxadiene synthase genes responsible for the downstream pathway. The K-derivative produced taxadiene roughly 2.5-fold higher than the B-derivative. A transcriptomics study revealed significant differences in pyruvate metabolism between the K and B strains, providing insight into the differences observed in taxadiene biosynthesis and targets for future metabolic engineering efforts. Next, the effect of temperature on cell growth and taxadiene production was analyzed in these two strains, revealing similar phenotypes between the two with 22°C as the optimal production temperature. Lastly, the effect of indole on cell growth was investigated between the two strains, showing that the K-derivative demonstrated greater growth inhibition compared to the B-derivative.

Computational identification of gene over-expression targets for metabolic engineering of taxadiene production.

Taxadiene is the first dedicated intermediate in the biosynthetic pathway of the anticancer compound Taxol. Recent studies have taken advantage of heterologous hosts to produce taxadiene and other isoprenoid compounds, and such ventures now offer research opportunities that take advantage of the engineering tools associated with the surrogate host. In this study, metabolic engineering was applied in the context of over-expression targets predicted to improve taxadiene production. Identified targets included genes both within and outside of the isoprenoid precursor pathway. These targets were then tested for experimental over-expression in a heterologous Escherichia coli host designed to support isoprenoid biosynthesis. Results confirmed the computationally predicted improvements and indicated a synergy between targets within the expected isoprenoid precursor pathway and those outside this pathway. The presented algorithm is broadly applicable to other host systems and/or product choices.

Simultaneous production and partitioning of heterologous polyketide and isoprenoid natural products in an Escherichia coli two-phase bioprocess.

Natural products have long served as rich sources of drugs possessing a wide range of pharmacological activities. The discovery and development of natural product drug candidates is often hampered by the inability to efficiently scale and produce a molecule of interest, due to inherent qualities of the native producer. Heterologous biosynthesis in an engineering and process-friendly host emerged as an option to produce complex natural products. Escherichia coli has previously been utilized to produce complex precursors to two popular natural product drugs, erythromycin and paclitaxel. These two molecules represent two of the largest classes of natural products, polyketides and isoprenoids, respectively. In this study, we have developed a platform E. coli strain capable of simultaneous production of both product precursors at titers greater than 15 mg l(-1). The utilization of a two-phase batch bioreactor allowed for very strong in situ separation (having a partitioning coefficient of greater than 5,000), which would facilitate downstream purification processes. The system developed here could also be used in metagenomic studies to screen environmental DNA for natural product discovery and preliminary production experiments.

Multi-factorial engineering of heterologous polyketide production in Escherichia coli reveals complex pathway interactions.

Polyketides represent a significant fraction of all natural products. Many possess pharmacological activity which makes them attractive drug candidates. The production of the parent macrocyclic aglycones is catalyzed by multi-modular polyketide synthases utilizing short-chain acyl-CoA monomers. When producing polyketides through heterologous hosts, one must not only functionally express the synthase itself, but activate the machinery used to generate the required substrate acyl-CoA's. As a result, metabolic engineering of these pathways is necessary for high-level production of heterologous polyketides. In this study, we over-express three different pathways for provision of the two substrates (propionyl-CoA and (2S)-methylmalonyl-CoA) utilized for the biosynthesis of 6-deoxyerythronolide B (6-dEB; the macrolactone precursor of erythromycin): (1) a propionate → propionyl-CoA → (2S)-methylmalonyl-CoA pathway, (2) a methylmalonate → methylmalonyl-CoA → propionyl-CoA pathway, and (3) a succinate → succinyl-CoA → (2R)-methylmalonyl-CoA → (2S)-methylmalonyl-CoA → propionyl-CoA pathway. The current study revealed that propionate is a necessary component for greater than 5 mg L(-1) titers. Deletion of the propionyl-CoA:succinate CoA transferase (ygfH) or over-expression of the transcriptional activator of short chain fatty acid uptake improved titer to over 100 mg L(-1), while the combination of the two improved titer to over 130 mg L(-1). The addition of exogenous methylmalonate could also improve titer to over 100 mg L(-1). Expression of a Streptomyces coelicolor A3(2) methylmalonyl-CoA epimerase, in conjunction with over-expression of Escherichia coli's native methylmalonyl-CoA mutase, allowed for the incorporation of exogenously fed succinate into the 6-dEB core.

Methods and options for the heterologous production of complex natural products.

This review will detail the motivations, experimental approaches, and growing list of successful cases associated with the heterologous production of complex natural products.

Utilizing elementary mode analysis, pathway thermodynamics, and a genetic algorithm for metabolic flux determination and optimal metabolic network design.

BACKGROUND: Microbial hosts offer a number of unique advantages when used as production systems for both native and heterologous small-molecules. These advantages include high selectivity and benign environmental impact; however, a principal drawback is low yield and/or productivity, which limits economic viability. Therefore a major challenge in developing a microbial production system is to maximize formation of a specific product while sustaining cell growth. Tools to rationally reconfigure microbial metabolism for these potentially conflicting objectives remain limited. Exhaustively exploring combinations of genetic modifications is both experimentally and computationally inefficient, and can become intractable when multiple gene deletions or insertions need to be considered. Alternatively, the search for desirable gene modifications may be solved heuristically as an evolutionary optimization problem. In this study, we combine a genetic algorithm and elementary mode analysis to develop an optimization framework for evolving metabolic networks with energetically favorable pathways for production of both biomass and a compound of interest. RESULTS: Utilization of thermodynamically-weighted elementary modes for flux reconstruction of E. coli central metabolism revealed two clusters of EMs with respect to their Delta Gp degrees. For proof of principle testing, the algorithm was applied to ethanol and lycopene production in E. coli. The algorithm was used to optimize product formation, biomass formation, and product and biomass formation simultaneously. Predicted knockouts often matched those that have previously been implemented experimentally for improved product formation. The performance of a multi-objective genetic algorithm showed that it is better to couple the two objectives in a single objective genetic algorithm. CONCLUSION: A computationally tractable framework is presented for the redesign of metabolic networks for maximal product formation combining elementary mode analysis (a form of convex analysis), pathway thermodynamics, and a genetic algorithm to optimize the production of two industrially-relevant products, ethanol and lycopene, from E. coli. The designed algorithm can be applied to any small-scale model of cellular metabolism theoretically utilizing any substrate and applied towards the production of any product.

Metabolic flux analysis and pharmaceutical production.

Rational engineering of biological systems is an inherently complex process due to their evolved nature. Metabolic engineering emerged and developed over the past 20 years as a field in which methodologies for the rational engineering of biological systems is now being applied to specific industrial, medical, or scientific problems. Of considerable interest is the determination of metabolic fluxes within the cell itself, called metabolic flux analysis. This special issue and this review have a particular interest in the application of metabolic flux analysis for improving the pharmaceutical production process (for both small and large molecules). Though metabolic flux analysis has been somewhat limited in application towards pharmaceutical production, the overall goal is to: (1) have a better understanding of the organism and/or process in question, and (2) provide a rational basis to further engineer (on both metabolic and process scales) improved pharmaceutical production in these organisms. The focus of this review article is to present how experimental and computational methods of metabolic flux analysis have matured, mirroring the maturation of the metabolic engineering field itself, while highlighting some of the successful applications towards both small- and large-molecule pharmaceuticals.

Investigating the role of native propionyl-CoA and methylmalonyl-CoA metabolism on heterologous polyketide production in Escherichia coli.

6-Deoxyerythronolide B (6dEB) is the macrocyclic aglycone precursor of the antibiotic natural product erythromycin. Heterologous production of 6dEB in Escherichia coli was accomplished, in part, by designed over-expression of a native prpE gene (encoding a propionyl-CoA synthetase) and heterologous pcc genes (encoding a propionyl-CoA carboxylase) to supply the needed propionyl-CoA and (2S)-methylmalonyl-CoA biosynthetic substrates. Separate E. coli metabolism includes three enzymes, Sbm (a methylmalonyl-CoA mutase), YgfG (a methylmalonyl-CoA decarboxylase), and YgfH (a propionyl-CoA:succinate CoA transferase), also involved in propionyl-CoA and methylmalonyl-CoA metabolism. In this study, the sbm, ygfG, and ygfH genes were individually deleted and over-expressed to investigate their effect on heterologous 6dEB production. Our results indicate that the deletion and over-expression of sbm did not influence 6dEB production; ygfG over-expression reduced 6dEB production by fourfold while ygfH deletion increased 6dEB titers from 65 to 129 mg/L in shake flask experiments. It was also found that native E. coli metabolism could support 6dEB biosynthesis in the absence of exogenous propionate and the substrate provision pcc genes. Lastly, the effect of the ygfH deletion was tested in batch bioreactor cultures in which 6dEB titers improved from 206 to 527 mg/L.

Computational analysis of phenotypic space in heterologous polyketide biosynthesis--applications to Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae.

Polyketides represent a class of natural product small molecules with an impressive range of medicinal activities. In order to improve access to therapeutic polyketide compounds, heterologous metabolic engineering has been applied to transfer polyketide genetic pathways from often fastidious native hosts to more industrially-amenable heterologous hosts such as Escherichia coli, Saccharomyces cerevisiae, or Streptomyces coelicolor. Efforts thus far have resulted in titers either inferior to the native host and significantly below the theoretical yield, emphasizing the need to computationally investigate and engineer the interaction between native and heterologous metabolism for the improved production of heterologous polyketide compounds. In this work, we applied flux balance analysis on genome-scale models to simulate cellular metabolism and 6-deoxyerythronolide B (the cyclized polyketide precursor to erythromycin) production in three common heterologous hosts (E. coli, Bacillus subtilis, and S. cerevisiae) under a variety of carbon-source and medium compositions. We then undertook minimization of metabolic adjustment optimization to identify single and double gene-knockouts that resulted in increased polyketide production while maintaining cellular growth. For the production of 6-deoxyerythronolide B, the results suggest B. subtilis and E. coli are better heterologous hosts when compared to S. cerevisiae and that several single and multiple gene-knockout mutants are computationally predicted to improve specific production, in some cases, over 25-fold.

Current status, strategies, and potential for the metabolic engineering of heterologous polyketides in Escherichia coli.

Heterologous natural product biosynthesis has emerged as a strategy to produce medicinal compounds that pose challenges to conventional production routes. Polyketide compounds, an important class of natural products with wide-ranging therapeutic value, have been heterologously produced through Escherichia coli, presenting new opportunities to realize the medicinal potential of polyketide natural products. However, current production levels are often suboptimal when compared to native strain producers or heterologous theoretical yields. This problem provides an excellent opportunity to apply and further develop current metabolic engineering tools.

Improving heterologous polyketide production in Escherichia coli by overexpression of an S-adenosylmethionine synthetase gene.

An S-adenosylmethionine synthetase gene (metK) from Streptomyces spectabilis was cloned into an expression plasmid under the control of an inducible T7 promoter and introduced into a strain of Escherichia coli (BAP1(pBP130/pBP144)) capable of producing the polyketide product 6-deoxyerythronolide B (6-dEB). The metK coexpression in BAP1(pBP130/pBP144) improved the specific production of 6-dEB from 10.86 to 20.08 mg l(-1) OD(600)(-1). In an effort to probe the reason for this improvement, a series of gene deletion and expression experiments were conducted based on a metK metabolic pathway that branches between propionyl-CoA (a 6-dEB precursor) and autoinducer compounds. The deletion and expression studies suggested that the autoinducer pathway had a larger impact on improved 6-dEB biosynthesis. Supporting these results were experiments demonstrating the positive effect conditioned media (the suspected location of the autoinducer compounds) had on 6-dEB production. Taken together, the results of this study show an increase in heterologous 6-dEB production concomitant with heterologous metK gene expression and suggest that the mechanism for this improvement is linked to native autoinducer compounds.

Performance validation of an improved Xenon-arc lamp-based CCD camera system for multispectral imaging in proteomics.

Advances in gel-based nonradioactive protein expression and PTM detection using fluorophores has served as the impetus for developing analytical instrumentation with improved imaging capabilities. We describe a CCD camera-based imaging instrument, equipped with both a high-pressure Xenon arc lamp and a UV transilluminator, which provides broad-band wavelength coverage (380-700 nm and UV). With six-position filter wheels, both excitation and emission wavelengths may be selected, providing optimal measurement and quantitation of virtually any dye and allowing excellent spectral resolution among different fluorophores. While spatial resolution of conventional fixed CCD camera imaging systems is typically inferior to laser scanners, this problem is circumvented with the new instrument by mechanically scanning the CCD camera over the sample and collecting multiple images that are subsequently automatically reconstructed into a complete high-resolution image. By acquiring images in succession, as many as four different fluorophores may be evaluated from a gel. The imaging platform is suitable for analysis of the wide range of dyes and tags commonly encountered in proteomics investigations. The instrument is unique in its capabilities of scanning large areas at high resolution and providing accurate selectable illumination over the UV/visible spectral range, thus maximizing the efficiency of dye multiplexing protocols.