Drug Discovery Applications: Metabolic Engineering and Drug Bioproduction
Natural drugs are often in minimum amounts and expensively obtained. Excitingly, synthetic biology technologies are helpful to expand the range of drugs with high efficiency. The combination of metabolic engineering and synthetic biology technologies provides innovative solutions for medical biotechnology through enzymatic bioprocessing by modifying and introducing enzymatic pathways into industrial organisms to transform natural raw materials and/or supplement chemical precursors.
Microbial bioproduction is often preferred because of its relatively easy manipulation, well-controlled fermentation process, fast growth rate, and inexpensive substrates. Microbial production becomes a promising solution for drug production, however, pharmaceutically-significant compounds that have reached an industrial scale for biological production (greater than 50 g/L) are limited to some amino acids and isoprenoids. Artemisinin and several antibiotics have just reached a moderate scale (5-50 g/L). Therefore, the number of drugs that can currently be produced biologically on an industrial scale is a challenge. Synthetic biology is expected to overcome these limitations and contribute to bringing drugs to market by providing engineering approaches that combine modeling and simulation of metabolic pathways with the design, construction, testing, and optimization of host strains.
The main natural products of biological manufacturing drugs include isoprenoids, polyketides, non-ribosomal peptides, and other natural polyphenols (flavonoids, astragalus, etc.). Isoprenoids are a large group of natural products (more than 40,000 structurally unique compounds) that encompass many drug-related compounds, such as antioxidants, cancer drugs, or antimalarials. The isoprenoid pathway has been expressed in multiple hosts and is assembled from genes imported from multiple sources.
Several synthetic biology techniques have been used to improve the performance of the isoprenoid pathway, such as modularized tuning of gene expression, increased flux, control of substrate toxicity, or colocalization by synthetic protein scaffoldings. Perhaps the best-known case is the semi-synthetic production of the antimalarial isoprenoid artemisinin, achieved through a combination of metabolic engineering and synthetic biology. The balance of restriction enzyme and gene expression is identified by plasmid copy number and promoter strength, and the production of artemisinin precursor amorpha-4,11-diene could reach 25 g/L in Escherichia coli and 40 g/L in yeast.
Similarly, synthetic biology technologies have been applied to produce the anti-cancer and chemotherapy drug paclitaxel. Paclitaxel is difficult to chemically synthesize and very inefficient to extract from its natural producer, Pacific Yew. By using a modular approach, the optimal combination of expression levels for different parts of the paclitaxel precursor pathway is determined to achieve a titer of 1 g/L in Escherichia coli.
In addition, the design of combinatorial biosynthesis for drug compounds involves synthetic biology as well, such as cyclic lipopeptide antibiotics. For example, by implementing a pathway to introduce fluorine into the polyketide compound backbone, fluorinated natural products can be produced with improved absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties.
However, synthetic bio-manufacturing is a complex process whose success depends not only on appropriate pathway selection but also on many other factors, such as cofactor and REDOX equilibria, thermodynamic feasibility, flux coupling, or regulatory elements. Synthetic pathways are often inefficient and need to be enhanced by protein engineering and directed evolution, which can be applied to both enzymes and complete pathways.
Automated computer-aided design of metabolic pathways helps to find the most effective pathway among a large number of potential pathways. With this strategy, a full-automatic framework based on the inverse synthesis method has been applied to the production of flavonoids. Flavonoids have medicinal potential due to their health-promoting activity, among which naringenin and coniferin are key molecular scaffolds and precursors of flavonoids. In addition to these proof-of-concept applications, the integration of computer-aided design with automated DNA assembly and genome compilation methods and robotic manufacturing will accelerate future drug development.
One of the most promising contributions of synthetic biology to drug bioproduction is the use of biological elements constructed from adequately characterized and standardized genetic elements for dynamic pathway regulation and metabolic control. Metabolite-responsive transcriptional modulators and riboswitches are two genetic elements that can be used to design synthetic and dynamically regulate metabolic pathways. Zhang et al. designed a fatty acid synthesis pathway that is regulated by transcriptional repressors and is inactivated when bound to fatty acids. This feedback loop resulted in a threefold increase in fatty acid ethyl ester production, increased pathway gene stability, and promoted the production of other malonyl-CoA derivatives at higher yields. Another area where synthetic biology is used for biological production is cell-free metabolic engineering, consisting of enzyme populations in vitro. Some groups have been able to recreate biosynthetic pathways in vitro for isoprenoid or protein production.
Reference
- Trosset J, Carbonell P, Synthetic biology for pharmaceutical drug discovery, Drug Des. Devel. Ther., 2015, 9, 6285-6302.
