Part a of the figure shows a synthetic mammalian gene circuit that enabled drug discovery for antituberculosis compounds (Weber etal., 2008). The antibiotic ethionamide is rendered cytotoxic to Mycobacterium tuberculosis by the enzyme EthA in infected cells. Because EthA is natively repressed by EthR, resistance to ethionamide treatment is common. In the gene circuit, a fusion of EthR and the mammalian transactivator VP16 binds a minimal promoter (Pmin) with a synthetic EthR operator site and activates expression of the reporter gene SEAP (human placental secreted alkaline phosphatase). This platform allows for the rapid screening of EthR inhibitors in mammalian cells.
Part b shows a synthetic mammalian genetic switch for tight, tunable and reversible control of a desired gene for therapeutic or gene-delivery applications. In the OFF configuration (upper panel), expression of the gene of interest (green) is repressed at the levels of both transcription and translation. Constitutively expressed LacI repressor (red) binds to the lac operator sites in the transgene module of the gene of interest, therefore repressing its transcription. Any transcriptional leakage is repressed at the level of translation by an interfering RNA (blue), which targets the gene’s 3′ UTR. The system is switched ON (lower panel) by the addition of isopropyl-β-d-thiogalactopyranoside (IPTG), which binds LacI repressor proteins and consequently relieves both forms of repression.
The discovery of drugs does not always translate to the people who need them the most because drug production processes can be difficult and costly. Antibiotics are industrially produced from microbes and fungi, and are therefore widespread and cheap. Conversely, many other drugs are isolated from hosts that are not as amenable to large-scale production and are therefore costly and in short supply. Such drugs include the antimalaria drug artemisinin and the anticancer drug taxol. Fortunately, global access to drugs is being enabled by hybrid synthetic biology and metabolic engineering strategies for the microbial production of rare natural products. In the case of artemisinin (part c), there exist two biosynthetic pathways for the synthesis of the universal precursors to all isoprenoids, the large and diverse family of natural products of which artemisinin is a member. The native isoprenoid pathway found in Escherichia coli (the deoxyxylulose 5-phosphate (DXP) pathway) has been difficult to optimize, so instead researchers have synthetically constructed and tested the entire Saccharomyces cerevisiae mevalonate-dependent (MEV) pathway in E. coli in a piece-wise fashion (for example, by separating the ‘top’ and ‘bottom’ operons). The researchers initially used E. coli as a simple, orthogonal host platform to construct, debug and optimize the large metabolic pathway (Martin et al., 2003). They then linked the optimized heterologous pathway to a codon-optimized form of the plant terpene synthase ADS to funnel metabolic production to the specific terpene precursor to artemisinin. This work allowed them to build a full, optimized solution that could be ultimately and seamlessly deployed back into S. cerevisiae for cost-effective synthesis and purification of industrial quantities of the immediate drug precursor of artemisinin (Ro et al., 2006). FPP, farnesyl pyrophosphate.
From: A2, SYNTHETIC BIOLOGY: APPLICATIONS COME OF AGE
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