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Genetic Engineering of Clostridial Strains for Cancer Therapy
Published in Ananda M. Chakrabarty, Arsénio M. Fialho, Microbial Infections and Cancer Therapy, 2019
Maria Zygouropoulou, Aleksandra Kubiak, Adam V. Patterson, Nigel P. Minton
In this context, a seminal moment in the development of genetic tools for clostridia was the recent implementation of an allele-coupled exchange (ACE) method, which allows the insertion of heterologous cargos into the host chromosome without the use of antibiotic resistance markers [62]. This method is based on the principle that the integration event yields a selectable phenotype by the means of pyrE inactivation. The pyrE gene is involved in pyrimidine biosynthesis, and its inactivation eliminates the ability of cells to metabolize 5-fluoroorotic acid (5-FOA) into the toxic metabolite 5-fluorouracil (5-FU). This serves primarily as a counterselection marker, rendering recombinant cells the ability to survive in the presence of 5-FOA, but it simultaneously confers uracil auxotrophy upon them. This is of particular value in cancer treatment, since it automatically provides an additional containment measure; uracil is abundant at the site of the tumor but not readily found in other environments [41]. An outline of the genetic events that take place during ACE can be found in Fig. 3.1.
Yeast: bridging the gap between phenotypic and biochemical assays for high-throughput screening
Published in Expert Opinion on Drug Discovery, 2018
Lethal expression-based platform readout is reliant on positive growth changes in response to the direct chemical modulation of the target protein. However, given the extreme genetic tractability of both S. cerevisiae and S. pombe, it is possible to engineer these model yeast to provide an indirect HTS readout through transactivation. Several examples have been reported [22,23] (Table 2), but the most extensively described and HTS utilized example of a yeast transactivation platform has been directed toward the discovery of mammalian phosphodiesterase (PDE) inhibitors [24–28]. The approximately 100, tissue specific, mammalian PDE isoforms all catalyze the conversion of cAMP and cGMP to the cyclic secondary messengers 5ʹAMP and 5ʹGMP. Given the tissue specificity of many of these isoforms, selective modulation has been recognized as having therapeutic potential for a wide range of human diseases [29,30] (Table 2). S. pombe can tolerate deletion of adenylate cyclase (cyr1) and PDE (csg2). In their absence, exogenous cAMP or cGMP activates Protein Kinase A (PKA) and ura4 expression from an introduced selectable marker is repressed. In the absence of ura4 expression, the yeast can grow in the presence of 5-fluoroorotic acid (5FOA), which is converted to toxic fluoroorotidine monophosphate in the presence of URA4 [28,31]. However, expression of yeast (Cyr1) or a mammalian PDE facilitated the conversion of cAMP or cGMP to 5ʹAMP or 5ʹGMP, leading to ura4 expression and 5FOA sensitivity. Inhibition of PDE function rescued growth and the system has been formatted into a positive selection HTS used to screen libraries >200k in size [24–28]. To ensure specificity against single or small numbers of PDE isoforms screening against yeast expressing different isoforms can be undertaken, so-called counter screening. Using this approach, many of the hits were demonstrated to be specific for one or more PDE isoforms [24–28]. Again, like the lethal expression platforms, these phenotypic rescue assays reduce the likelihood of false-positive hits.