The solventogenic Clostridia are of interest to the chemical industry because of their natural ability to produce chemicals such as butanol, acetone and ethanol from diverse feedstocks. Their use as whole cell factories presents multiple metabolic engineering targets that could lead to improved sustainability and profitability of Clostridium industrial processes. However, engineering efforts have been held back by the scarcity of genetic and synthetic biology tools. Over the past decade, genetic tools to enable transformation and chromosomal modifications have been developed, but the lack of a broad palette of synthetic biology parts remains one of the last obstacles to the rapid engineered improvement of these species for bioproduction. We have systematically reviewed existing parts that have been used in the modification of solventogenic Clostridia, revealing a narrow range of empirically chosen and nonengineered parts that are in current use. The analysis uncovers elements, such as promoters, transcriptional terminators and ribosome binding sites where increased fundamental knowledge is needed for their reliable use in different applications. Together, the review provides the most comprehensive list of parts used and also presents areas where an improved toolbox is needed for full exploitation of these industrially important bacteria.

Summary 26 The explosive 2,4,6-trinitrotoluene (TNT) is a significant, global environmental 27 pollutant that is both toxic and recalcitrant to degradation. Given the sheer scale, and 28 inaccessible nature of contaminated areas, phytoremediation may be a viable clean-up 29 approach. Here, we have characterised a Drosophila melanogaster (Meigen, 1830) 30 glutathione transferase (DmGSTE6) which has activity towards TNT. 31  Recombinantly-expressed, purified DmGSTE6 produces predominantly 2-32 glutathionyl-4,6-dinitrotoluene, and has a 2.5-fold higher V max , and 5-fold lower K m 33 than previously characterised TNT-active Arabidopsis thaliana (L. 2004), and many contaminated sites in Europe and Asia (Kalderis et al., 2011; Pichtel, 2012). 59)For example, the Werk Tanne former ammunition site in Germany, detonated in 1944, is 60 heavily contaminated with TNT (Eisentraeger et al., 2007). Increased environmental 61 awareness is now compelling governments to identify sites of explosives contamination and 62 put together remediation strategies (Lima et al., 2011). However, a major challenge to 63 cleaning-up these sites is the sheer scale and hazardous nature of many contaminated sites, 64 which rules-out many strategies such as excavation, land fill and off-site treatments, as 65 prohibitively expensive. Phytoremediation may be a viable alternative approach. 66TNT is not readily degraded in the environment due to the electron-withdrawing properties of 67 the three nitro groups of TNT which render the aromatic ring particularly resistant to 68 oxidative attack and ring cleavage (Qasim et al., 2009); the main route of aromatic 69 compounds by soil microbes. Instead microbial flora catalyse a series of reductive reactions, 70 producing predominantly hydroxylamino dinitrotoluene (HADNT) and amino dinitrotoluene 71 (ADNT) and further reduced derivatives (Rylott et al., 2011b). In plants, HADNT and ADNT 72 can be conjugated to sugars, for example, to glucose by UDP-glucosyltransferases (Gandia-73 Herrero et al., 2008), and it has recently been shown that glutathione transferases can 74 conjugate the TNT molecule directly (Gunning et al., 2014; Rylott et al., 2015). Two 75Arabidopsis thaliana (L.) Heynh (Arabidopsis) glutathione transferase (GST) genes, AtGST-76 U24 and AtGST-U25, are specifically upregulated in response to TNT exposure, and their 77 gene products catalyse the formation of three characterised TNT glutathionyl-products 78 (Gunning et al., 2014). The removal of a nitro group in one of the three products, 2-79 glutathionyl-4,6-dinitrotoluene, has the potential to be more amenable to subsequent 80 biodegradation in the environment, a property that could be applied in planta for the 81 5 detoxification of TNT in the field. Expression of AtGST-U24 and AtGST-U25 in Arabidopsis 82 conferred increased ability to take up and detoxify TNT; however, in the absence of TNT, 83 overexpression of these GSTs caused a reduction in plant biomass; an effect with deleterious 84 implications for xenobiotic detoxification...

Tethered β-lactam antibiotics provide insights into designing chemical tools to target specific β-lactamases.

Planobispora rosea is a genetically intractable bacterium used for the production of GE2270A on an industrial scale. GE2270A is a potent thiopeptide antibiotic currently used as a precursor for the synthesis of two compounds under clinical studies for the treatment of Clostridium difficile infection and acne.