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T al. AMB Express 2013, three:66 amb-express/content/3/1/ORIGINAL ARTICLEOpen AccessOptimisation of engineered Escherichia coli biofilms for enzymatic biosynthesis of L-halotryptophansStefano Perni1, Louise Hackett1, Rebecca JM Goss2, Mark J Simmons1 and Tim W Overton1AbstractEngineered biofilms comprising a single recombinant species have demonstrated remarkable activity as novel biocatalysts for a range of applications. Within this work, we focused SFRP2 Protein medchemexpress around the biotransformation of 5-haloindole into 5-halotryptophan, a pharmaceutical intermediate, employing Escherichia coli expressing a recombinant tryptophan synthase enzyme encoded by plasmid pSTB7. To optimise the reaction we compared two E. coli K-12 strains (MC4100 and MG1655) and their ompR234 mutants, which overproduce the adhesin curli (PHL644 and PHL628). The ompR234 mutation increased the quantity of biofilm in both MG1655 and MC4100 backgrounds. In all circumstances, no conversion of 5-haloindoles was observed using cells with no the pSTB7 plasmid. Engineered biofilms of strains Annexin V-PE Apoptosis Detection Kit site PHL628 pSTB7 and PHL644 pSTB7 generated more 5-halotryptophan than their corresponding planktonic cells. Flow cytometry revealed that the vast majority of cells had been alive just after 24 hour biotransformation reactions, both in planktonic and biofilm types, suggesting that cell viability was not a major factor within the greater overall performance of biofilm reactions. Monitoring 5-haloindole depletion, 5-halotryptophan synthesis and the percentage conversion in the biotransformation reaction suggested that there had been inherent variations involving strains MG1655 and MC4100, and in between planktonic and biofilm cells, when it comes to tryptophan and indole metabolism and transport. The study has reinforced the want to completely investigate bacterial physiology and make informed strain selections when developing biotransformation reactions. Keyword phrases: E. coli; Biofilm; Biotransformation; Haloindole; HalotryptophanIntroduction Bacterial biofilms are renowned for their enhanced resistance to environmental and chemical stresses for example antibiotics, metal ions and organic solvents when in comparison with planktonic bacteria. This property of biofilms is really a reason for clinical concern, in particular with implantable healthcare devices (like catheters), since biofilm-mediated infections are frequently harder to treat than those triggered by planktonic bacteria (Smith and Hunter, 2008). However, the increased robustness of biofilms might be exploited in bioprocesses where cells are exposed to harsh reaction conditions (Winn et al., 2012). Biofilms, typically multispecies, have been utilized for waste water treatment (biofilters) (Purswani et al., 2011; Iwamoto and Nasu, 2001; Correspondence: [email protected] 1 College of Chemical Engineering, University of Birmingham, Birmingham B15 2TT, UK Full list of author data is offered at the end in the articleCortes-Lorenzo et al., 2012), air filters (Rene et al., 2009) and in soil bioremediation (Zhang et al., 1995; Singh and Cameotra, 2004). Most recently, single species biofilms have found applications in microbial fuel cells (Yuan et al., 2011a; Yuan et al., 2011b) and for particular biocatalytic reactions (Tsoligkas et al., 2011; Gross et al., 2010; Kunduru and Pometto, 1996). Current examples of biotransformations catalysed by single-species biofilms consist of the conversion of benzaldehyde to benzyl alcohol (Zymomonas mobilis; Li et al., 2006), ethanol production (Z. mobilis and Saccharomyces cerevisiae; Kunduru and Pomett.