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 exceptional activity as novel biocatalysts for a array of applications. Within this work, we focused on the biotransformation of 5-haloindole into 5-halotryptophan, a pharmaceutical intermediate, applying 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 enhanced the quantity of biofilm in each MG1655 and MC4100 backgrounds. In all circumstances, no conversion of 5-haloindoles was observed utilizing cells Guanylate Cyclase Activator medchemexpress without having the pSTB7 plasmid. Engineered biofilms of strains PHL628 pSTB7 and PHL644 pSTB7 generated far more 5-halotryptophan than their corresponding planktonic cells. Flow cytometry revealed that the vast majority of cells were alive soon after 24 hour biotransformation reactions, both in planktonic and biofilm types, suggesting that cell viability was not a significant factor inside the higher functionality of biofilm reactions. Monitoring 5-haloindole depletion, 5-halotryptophan synthesis and the percentage conversion of the biotransformation reaction recommended that there were inherent differences among strains MG1655 and MC4100, and among planktonic and biofilm cells, when it comes to tryptophan and indole metabolism and transport. The study has reinforced the have to have to thoroughly Dopamine Receptor Antagonist Synonyms investigate bacterial physiology and make informed strain selections when creating biotransformation reactions. Keywords and 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 compared to planktonic bacteria. This house of biofilms is really a reason for clinical concern, particularly with implantable healthcare devices (for instance catheters), considering the fact that biofilm-mediated infections are often harder to treat than those caused by planktonic bacteria (Smith and Hunter, 2008). However, the elevated robustness of biofilms is usually exploited in bioprocesses exactly where cells are exposed to harsh reaction situations (Winn et al., 2012). Biofilms, normally multispecies, have been applied for waste water treatment (biofilters) (Purswani et al., 2011; Iwamoto and Nasu, 2001; Correspondence: [email protected] 1 School of Chemical Engineering, University of Birmingham, Birmingham B15 2TT, UK Full list of author information is offered at the finish 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 certain biocatalytic reactions (Tsoligkas et al., 2011; Gross et al., 2010; Kunduru and Pometto, 1996). Recent examples of biotransformations catalysed by single-species biofilms include the conversion of benzaldehyde to benzyl alcohol (Zymomonas mobilis; Li et al., 2006), ethanol production (Z. mobilis and Saccharomyces cerevisiae; Kunduru and Pomett.