T al. AMB Express 2013, 3:66 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 CGRP Receptor Antagonist web species have demonstrated outstanding activity as novel biocatalysts to get a range of applications. In this perform, we focused on the biotransformation of 5-haloindole into 5-halotryptophan, a pharmaceutical intermediate, utilizing 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 working with cells without 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 have been alive soon after 24 hour biotransformation reactions, each in planktonic and biofilm forms, suggesting that cell viability was not a significant aspect in the greater efficiency of biofilm reactions. Monitoring 5-haloindole depletion, 5-halotryptophan synthesis as well as the percentage conversion of your biotransformation reaction recommended that there have been inherent variations involving strains MG1655 and MC4100, and between planktonic and biofilm cells, when it comes to tryptophan and indole metabolism and transport. The study has reinforced the want to completely investigate DAPK list bacterial physiology and make informed strain selections when establishing biotransformation reactions. Keywords: E. coli; Biofilm; Biotransformation; Haloindole; HalotryptophanIntroduction Bacterial biofilms are renowned for their enhanced resistance to environmental and chemical stresses including antibiotics, metal ions and organic solvents when compared to planktonic bacteria. This house of biofilms is usually a reason for clinical concern, specially with implantable medical devices (like catheters), since biofilm-mediated infections are often tougher to treat than these brought on by planktonic bacteria (Smith and Hunter, 2008). Even so, the elevated robustness of biofilms may be exploited in bioprocesses where cells are exposed to harsh reaction situations (Winn et al., 2012). Biofilms, typically multispecies, have been employed for waste water therapy (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 information and facts is obtainable at the finish with 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 lately, single species biofilms have located applications in microbial fuel cells (Yuan et al., 2011a; Yuan et al., 2011b) and for distinct biocatalytic reactions (Tsoligkas et al., 2011; Gross et al., 2010; Kunduru and Pometto, 1996). Current examples of biotransformations catalysed by single-species biofilms include things like the conversion of benzaldehyde to benzyl alcohol (Zymomonas mobilis; Li et al., 2006), ethanol production (Z. mobilis and Saccharomyces cerevisiae; Kunduru and Pomett.