T al. AMB Express 2013, 3:66 amb-express/content/3/1/ORIGINAL ARTICLEOpen AccessOptimisation of engineered Escherichia coli biofilms for

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 species have demonstrated exceptional activity as novel biocatalysts to get a selection of applications. Within this operate, we focused on 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 elevated the quantity of biofilm in both MG1655 and MC4100 backgrounds. In all circumstances, no conversion of 5-haloindoles was observed using 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 were alive soon after 24 hour biotransformation reactions, both in planktonic and biofilm forms, suggesting that cell viability was not a significant aspect within the greater functionality of biofilm reactions. Monitoring 5-haloindole depletion, 5-halotryptophan synthesis along with the percentage conversion from the biotransformation reaction recommended that there were 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 will need to thoroughly investigate 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 in comparison to planktonic bacteria. This house of biofilms is TRAIL/TNFSF10 Protein custom synthesis usually a reason for clinical concern, especially with implantable medical devices (like catheters), since biofilm-mediated infections are regularly tougher to treat than these triggered by planktonic bacteria (Smith and Hunter, 2008). Having said that, the elevated robustness of biofilms could be exploited in bioprocesses exactly where cells are exposed to harsh reaction conditions (Winn et al., 2012). Biofilms, typically multispecies, happen to be applied for waste water remedy (biofilters) (Purswani et al., 2011; Iwamoto and Nasu, 2001; Siglec-10 Protein Gene ID Correspondence: [email protected] 1 College of Chemical Engineering, University of Birmingham, Birmingham B15 2TT, UK Complete list of author info is offered at the end 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 not too long ago, single species biofilms have found 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 the conversion of benzaldehyde to benzyl alcohol (Zymomonas mobilis; Li et al., 2006), ethanol production (Z. mobilis and Saccharomyces cerevisiae; Kunduru and Pomett.