Optimisation of engineered Escherichia coli biofilms for enzymatic biosynthesis of l-halotryptophans

Abstract

Engineered biofilms comprising a single recombinant species have demonstrated remarkable activity as novel biocatalysts for a range of applications. In this work, we focused on the biotransformation of 5-haloindole into 5-halotryptophan, a pharmaceutical intermediate, using 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 cases, no conversion of 5-haloindoles was observed using cells without the pSTB7 plasmid. Engineered biofilms of strains PHL628 pSTB7 and PHL644 pSTB7 generated more 5-halotryptophan than their corresponding planktonic cells. Flow cytometry revealed that the vast majority of cells were alive after 24 hour biotransformation reactions, both in planktonic and biofilm forms, suggesting that cell viability was not a major factor in the greater performance of biofilm reactions. Monitoring 5-haloindole depletion, 5-halotryptophan synthesis and the percentage conversion of the biotransformation reaction suggested that there were inherent differences between strains MG1655 and MC4100, and between planktonic and biofilm cells, in terms of tryptophan and indole metabolism and transport. The study has reinforced the need to thoroughly investigate bacterial physiology and make informed strain selections when developing biotransformation reactions.

Optogenetic Modulation of a Catalytic Biofilm for the Biotransformation of Indole into Tryptophan

“…The biotransformation experiment with planktonic cells was conducted by using a previously described method with slight modifications . The overnight cultures grown in LB media at 37 °C were inoculated in M9 minimal media or 50 % LB media.…”

“…The concentration of tryptophan was quantified by using HPLC (Shimadzu, Singapore) equipped with a ZORBAX (SB‐C18 4.6 mm×15 cm) column. A mixture of methanol (solvent A) and water (solvent B) was applied as the mobile phase (1 mL min −1 at 30 °C) with controlled gradients as follows: 0–0.5 min, 0–10 % A; 0.5–12.5 min, 10–90 % A; 15–16 min, 90–10 % A; 16–21 min, 10 % A . The mobile phase was acidified with 0.1 % formic acid.…”

Influence of csgD and ompR on Nanomechanics, Adhesion Forces, and Curli Properties of E. coli

“…Because of their resistance to sanitizing agents and consequent involvement in many infections or contaminations, biofilms are generally perceived negatively 4 . However, properties such higher resistance to toxic chemicals, have been exploited in biotechnological processes as biofilm cells can survive the unfavorable environment, from a biological standpoint, that various chemical reactions require 5,6 .…”

“…Moreover, curli expression is triggered at low osmolality as controlled by the OmpR/EnvZ systems 16 , is stationary phase dependent as controlled by the  factor RpoS 17 and is under regulation by CpxA/CpxR that are involved in the response of cellular proteins to acidic pH 18,19 . OmpR binds the csgDEFG operon positively controlling curli expression 20 and the specific mutation of this gene ompR234 (G to T at position 43 of OmpR, corresponding leucine to arginine substitution) has been shown to increase E. coli adhesion to abiotic surfaces through increased curli production 6,[20][21][22][23] . We have chosen to use in this work E. coli MG1655 as it is a widely used curli non-producing strain whereas mutants producing curli through overexpression of ompR and csgD have been prepared.…”

Non-pathogenic Escherichia coli biofilms: effects of growth conditions and surface properties on structure and curli gene expression

“…methods was found to rapidly generate robust biocatalytic biofilms (Tsoligkas et al 2012); subsequent work focused on optimising the tryptophan synthase reaction (Perni et al 2013) and revealed that enzyme turnover was at least partially responsible for the improved biocatalytic potential of biofilms (Tong et al 2016).…”