The efficiency of recombinant Escherichia coli as biocatalyst for stereospecific epoxidation.
Styrene is efficiently converted into (S)-styrene oxide by growing Escherichia coli expressing the styrene monooxygenase genes styAB of Pseudomonas sp. strain VLB120 in an organic/aqueous emulsion. Now, we investigated factors influencing the epoxidation activity of recombinant E. coli with the aim to improve the process in terms of product concentration and volumetric productivity. The catalytic activity of recombinant E. coli was not stable and decreased with reaction time. Kinetic analyses and the independence of the whole-cell activity on substrate and biocatalyst concentrations indicated that the maximal specific biocatalyst activity was not exploited under process conditions and that substrate mass transfer and enzyme inhibition did not limit bioconversion performance. Elevated styrene oxide concentrations, however, were shown to promote acetic acid formation, membrane permeabilization, and cell lysis, and to reduce growth rate and colony-forming activity. During biotransformations, when cell viability was additionally reduced by styAB overexpression, such effects coincided with decreasing specific epoxidation rates and metabolic activity. This clearly indicated that biocatalyst performance was reduced as a result of product toxicity. The results point to a product toxicity-induced biological energy shortage reducing the biocatalyst activity under process conditions. By reducing exposure time of the biocatalyst to the product and increasing biocatalyst concentrations, volumetric productivities were increased up to 1,800 micromol/min/liter aqueous phase (with an average of 8.4 g/L(aq) x h). This represents the highest productivity reported for oxygenase-based whole-cell biocatalysis involving toxic products.
Park
Jin-Byung
JB
Institute of Biotechnology, ETH Zurich, Zurich, Switzerland.
Bühler
Bruno
B
Habicher
Tilo
T
Hauer
Bernhard
B
Panke
Sven
S
Witholt
Bernard
B
Schmid
Andreas
A
D001711:Biotransformation / Q000235:genetics / Q000502:physiology; D002384:Catalysis; D002463:Cell Membrane Permeability / Q000187:drug effects; D004852:Epoxy Compounds / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D005822:Genetic Vectors; D007700:Kinetics; D010105:Oxygenases / Q000378:metabolism*; D020058:Styrene / Q000378:metabolism
D004852:Epoxy Compounds; D020058:Styrene; C013690:styrene oxide; D010105:Oxygenases; C026721:styrene monooxygenase