Pilot‐scale production of (S)‐styrene oxide from styrene by recombinant Escherichia coli synthesizing styrene monooxygenase

Abstract

Recombinant Escherichia coli JM101(pSPZ10) cells produce the styrene monooxygenase of Pseudomonas sp. strain VLB120, which catalyzes the oxidation of styrene to (S)-styrene oxide at an enantiomeric excess larger than 99%. This biocatalyst was used to produce 388 g of styrene oxide in a two-liquid phase 30-L fed-batch bioconversion. The average overall volumetric activity was 170 U per liter over a period of more than 10 h, equivalent to mass transfer rates of 10.2 mmoles per liter per hour at a phase ratio of 0.5. At this transfer rate, the biotransformation system appeared to be substrate mass-transfer limited. The reactor had an estimated power input in the order of 5 W. L(-1), which is close to values typically obtained with commercially operating units. The product could be easily purified by fractional distillation to a purity in excess of 97%. The process illustrates the feasibility of recombinant whole cell biotransformations in two-liquid phase systems with toxic substrates and products.

Mutations at the putative active cavity of styrene monooxygenase: Enhanced activity and reversed enantioselectivity

“…The native substrate of SMO is styrene, which can undergo asymmetric epoxidation to form the single enantiomer of (S)-styrene oxide ( Fig. 1) (Bernasconi et al, 2000;Di Gennaro et al, 1999;Lin et al, 2010;Panke et al, 2002;Park et al, 2006;Tischler et al, 2010;Toda et al, 2012).…”

“…The process for the production of (S)-styrene oxide has been scaled up by using recombinant E. coli cells expressing the SMO from Pseudomonas fluorescens VLB120 and economic assessment shows that bioprocess performs best in terms of production costs compared with other three chemical alternatives (Kuhn et al, 2010;Panke et al, 2002;Schmid et al, 2001). In addition, with continuous effort in the functional studies and the identification of novel SMOs, the substrate spectrum of SMO has been expanded.…”

NADH Availability Limits Asymmetric Biocatalytic Epoxidation in a Growing Recombinant Escherichia coli Strain

“…Yet, microbial cells, especially in a nongrowing state, often lose biooxidation activity over time, which may be due to oxygenase inactivation and/or the metabolic burden imposed by redox-coenzyme withdrawal and reactive oxygen species formed via uncoupled oxygen reduction. This led to the use of growing cells as biocatalysts, the metabolism of which supports not only coenzyme regeneration but also continuous oxygenase synthesis and cell reproduction (5,19,27,32,49). However, during cell growth, various reactions-especially oxidative phosphorylation-consume high amounts of reduction equivalents (59).…”

“…It is clear that, depending on the reaction conditions (induction level, glucose and oxygen availability, growth rate, and substrate and product concentrations), the NADH flux distribution to the different NADH-dependent reactions and thus the achievable specific epoxidation rate may vary considerably. This explains why substantially higher epoxidation rates of up to 60 U (g CDW) Ϫ1 have been achieved during fed-batch cultivation of E. coli JM101(pSPZ10) (49,51,52).…”

Bioproduction of lauryl lactone and 4-vinyl guaiacol as value-added chemicals in two-phase biotransformation systems

“…The cell pellet was recovered by centrifugation and inoculated in a 3-l bioreactor (Biobundles, Applikon Inc, USA) that contains two Rushton turbine impellers and four baffles. The 1-l culture medium contains 4.0 g Na 2 HPO 4 ; 2.0 g KH 2 PO 4 ; 3.0 g (NH 4 ) 2 SO 4 ; 0.5 g NaCl; 1.0 g casamino acid; 0.12 g MgSO 4 ; 58.0 mg CaCl 2 ·2H 2 O; 50.0 mg thiamine; 50.0 mg ampicillin; 6.0 mg FeSO 4 ·7H 2 O; 20 g glucose, and 4.5 ml trace element solution as described by Panke et al (2002). An additional carbon source (glucose 500 g/l) was fed at a rate of 10 ml/h when the OD 600 reached a value of 10.…”