Interfacing microbial styrene production with a biocompatible cyclopropanation reaction

Abstract

The introduction of new reactivity into living organisms is a major challenge in synthetic biology. Despite an increasing interest in both the development of small-molecule catalysts that are compatible with aqueous media and the engineering of enzymes to perform new chemistry in vitro, the integration of non-native reactivity into metabolic pathways for small-molecule production has been underexplored. Herein we report a biocompatible iron(III) phthalocyanine catalyst capable of efficient olefin cyclopropanation in the presence of a living microorganism. By interfacing this catalyst with E. coli engineered to produce styrene, we synthesized non-natural phenyl cyclopropanes directly from D-glucose in single-vessel fermentations. This process is the first example of the combination of nonbiological carbene-transfer reactivity with cellular metabolism for small-molecule production.

Keywords: iron; metabolism; phthalocyanine; synthetic biology; synthetic methods.

Figure 1

Design of a biocompatible cyclopropanation reaction. A) Carbene intermediates in biological catalysis. B) Current approaches for accessing ethyl-2-phenylcyclopropane-1-carboxylate (1) from styrene using transition metal-mediated carbene chemistry. C) Phenyl cyclopropane production from D-glucose by combining in vivo styrene production with biocompatible chemistry.

Figure 2

A reaction byproduct reports on E. coli survival under the cyclopropantion reaction conditions. Reactions were performed as described in Table 1.

Figure 3

The biocompatible cyclopropanation reaction can be interfaced with microbial styrene production. A) Engineered pathway for styrene production in the L-phenylalanine overproducer E. coli NST74. B) Cyclopropanation using metabolically generated styrene. C) Cyclopropane production requires all reaction components and living E. coli. D) Metabolite production during fermentations. E) Additional cyclopropanes accessed via this approach. Metabolite concentrations were determined by GC relative to an internal standard of 1,3,5-trimethoxybenzene. Yields in Section E are of isolated material from 800 mL cultures. All data is shown as an average of three independent experiments to one standard deviation. [a] 93% isolated yield. [b] 72 h reaction.