Styrene oxide and 2-phenylethanol metabolism in the styrene-degrading Xanthobacter sp. strain 124X was shown to proceed via phenylacetaldehyde and phenylacetic acid. In cell extracts 2-phenylethanol was oxidized by a phenazine methosulfate-dependent enzyme, probably a pyrroloquinoline quinone enzyme. Xanthobacter sp. strain 124X also contains a novel enzymatic activity designated as styrene oxide isomerase. Styrene oxide isomerase catalyzes the isomerization of styrene oxide to phenylacetaldehyde. The enzyme was partially purified and shown to have a very high substrate specificity. Of the epoxides tested, styrene oxide was the only substrate transformed. The initial step in styrene metabolism in Xanthobacter sp. strain 124X is oxygen dependent and probably involves oxidation of the aromatic nucleus.
Mycobacterium aurum Li, capable of growth on vinyl chloride as a sole carbon and energy source, was previously isolated from soil contaminated with vinyl chloride (S. Hartmans et al., Biotechnol. Lett. 7:383-388, 1985). The initial step in vinyl chloride metabolism in strain Li is catalyzed by alkene monooxygenase, transforming vinyl chloride into the reactive epoxide chlorooxirane. The enzyme responsible for chlorooxirane degradation appeared to be very unstable and thus hampered the characterization of the second step in vinyl chloride metabolism. Dichloroethenes are also oxidized by vinyl chloride-grown cells of strain Li, but they are not utilized as growth substrates. Three additional bacterial strains which utilize vinyl chloride as a sole carbon and energy source were isolated from environments with no known vinyl chloride contamination. The three new isolates were similar to strain Li and were also identified as Mycobacterium aurum. Vinyl chloride is carcinogenic in experimental animals and humans (4, 25). Consequently, the U.S. Environmental Protection Agency has classified vinyl chloride as a priority pollutant. The compound is produced on a very large scale by the chemicals industry, mainly for use in the production of the polymer polyvinyl chloride. Associated with these largescale processes are inevitable losses to the environment. Vinyl chloride is a gas at ambient conditions (boiling point, 1220
Synthetic genetic programs promise to enable novel applications in industrial processes. For such applications, the genetic circuits that compose programs will require fidelity in varying and complex environments. In this work, we report the performance of two synthetic circuits in Escherichia coli under industrially relevant conditions, including the selection of media, strain, and growth rate. We test and compare two transcriptional circuits: an AND and a NOR gate. In E. coli DH10B, the AND gate is inactive in minimal media; activity can be rescued by supplementing the media and transferring the gate into the industrial strain E. coli DS68637 where normal function is observed in minimal media. In contrast, the NOR gate is robust to media composition and functions similarly in both strains. The AND gate is evaluated at three stages of early scale-up: 100 ml shake-flask experiments, a 1 ml MTP microreactor, and a 10 L bioreactor. A reference plasmid that constitutively produces a GFP reporter is used to make comparisons of circuit performance across conditions. The AND gate function is quantitatively different at each scale. The output deteriorates late in fermentation after the shift from exponential to constant feed rates, which induces rapid resource depletion and changes in growth rate. In addition, one of the output states of the AND gate failed in the bioreactor, effectively making it only responsive to a single input. Finally, cells carrying the AND gate show considerably less accumulation of biomass. Overall, these results highlight challenges and suggest modified strategies for developing and characterizing genetic circuits that function reliably during fermentation.
By using styrene as the sole source of carbon and energy in concentrations of 10 to 500 ,uM, 14 strains of aerobic bacteria and two strains of fungi were isolated from various soil and water samples. In cell extracts of 11 of the bacterial isolates, a novel flavin adenine dinucleotide-requiring styrene monooxygenase activity that oxidized styrene to styrene oxide (phenyl oxirane) was detected. In one bacterial strain (S5), styrene metabolism was studied in more detail. In addition to styrene monooxygenase, cell extracts from strain S5 contained styrene oxide isomerase and phenylacetaldehyde dehydrogenase activities. A pathway for styrene degradation via styrene oxide and phenylacetaldehyde to phenylacetic acid is proposed.
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