Reconstitution of Active Mycobacterial Binuclear Iron Monooxygenase Complex in Escherichia coli

Furuya, Toshiki; Hayashi, Mitsuko; Kino, Kuniki

doi:10.1128/aem.01856-13

Cited by 12 publications

(17 citation statements)

References 34 publications

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“…These binuclear iron monooxygenases are able to epoxidize styrene. Homologous clusters can be found in several actinobacteria (28)(29)(30), e.g., in R. opacus 1CP and Gordonia sp. i37 (21).…”

Section: Resultsmentioning

confidence: 99%

On the Enigma of Glutathione-Dependent Styrene Degradation in Gordonia rubripertincta CWB2

Heine

Zimmerling

Ballmann

et al. 2018

Appl Environ Microbiol

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Among bacteria, only a single styrene-specific degradation pathway has been reported so far. It comprises the activity of styrene monooxygenase, styrene oxide isomerase, and phenylacetaldehyde dehydrogenase, yielding phenylacetic acid as the central metabolite. The alternative route comprises ring-hydroxylating enzymes and yields vinyl catechol as central metabolite, which undergoes -cleavage. This was reported to be unspecific and also allows the degradation of benzene derivatives. However, some bacteria had been described to degrade styrene but do not employ one of those routes or only parts of them. Here, we describe a novel "hybrid" degradation pathway for styrene located on a plasmid of foreign origin. As putatively also unspecific, it allows metabolizing chemically analogous compounds (e.g., halogenated and/or alkylated styrene derivatives). CWB2 was isolated with styrene as the sole source of carbon and energy. It employs an assembled route of the styrene side-chain degradation and isoprene degradation pathways that also funnels into phenylacetic acid as the central metabolite. Metabolites, enzyme activity, genome, transcriptome, and proteome data reinforce this observation and allow us to understand this biotechnologically relevant pathway, which can be used for the production of ibuprofen. The degradation of xenobiotics by bacteria is not only important for bioremediation but also because the involved enzymes are potential catalysts in biotechnological applications. This study reveals a novel degradation pathway for the hazardous organic compound styrene in CWB2. This study provides an impressive illustration of horizontal gene transfer, which enables novel metabolic capabilities. This study presents glutathione-dependent styrene metabolization in an (actino-)bacterium. Further, the genomic background of the ability of strain CWB2 to produce ibuprofen is demonstrated.

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Section: Resultsmentioning

confidence: 99%

On the Enigma of Glutathione-Dependent Styrene Degradation in Gordonia rubripertincta CWB2

Heine

Zimmerling

Ballmann

et al. 2018

Appl Environ Microbiol

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“…The genome sequences do not provide any obvious answers; for example, in strain NBB4, the ethene MO and propene MO gene clusters are not associated with any ferredoxins or flavodoxins or other obvious helper proteins. These gene clusters also lack chaperonin genes, which are frequently found immediately adjacent to SDIMO gene clusters (13,61,75,76). Strain NBB4 does possess chaperonin genes adjacent to its group 3 and 6 SDIMO gene clusters; could this be a prerequisite to later acquisition of the alkene monooxygenases?…”

Section: Discussionmentioning

confidence: 99%

Heterologous Expression of Mycobacterium Alkene Monooxygenases in Gram-Positive and Gram-Negative Bacterial Hosts

McCarl

Somerville

et al. 2018

Appl Environ Microbiol

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Alkene monooxygenases (MOs) are soluble di-iron-containing enzymes found in bacteria that grow on alkenes. Here, we report improved heterologous expression systems for the propene MO (PmoABCD) and ethene MO (EtnABCD) from strain NBB4. Strong functional expression of PmoABCD and EtnABCD was achieved in mc155, yielding epoxidation activities (62 and 27 nmol/min/mg protein, respectively) higher than any reported to date for heterologous expression of a di-iron MO system. Both PmoABCD and EtnABCD were specialized for the oxidation of gaseous alkenes (C to C), and their activity was much lower on liquid alkenes (C to C). Despite intensive efforts to express the complete EtnABCD enzyme in , this was not achieved, although recombinant EtnB and EtnD proteins could be purified individually in soluble form. The biochemical function of EtnD as an oxidoreductase was confirmed (1.36 μmol cytochrome reduced/min/mg protein). Cloning the EtnABCD gene cluster into KT2440 yielded detectable epoxidation of ethene (0.5 nmol/min/mg protein), and this could be stimulated (up to 1.1 nmol/min/mg protein) by the coexpression of chaperonins from either spp. or Successful expression of the ethene MO in a Gram-negative host was validated by both whole-cell activity assays and peptide mass spectrometry of induced proteins seen on SDS-PAGE gels. Alkene MOs are of interest for their potential roles in industrial biocatalysis, most notably for the stereoselective synthesis of epoxides. Wild-type bacteria that grow on alkenes have high activities for alkene oxidation but are problematic for biocatalysis, since they tend to consume the epoxide products. Using recombinant biocatalysts is the obvious alternative, but a major bottleneck is the low activities of recombinant alkene MOs. Here, we provide new high-activity recombinant biocatalysts for alkene oxidation, and we provide insights into how to further improve these systems.

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“…It has been pointed out that protein GroEL-like chaperone protein encoded by mmoG is essential for the maturation and functional expression of sMMO (Lloyd 1997). Similarly, a GroEL-like chaperonin protein was required for the expression of a binuclear iron monooxygenase of mycobacteria strains (Furuya et al 2012(Furuya et al , 2013.…”

Section: Challenges With Establishing Functional Heterologous Expressmentioning

confidence: 99%

Methane monooxygenases: central enzymes in methanotrophy with promising biotechnological applications

Khider

Brautaset

Irla

2021

World J Microbiol Biotechnol

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Worldwide, the use of methane is limited to generating power, electricity, heating, and for production of chemicals. We believe this valuable gas can be employed more widely. Here we review the possibility of using methane as a feedstock for biotechnological processes based on the application of synthetic methanotrophs. Methane monooxygenase (MMO) enables aerobic methanotrophs to utilize methane as a sole carbon and energy source, in contrast to industrial microorganisms that grow on carbon sources, such as sugar cane, which directly compete with the food market. However, naturally occurring methanotrophs have proven to be difficult to manipulate genetically and their current industrial use is limited to generating animal feed biomass. Shifting the focus from genetic engineering of methanotrophs, towards introducing metabolic pathways for methane utilization in familiar industrial microorganisms, may lead to construction of efficient and economically feasible microbial cell factories. The applications of a technology for MMO production are not limited to methane-based industrial synthesis of fuels and value-added products, but are also of interest in bioremediation where mitigating anthropogenic pollution is an increasingly relevant issue. Published research on successful functional expression of MMO does not exist, but several attempts provide promising future perspectives and a few recent patents indicate that there is an ongoing research in this field. Combining the knowledge on genetics and metabolism of methanotrophy with tools for functional heterologous expression of MMO-encoding genes in non-methanotrophic bacterial species, is a key step for construction of synthetic methanotrophs that holds a great biotechnological potential.

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Reconstitution of Active Mycobacterial Binuclear Iron Monooxygenase Complex in Escherichia coli

Cited by 12 publications

References 34 publications

On the Enigma of Glutathione-Dependent Styrene Degradation in Gordonia rubripertincta CWB2

On the Enigma of Glutathione-Dependent Styrene Degradation in Gordonia rubripertincta CWB2

Heterologous Expression of Mycobacterium Alkene Monooxygenases in Gram-Positive and Gram-Negative Bacterial Hosts

Methane monooxygenases: central enzymes in methanotrophy with promising biotechnological applications

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