Discovery of a Novel Styrene Monooxygenase Originating from the Metagenome

Abstract

Oxygenases form an interesting class of biocatalysts, as they typically perform oxygenations with exquisite chemo-, regio-, and/or enantioselectivity. It has been observed that, once heterologously expressed in Escherichia coli, some oxygenases are able to form the blue pigment indigo. We have exploited this characteristic to screen a metagenomic library derived from loam soil and identified a novel oxygenase. This oxygenase shows 50% sequence identity with styrene monooxygenases from pseudomonads (StyA). Only a limited number of homologs can be found in the genome sequence database, indicating that this biocatalyst is a member of a relatively small family of bacterial monooxygenases. The newly identified monooxygenase catalyzes the epoxidation of styrene and styrene derivatives and forms the corresponding (S)-epoxides with excellent enantiomeric excess [e.g., (S)-styrene oxide is formed with >99% enantiomeric excess, ee] and therefore is named styrene monooxgenase subunit A (SmoA). SmoA shows high enantioselectivity towards aromatic sulfides [e.g., (R)-ethyl phenyl sulfoxide is formed with 92% ee]. This excellent enantioselectivity in combination with the moderate sequence identity forms a clear indication that SmoA from a metagenomic origin represents a new enzyme within the small family of styrene monooxygenases.Oxygenases are of growing interest for biotechnological applications (5, 38). These oxidative biocatalysts are able to insert one or two oxygen atoms into a substrate molecule. In order to do so, they usually need NAD(P)H as an electron donating coenzyme, while a metal or organic cofactor is required for oxygen functionalization. A feature that makes these biocatalysts of special interest is their ability to perform oxygenations in a chemo-, regio-, and/or enantioselective manner (13, 38). Often, selectivities are observed that cannot be rivaled by chemical approaches. Due to their selectivity and ability to use oxygen as a cheap and environmentally friendly oxidant, oxygenases form an important class of enzymes which can be applied for the biocatalytic oxidation of several compounds. The need for expensive coenzymes is no longer considered a hurdle for such industrial applications, since biocatalytic systems employing whole cells can be used which circumvent expensive coenzyme regeneration procedures (36).Oxygenases are found in all kingdoms of life but especially in bacteria. Their physiological role is often related to degradation of toxic compounds or the synthesis of secondary metabolites (40). The classical approach to finding novel oxygenases with biocatalytic potential is therefore to screen for organisms which are able to grow on toxic compounds, like aromatics, or to use cultures which are enriched with these kinds of compounds. However, this method only focuses on the culturable portion of organisms which contain putative oxygenases, and it is estimated that more than 99% of all microbes are not cultivable (1) and therefore not accessible as a source for finding novel biocatalysts. With the...

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

“…Amino acid residues at positions 73 and 96 are the same for all SMOs originating from the genus Pseudomonas, as well as the one from the metagenome (van Hellemond et al, 2007), i.e. Tyr and Ser, respectively.…”

“…Therefore, we assumed that the putative active cavity of SMO should be strictly shaped by surrounding residues and the mutations could hardly change the orientation of the substrate. The reversal of enantioselectivity for the mutant Y73V during the epoxidation of 1-phenylcyclohexene was unexpected because all other experimentally assigned SMOs display the same enantioselectivity (Bernasconi et al, 2000;Di Gennaro et al, 1999;Lin et al, 2010;Panke et al, 1998;Park et al, 2005;Tischler et al, 2009;van Hellemond et al, 2005), and the overall structure of the active cavity of SMOs should not be flexible enough to generate complementary enantiomers. Moreover, the mutants from directed evolution were also reported to produce (S)-enantiomers (Gursky et al, 2010).…”

“…The majority of SMOs characterized so far have shown epoxidation activity toward a variety of substituted styrene derivatives and heterocyclic styrene analogues, and the SMO from Pseudomonas sp. LQ26 also takes non-conjugated secondary allylic alcohols as substrates (Bernasconi et al, 2000(Bernasconi et al, , 2004Lin et al, 2011a,b,c;Park et al, 2005;van Hellemond et al, 2005). Although SMOs display excellent enantioselectivity in most cases and could serve as a good complementary or better approach to existing chemo-catalytic methods, it remains highly desired to improve their catalytic activity in order to enhance its potential for scaled-up production.…”

Isolation and Characterization of a 4-Nitrotoluene-Oxidizing Enzyme from Activated Sludge by a Metagenomic Approach

“…On the other hand, icpA was able to convert 4-NT in the absence of icpB. Likewise, styrene monooxygenase, also a flavoprotein monooxygenase, does not need a flavin reductase component to be active (50). Apparently, no specific interaction is required for enzymatic activity between icpA and icpB.…”

“…Our genes were not at all similar to the gene for indigo production from another metagenome (26,28,50). Nor did they align with the gene products which yielded indigo in E. coli (6,9).…”