Tandem-yeast expression system for engineering and producing unspecific peroxygenase

Molina‐Espeja, Patricia; Ma, Su; Maté, Diana M.; Ludwig, Roland; Alcalde, Miguel

doi:10.1016/j.enzmictec.2015.03.004

Cited by 150 publications

(187 citation statements)

References 19 publications

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“…Such an approach was recently described for an A. aegerita UPO that was expressed in S. cerevisiae and mutated in nine positions, and then optimized for recombinant protein production and secretion in P. pastoris 25, 55, 56. Moreover, modern protein engineering techniques might help to design tailor‐made UPOs for specific steroid hydroxylations and to overcome catalytic bottlenecks such as solvent and peroxide instability.…”

Section: Discussionmentioning

confidence: 99%

A Peroxygenase from Chaetomium globosum Catalyzes the Selective Oxygenation of Testosterone

et al. 2017

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Unspecific peroxygenases (UPO, EC 1.11.2.1) secreted by fungi open an efficient way to selectively oxyfunctionalize diverse organic substrates, including less‐activated hydrocarbons, by transferring peroxide‐borne oxygen. We investigated a cell‐free approach to incorporate epoxy and hydroxyl functionalities directly into the bulky molecule testosterone by a novel unspecific peroxygenase (UPO) that is produced by the ascomycetous fungus Chaetomium globosum in a complex medium rich in carbon and nitrogen. Purification by fast protein liquid chromatography revealed two enzyme fractions with the same molecular mass (36 kDa) and with specific activity of 4.4 to 12 U mg−1. Although the well‐known UPOs of Agrocybe aegerita (AaeUPO) and Marasmius rotula (MroUPO) failed to convert testosterone in a comparative study, the UPO of C. globosum (CglUPO) accepted testosterone as substrate and converted it with total turnover number (TTN) of up to 7000 into two oxygenated products: the 4,5‐epoxide of testosterone in β‐configuration and 16α‐hydroxytestosterone. The reaction performed on a 100 mg scale resulted in the formation of about 90 % of the epoxide and 10 % of the hydroxylation product, both of which could be isolated with purities above 96 %. Thus, CglUPO is a promising biocatalyst for the oxyfunctionalization of bulky steroids and it will be a useful tool for the synthesis of pharmaceutically relevant steroidal molecules.

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Section: Discussionmentioning

confidence: 99%

A Peroxygenase from Chaetomium globosum Catalyzes the Selective Oxygenation of Testosterone

et al. 2017

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“…[23] PaDa-I harbors 9 mutations (F12Y-A14V-R15G-A21D-V57A-L67F-V75I-I248V-F311L: the mutations in the signal peptide are underlined) that enhance its functional expression in yeast (8 mg/L in S. cerevisiae and over 200 mg/L in Pichia pastoris). [24] At the same time this mutant retains strong activity and stability, particularly in terms of temperature and the presence of co-solvents. Here, mutant libraries of UPO were constructed by random mutagenesis, StEP recombination and in vivo shuffling and they were explored for 1-naphthol synthesis.…”

Section: Directed Evolution Approachmentioning

confidence: 99%

Synthesis of 1‐Naphthol by a Natural Peroxygenase Engineered by Directed Evolution

et al. 2016

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Abstract:There is an increasing interest in enzymes that catalyze the hydroxylation of naphthalene under mild conditions and with minimal requirements. To address this challenge, an extracellular fungal aromatic peroxygenase with mono(per)oxygenase activity was engineered to selectively convert naphthalene into 1-naphthol. Mutant libraries constructed by random mutagenesis and DNA recombination were screened for peroxygenase activity on naphthalene while quenching the undesired peroxidative activity on 1-naphthol (one-electron oxidation). The resulting double mutant (G241D-R257K) of this process was characterized biochemically and computationally. The conformational changes produced by directed evolution improved the substrate´s catalytic position. Powered exclusively by catalytic concentrations of H2O2, this soluble and stable biocatalyst has total turnover numbers of 50,000, with high regioselectivity (97%) and reduced peroxidative activity.

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“…Whether this tyrosine is responsible for the preferred epoxidation of isolated double bonds within complex moleculesin contrast to other UPOs, CglUPO also specifically epoxidized testosterone 18 -will have to be clarified in future mutagenesis studies. Although a yeast expression system for AaeUPO has been developed after enzyme directed evolution, 33 new procedures and hosts for expressing wild-type genes of this and other UPOs are required for investigating active-site residues and engineering UPO biocatalysts for selective oxygenation reactions of biotechnological interest. 34 …”

Section: View Article Onlinementioning

confidence: 99%

Selective synthesis of the resveratrol analogue 4,4′-dihydroxy-trans-stilbene and stilbenoids modification by fungal peroxygenases

Aranda

Ullrich

Kiebist

et al. 2018

Catal. Sci. Technol.

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aThis work gives first evidence that the unspecific peroxygenases (UPOs) from the basidiomycetes Agrocybe aegerita (AaeUPO), Coprinopsis cinerea (rCciUPO) and Marasmius rotula (MroUPO) are able to catalyze the regioselective hydroxylation of trans-stilbene to 4,4′-dihydroxy-trans-stilbene (DHS), a resveratrol (RSV) analogue whose preventive effects on cancer invasion and metastasis have very recently been shown. Nearly complete transformation of substrate (yielding DHS) was achieved with the three enzymes tested, using H 2 O 2 as the only co-substrate, with AaeUPO showing exceptionally higher total turnover number (200 000) than MroUPO (26 000) and rCciUPO (1400). Kinetic studies demonstrated that AaeUPO was the most efficient enzyme catalyzing stilbene dihydroxylation with catalytic efficiencies (k cat /K m ) one and two orders of magnitude higher than those of MroUPO and rCciUPO, so that 4-hydroxystilbene appears to be the best UPO substrate reported to date. In contrast, the peroxygenase from the ascomycete Chaetomium globosum (CglUPO) failed to hydroxylate trans-stilbene at the aromatic ring and instead produced the trans-epoxide in the alkenyl moiety. In addition, stilbenoids such as pinosylvin (Pin) and RSV were tested as substrates for the enzymatic synthesis of RSV from Pin and oxyresveratrol (oxyRSV) from both RSV and Pin. Overall, lower conversion rates and regioselectivities compared with trans-stilbene were accomplished by three of the UPOs, and no conversion was observed with CglUPO. The highest amount of RSV (63% of products) and oxyRSV (78%) were again attained with AaeUPO. True peroxygenase activity was demonstrated by incorporation of 18 O from H 2 18O 2 into the stilbene hydroxylation products. Differences in the number of phenylalanine residues at the heme access channels seems related to differences in aromatic hydroxylation activity, since they would facilitate substrate positioning by aromatic-aromatic interactions. The only ascomycete UPO tested (that of C. globosum) turned out to have the most differing active site (distal side of heme cavity) and reactivity with stilbenes resulting in ethenyl epoxidation instead of aromatic hydroxylation. The above oxyfunctionalizations by fungal UPOs represent a novel and simple alternative to chemical synthesis for the production of DHS, RSV and oxyRSV.

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Tandem-yeast expression system for engineering and producing unspecific peroxygenase

Cited by 150 publications

References 19 publications

A Peroxygenase from Chaetomium globosum Catalyzes the Selective Oxygenation of Testosterone

A Peroxygenase from Chaetomium globosum Catalyzes the Selective Oxygenation of Testosterone

Synthesis of 1‐Naphthol by a Natural Peroxygenase Engineered by Directed Evolution

Selective synthesis of the resveratrol analogue 4,4′-dihydroxy-trans-stilbene and stilbenoids modification by fungal peroxygenases

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