Patent application EP19382479A (EP), filed 2019-06-07
Publication number: EP3748000A1
Title: UNSPECIFIC PEROXYGENASE ENZYME VARIANTS FOR SELECTIVE FATTY ACID EPOXIDATION OR HYDROXYLATION
Abstract: The invention relates to a recombinant Marasmius rotula unspecific peroxygenase (rMroUPO) and two mutants thereof, wherein said mutants show enhanced selectivity towards either the epoxidation or the (sub)terminal ω/(ω-1)-hydroxylation of unsaturated fatty acids. The invention also refers to the use of these enzyme variants for the specific epoxidation or hydroxylation of fatty acids such as oleic acid, linoleic acid and/or alpha-linolenic acid.
IPC codes: C12N  15/    52            A I
C12N   9/    08            A I
Claims: number: 1
text: A recombinant peroxygenase enzyme that comprises the amino acid sequence shown in SEQ ID NO: 1, wherein said enzyme is encoded by a polynucleotide sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 9, preferably by the polynucleotide sequence shown in SEQ ID NO: 9.
number: 2
text: The enzyme according to claim 1, which consists of the amino acid sequence shown in SEQ ID NO: 1.
number: 3
text: The enzyme according to any of claims 1 or 2, which comprises the amino acid substitution I153T.
number: 4
text: The enzyme according to any of claims 1 or 2, which comprises the amino acid substitutions I153F and S156F.
number: 5
text: A polynucleotide sequence that encodes the enzyme according to any of claims 1 to 4.
number: 6
text: A genetic construct that comprises the polynucleotide sequence according to claim 5.
number: 7
text: A host cell that comprises the polynucleotide sequence according to claim 5 or the genetic construct according to claim 6.
number: 8
text: The host cell according to claim 7 which is an Escherichia coli cell.
number: 9
text: Use of the host cell according to any of claims 7 or 8 for producing the enzyme according to any of claims 1 to 4.
number: 10
text: Use of the enzyme according to any of claims 1 or 2 for the epoxidation and hydroxylation of unsaturated fatty acids.
number: 11
text: Use of the enzyme according to claim 3 for the epoxidation of unsaturated fatty acids.
number: 12
text: Use of the enzyme according to claim 11, wherein the unsaturated fatty acids are polyunsaturated fatty acids.
number: 13
text: Use of the enzyme according to claim 4 for the terminal and sub-terminal hydroxylation of unsaturated fatty acids.
number: 14
text: Use according to any of claims 10 to 13, wherein the unsaturated fatty acids are selected from the list consisting of: oleic acid, linoleic acid, alpha-linolenic acid or any combination thereof.
Office action: search_report, dated 2020-12-09
Rejection reasons: novelty_epc_54
Prior art cited: ref_id: - AOUF, C. ET AL., GREEN CHEM., vol. 16, 2014, pages 1740 - 1754
ref_type: npl
severity: background
ref_id: - SUSTAITA-RODRIGUEZ, A. ET AL., CHEMISTRY CENTRAL JOURNAL, vol. 12, 2018, pages 39
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severity: background
ref_id: - HAITZ, F. ET AL., APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY, vol. 185, 2018, pages 13 - 33
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severity: background
ref_id: - RUETTINGER, R. T.FULCO, A. J., J. BIOL. CHEM., vol. 256, 1981, pages 5728 - 5734
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severity: background
ref_id: - KOCKRITZ, A.MARTIN, A., EUR. J. LIPID SCI. TECHNOL., vol. 113, 2011, pages 83 - 91
ref_type: npl
severity: background
ref_id: - HAN, L. ET AL., FRONTIERS IN MICROBIOLOGY, vol. 8, 2017, pages 2184
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severity: background
ref_id: - KIM, S. K.PARK, Y. C., APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 103, 2019, pages 191 - 199
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severity: background
ref_id: - ARANDA, C. ET AL., CHEMCATCHEM, vol. 10, 2018, pages 3964 - 3968
ref_type: npl
severity: background
ref_id: - MOLINA-ESPEJA, P. ET AL., APPL. ENVIRON. MICROBIOL., vol. 80, 2014, pages 3496 - 3507
ref_type: npl
severity: background
ref_id: - MOLINA-ESPEJA, P. ET AL., ENZYME MICROB. TECHNOL., vol. 73-74, 2015, pages 29 - 33
ref_type: npl
severity: background
ref_id: - ANDRES OLMEDO ET AL: "From Alkanes to Carboxylic Acids: Terminal Oxygenation by a Fungal Peroxygenase", ANGEWANDTE CHEMIE, INTERNATIONAL EDITION, vol. 55, no. 40, 30 August 2016 (2016-08-30), DE, pages 12248 - 12251, XP055415744, ISSN: 1433-7851, DOI: 10.1002/anie.201605430
ref_type: npl
severity: background
ref_id: EP3392341A1
ref_type: patent
severity: background
ref_id: WO2016207373A1
ref_type: patent
severity: background
ref_id: - CARMEN ARANDA ET AL: "Selective Epoxidation of Fatty Acids and Fatty Acid Methyl Esters by Fungal Peroxygenases", CHEMCATCHEM, vol. 10, no. 18, 26 July 2018 (2018-07-26), DE, pages 3964 - 3968, XP055640437, ISSN: 1867-3880, DOI: 10.1002/cctc.201800849
ref_type: npl
severity: background
ref_id: - GLENN GRÖBE ET AL: "High-yield production of aromatic peroxygenase by the agaric fungus Marasmius rotula", AMB EXPRESS, vol. 1, no. 31, 2011, pages 1 - 11, XP055640426
ref_type: npl
severity: background
ref_id: WO2016207373
ref_type: patent
severity: novelty_destroying
ref_id: EP3392341
ref_type: patent
severity: novelty_destroying
ref_id: - CARMEN ARANDA ET AL, "Selective Epoxidation of Fatty Acids and Fatty Acid Methyl Esters by Fungal Peroxygenases", CHEMCATCHEM, DE, (20180726), vol. 10, no. 18, doi:10.1002/cctc.201800849, ISSN 1867-3880, pages 3964 - 3968, XP055640437 [X] 1-4,9-14 * abstract * * page  3964, column  r, paragraph  2 *
ref_type: npl
severity: novelty_destroying
ref_id: - GLENN GRÖBE ET AL, "High-yield production of aromatic peroxygenase by the agaric fungus Marasmius rotula", AMB EXPRESS, (2011), vol. 1, no. 31, pages 1 - 11, XP055640426 [X] 1-4,9-14 * abstract *
ref_type: npl
severity: novelty_destroying
Final disposition: pending (2020-12-09)