Novel beta-carotene oxidases
AssigneeDsm IP Assets B.V.
InventorsPeter Louis HOUSTON, René Marcel DE JONG
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Abstract
The present invention is related to a method for increasing the trans-specificity of a beta-carotene oxidase (BCO), particularly insect BCO, to be used in the production of vitamin A aldehyde (retinal) from conversion of beta-carotene, with at least about 75 to 100% of retinal in the trans-isoform.
Claims14
A beta-carotene oxidase enzyme, preferably insect enzyme, more preferably enzyme originated from Drosophila, comprising one or more amino acid substitution(s) in a sequence with at least about 60%, such as 65, 70, 75, 80, 85, 90, 92, 95, 97, 98, 99% or up to 100% identity to SEQ ID NO:1, wherein the one or more amino acid substitution(s) are located at position(s) corresponding to amino acid residue(s) selected from 91 and/or 499 in the polypeptide according to SEQ ID NO:1 and wherein the amino acid of residue 91 being tryptophan or phenylalanine and/or amino acids of residue 499 being selected from methionine, leucine or isoleucine.
Method for increasing the trans-specificity of a beta-carotene oxidase enzyme comprising the steps of:
- (1) alignment of different beta-carotene oxidase enzymes, including but not limited to enzymes originated from insects, preferably from Drosophila, such as e.g. identified via BLAST search against UNIREF/UNIPROT databases, with SEQ ID NO:1, wherein the selected enzymes show high activity towards retinal production, i.e. in the range of at least about 5%, such as e.g. at least 2-fold higher than the BCO of Danio rerio,
- (2) identification of the positions in the selected enzymes corresponding to amino residue 91 and/or 499 in the polypeptide according to SEQ ID NO:1,
- (3) introduction of at least one or two amino acid substitution(s) on position(s) corresponding to amino acid residue(s) selected from 91, 499 and combinations thereof identified in SEQ ID NO:1 in the aligned sequences;
- and
- (4) screening for trans-retinal activity in a carotenoid-producing host cell, preferably selected from Yarrowia or Saccharomyces, with conversion rates of at least about 78 to 100% towards formation of trans-retinal based on total retinoids, whereby the activity of the enzyme, i.e. conversion of beta-carotene into retinal, is in the range of at least about 5%.
Dependencies are inferred from the claim text.