Acetyl-transferases
The present invention is related to production of retinyl acetate generated via enzymatic conversion of retinol, said process including the use of modified enzymes with improved activity.
1 . A modified acetyl transferase enzyme comprising an amino acid sequence having at least 90% identity to SEQ ID NO:1, wherein:
the amino acid sequence comprises one or more substitute residue(s) relative to SEQ ID NO:1,
the one or more substitute residue(s) are located at position(s) corresponding to position 68, 69, 72, 73, 171, 172, 173, 174, 176, 178, 291, 292, 294, 301, 307, 308, 311, 312, 320, 322, 334, 362, 405, 407, 409, 480, 483, 484, 490, 492, 520, 521, 522, 524, 525, 526 and combinations thereof according to SEQ ID NO:1, and
the one or more substitute residue(s) is/are selected from the group consisting of: substitute residue corresponding to position 68 according to SEQ ID NO:1 is threonine, substitute residue corresponding to position 69 according to SEQ ID NO:1 is asparagine or serine or alanine, substitute residue corresponding to position 72 according to SEQ ID NO:1 is asparagine or lysine, substitute residue corresponding to position 73 according to SEQ ID NO:1 is leucine, substitute residue corresponding to position 171 according to SEQ ID NO:1 is lysine or asparagine, substitute residue corresponding to position 172 according to SEQ ID NO:1 is glycine, substitute residue corresponding to position 173 according to SEQ ID NO: 1 is leucine, substitute residue corresponding to position 174 according to SEQ ID NO:1 is isoleucine, substitute residue corresponding to position 176 according to SEQ ID NO:1 is alanine or glycine, substitute residue corresponding to position 178 according to SEQ ID NO:1 is valine, substitute residue corresponding to position 291 according to SEQ ID NO:1 is serine or glycine, substitute residue corresponding to position 292 according to SEQ ID NO:1 is alanine or serine or asparagine, substitute residue corresponding to position 294 according to SEQ ID NO:1 is leucine or valine, substitute residue corresponding to position 301 according to SEQ ID NO:1 is phenylalanine, substitute residue corresponding to position 307 according to SEQ ID NO:1 is isoleucine, substitute residue corresponding to position 308 according to SEQ ID NO:1 is valine, substitute residue corresponding to position 311 according to SEQ ID NO:1 is methionine or isoleucine, substitute residue corresponding to position 312 according to SEQ ID NO:1 is alanine, substitute residue corresponding to position 320 according to SEQ ID NO:1 is asparagine, substitute residue corresponding to position 322 according to SEQ ID NO:1 is valine or phenylalanine, substitute residue corresponding to position 334 according to SEQ ID NO:1 is methionine or leucine, substitute residue corresponding to position 362 according to SEQ ID NO:1 is alanine, substitute residue corresponding to position 405 according to SEQ ID NO:1 is alanine, substitute residue corresponding to position 407 according to SEQ ID NO:1 is isoleucine, substitute residue corresponding to position 409 according to SEQ ID NO:1 is alanine, substitute residue corresponding to position 480 according to SEQ ID NO:1 is glutamic acid or leucine or methionine or phenylalanine or glutamine, substitute residue corresponding to position 483 according to SEQ ID NO:1 is valine, substitute residue corresponding to position 484 according to SEQ ID NO:1 is leucine, substitute residue corresponding to position 490 according to SEQ ID NO:1 is isoleucine, substitute residue corresponding to position 492 according to SEQ ID NO:1 is valine, substitute residue corresponding to position 520 according to SEQ ID NO:1 is alanine, substitute residue corresponding to position 521 according to SEQ ID NO:1 is alanine or valine, substitute residue corresponding to position 522 according to SEQ ID NO:1 is serine or threonine, substitute residue corresponding to position 524 according to SEQ ID NO:1 is asparagine or serine, substitute residue corresponding to position 525 according to SEQ ID NO:1 is valine or isoleucine or arginine, and substitute residue corresponding to position 526 according to SEQ ID NO:1 is serine, and combinations thereof.
2 . The modified enzyme according to claim 1 , wherein the amino acid sequence comprises one substituted residue relative to SEQ ID NO: 1, wherein the substituted residue is at a position corresponding to position 68, 69, 72, 73, 171, 172, 173, 174, 176, 178, 291, 292, 294, 301, 307, 308, 311, 312, 320, 322, 334, 362, 405, 407, 409, 480, 483, 484, 490, 492, 520, 521, 522, 524, 525, or 526 according to SEQ ID NO:1.
3 . The modified enzyme according to claim 1 , wherein the amino acid sequence comprises a first, second or third set of two substituted residues relative to SEQ ID NO: 1, wherein the first set of substituted residues is at positions 480 and 409, the second set of substituted residues is at positions 480 and 407, and the third set of substituted residues is at positions 407 and 409 according to SEQ ID NO:1.
4 . The modified enzyme according to claim 3 , wherein the amino acid sequence further comprises one or more substitute residue(s) at position(s) corresponding to position 68, 69, 72, 311, 334, 484, and combinations thereof according to SEQ ID NO:1.
5 . The modified enzyme according to claim 1 , wherein the one or more substitute residue(s) are selected from the group consisting of Q68T, H69A, H69N, H69S, Q72K, Q72N, I73L, G171K, G171N, N172G, Q173L, V174I, S176A, S176G, L178V, A291G, A291S, G292A, G292N, G292S, F294L, F294V, Y301F, P3071, T308V, T311I, T311M, S312A, H320N, Y322F, Y322V, V334L, V334M, S362A, S405A, V407I, G409A, S480E, S480F, S480L, S480M, S480Q, L483V, I484L, V490I, D492V, I520A, C521A, C521V, A522S, A522T, D524N, D524S, Q525V, Q525I, Q525R, G526S, and combinations thereof, wherein the substitute residue(s) position(s) is/are according to SEQ ID NO:1.
6 . The modified enzyme according to claim 1 , wherein the modified enzyme's activity towards conversion or acetylation of retinol into retinyl acetate is increased by about 0.2 to 4-fold compared to the corresponding unmodified acetyl transferase enzyme.
7 . The modified enzyme according to claim 1 , which is expressed in a retinol-producing host cell.
8 . The modified enzyme according to claim 1 , wherein the amino acid sequence comprises the [NDEHCS]-H-x(3)-D-[GA] motif at positions corresponding to positions 218 to 224 according to SEQ ID NO:1, wherein “x” denotes an arbitrary amino acid and the histidine at the second position of the motif is part of the enzyme's binding pocket.
9 . A retinoid-producing host cell expressing the modified enzyme according to claim 6 .
10 . A process for the production of retinoids, comprising cultivating the host cell according to claim 9 under suitable culture conditions using a carbon source comprising glucose, fructose, raffinose, lactose, galactose, glycerol, xylose, arabinose, sucrose and/or maltose.
11 . The process according to claim 10 , wherein the retinoids produced comprise retinyl acetate at a percentage of about 0.2 to 4× higher compared to retinoids produced by a corresponding reference host cell expressing the corresponding unmodified acetyl transferase enzyme without said one or more substitute residue(s).
12 . A process for production of retinyl acetate comprising providing the retinol-producing host cell according to claim 9 , cultivating said host cell in a suitable culture medium under suitable culture conditions, and isolating and/or purifying the retinyl acetate from the medium.
13 . The modified enzyme according to claim 8 , wherein the motif comprises the sequence of [NDE]-H-x(3)-D-[GA].
14 . The modified enzyme according to claim 8 , wherein the motif comprises the sequence of [ND]-H-x(3)-D-[GA].
15 . The modified enzyme according to claim 8 , wherein the motif comprises the sequence of N—H-x(3)-D-[GA].
16 . The process according to claim 10 , wherein the host cell is a Yarrowia or Saccharomyces cell.
17 . The process according to claim 11 , wherein the host cell is a Yarrowia or Saccharomyces cell.
18 . The process according to claim 12 , wherein the host cell is a Yarrowia or Saccharomyces cell.
19 . The modified enzyme according to claim 1 , wherein the one or more substitute residue(s) are selected from the group consisting of V407I and H69S, V407I and Q72K, V407I and T311I, V407I and V334L, V407I and S480L, V407I and I484L, G409A and H69N, G409A and H69S, G409A and H69A, G409A and Q72K, G409A and T311I, G409A and V334L, G409A and V407I, G409A and S480L-489K, S480Q and Q68T, S480Q and H69N, S480Q and H69S, S480Q and H69A, S480Q and Q72K, S480Q and T311I, S480Q and V334L, S480Q and V407I, S480Q and G409A, and S480Q and I484L, wherein the substitute residue(s) position(s) is/are according to SEQ ID NO:1.
20 . The modified enzyme according to claim 1 , wherein the one or more substitute residue(s) are selected from the group consisting of G409A/V407I/H69N, G409A/V407I/Q72K, G409A/V407I/T311I, G409A/V407I/V334L, G409A/V407I/S480L, G409A/V407I/I484L, S480Q/V407I/Q68T, S480Q/V407I/H69N, S480Q/V407I/H69S, S480Q/V407I/H69A, S480Q/V407I/Q72K, S480Q/V407I/T311I, S480Q/V407I/V334L, S480Q/V407I/I484L, S480Q/G409A/Q68T, S480Q/G409A/H69A, S480Q/G409A/H69N, S480Q/G409A/I484L, S480Q/G409A/V407I/H69N, S480Q/G409A/V407I/H69A, S480Q/G409A/V407I/H69S, S480Q/G409A/V407I/Q72K, S480Q/G409A/V407I/T311I, S480Q/G409A/V407I/V334L, S480Q/G409A/V407I/I484L, and S480L/G409A/V407I/T311I, wherein the substitute residue(s) position(s) is/are according to SEQ ID NO:1.