Recombinant polynucleotide sequence for producing astaxanthin and uses thereof

Abstract

Disclosed herein are recombinant polynucleotide sequences, vectors, host cells and methods for producing astaxanthin. The recombinant polynucleotide sequence is designed to provide a higher level of astaxanthin precursors via a shorter metabolic pathway, and thereby attains higher level of end products (e.g., astaxanthin) with desired stereoisomeric form and/or esterified form.

What is claimed is: 
     
         1 . A polypeptide having hydroxylase activity, comprising a first catalytic domain and a second catalytic domain, in which
 the first catalytic domain comprises the amino acid sequence of SEQ ID NO: 100, or a first variant thereof, wherein the amino acid residues of the first variant are at least 80% identical to the amino acid residues of SEQ ID NO: 100 at positions 1, 9, 14, 16-17, 19-20, 22-23, 26-29, 31-32, 34-36, 39-40, 45-46, 48-53, 55-56 and 58-61; and   the second catalytic domain comprises the amino acid sequence of SEQ ID NO: 101, or a second variant thereof, wherein the amino acid residues of the second variant are at least 80% identical to the amino acid residues of SEQ ID NO: 101 at positions 1-2, 5-6, 9-10, 13, 17, 20-29, 31, 34-36, 38-43, 45-47, 49-52, 55, 57-59, 63-64, 67-68, 71, 73-74 and 76.   
     
     
         2 . The polypeptide of  claim 1 , wherein
 the first catalytic domain comprises the amino acid sequence of SEQ ID NO: 100, or a first variant thereof, wherein the amino acid residues of the first variant are at least 90% identical to the amino acid residues of SEQ ID NO: 100 at positions 1, 9, 14, 16-17, 19-20, 22-23, 26-29, 31-32, 34-36, 39-40, 45-46, 48-53, 55-56 and 58-61; and   the second catalytic domain comprises the amino acid sequence of SEQ ID NO: 101, or a second variant thereof, wherein the amino acid residues of the second variant are at least 90% identical to the amino acid residues of SEQ ID NO: 101 at positions 1-2, 5-6, 9-10, 13, 17, 20-29, 31, 34-36, 38-43, 45-47, 49-52, 55, 57-59, 63-64, 67-68, 71, 73-74 and 76.   
     
     
         3 . The polypeptide of  claim 1 , wherein
 the first catalytic domain comprises the amino acid sequence of SEQ ID NO: 100, or a first variant thereof, wherein the amino acid residues of the first variant are at least 95% identical to the amino acid residues of SEQ ID NO: 100 at positions 1, 9, 14, 16-17, 19-20, 22-23, 26-29, 31-32, 34-36, 39-40, 45-46, 48-53, 55-56 and 58-61; and   the second catalytic domain comprises the amino acid sequence of SEQ ID NO: 101, or a second variant thereof, wherein the amino acid residues of the second variant are at least 95% identical to the amino acid residues of SEQ ID NO: 101 at positions 1-2, 5-6, 9-10, 13, 17, 20-29, 31, 34-36, 38-43, 45-47, 49-52, 55, 57-59, 63-64, 67-68, 71, 73-74 and 76.   
     
     
         4 . The polypeptide of  claim 1 , wherein
 the first catalytic domain comprises the amino acid sequence at least 80% identical to the sequence of SEQ ID NO: 102, 103 or 104; and   the second catalytic domain comprises the amino acid sequence at least 80% identical to the sequence of SEQ ID NO: 105, 106 or 107.   
     
     
         5 . The polypeptide of  claim 1 , wherein
 the first catalytic domain comprises the amino acid sequence at least 90% identical to the sequence of SEQ ID NO: 102, 103 or 104; and   the second catalytic domain comprises the amino acid sequence at least 90% identical to the sequence of SEQ ID NO: 105, 106 or 107.   
     
     
         6 . The polypeptide of  claim 1 , wherein
 the first catalytic domain comprises the amino acid sequence at least 95% identical to the sequence of SEQ ID NO: 102, 103 or 104; and   the second catalytic domain comprises the amino acid sequence at least 95% identical to the sequence of SEQ ID NO: 105, 106 or 107.   
     
     
         7 . The polypeptide of  claim 1 , wherein the polypeptide catalyzes at least one of the following reactions:
 the formation of adonirubin from canthaxanthin,   the formation of astaxanthin from adonirubin,   the formation of β-cryptoxanthin from β-carotene,   the formation of zeaxanthin from β-cryptoxanthin.   
     
     
         8 . The polypeptide of  claim 7 , wherein the astaxanthin is 3S, 3S′-astaxanthin. 
     
     
         9 . A method of improving the tolerance of a host cell to a stress, comprising introducing into the host cell at least one polynucleotide encoding a polypeptide for producing a carotenoid. 
     
     
         10 . The method of  claim 9 , wherein the carotenoid is at least one selected from the group consisting of lypcopene, β-carotene, β-cryptoxanthin, zeaxanthin, β-doradexanthin, echinenone, canthaxanthin, adonirubin and astaxanthin. 
     
     
         11 . The method of  claim 9 , wherein the stress to the host cell is caused by being exposed to ethanol, butanol, UV exposure, furfural, or a drug precursor. 
     
     
         12 . The method of  claim 11 , wherein the drug precursor is 10-deacetyl baccatin III. 
     
     
         13 . The method of  claim 9 , wherein
 the polypeptide has the amino acid sequence of SEQ ID NO: 108, or is a first variant thereof, wherein the amino acid sequence of the first variant is at least 80% identical to the amino acid sequence of SEQ ID NO: 108;   the polypeptide has the amino acid sequence of SEQ ID NO: 109, or is a second variant thereof, wherein the amino acid sequence of the second variant is at least 80% identical to the amino acid sequence of SEQ ID NO: 109; or   the polypeptide has the amino acid sequence of SEQ ID NO: 110, or is a third variant thereof, wherein the amino acid sequence of the third variant is at least 80% to the amino acid sequence of SEQ ID NO: 110.   
     
     
         14 . The method of  claim 9 , wherein
 the polypeptide has the amino acid sequence of SEQ ID NO: 108, or is a first variant thereof, wherein the amino acid sequence of the first variant is at least 90% identical to the amino acid sequence of SEQ ID NO: 108;   the polypeptide has the amino acid sequence of SEQ ID NO: 109, or is a second variant thereof, wherein the amino acid sequence of the second variant is at least 90% identical to the amino acid sequence of SEQ ID NO: 109; or   the polypeptide has the amino acid sequence of SEQ ID NO: 110, or is a third variant thereof, wherein the amino acid sequence of the third variant is at least 90% to the amino acid sequence of SEQ ID NO: 110.   
     
     
         15 . The method of  claim 9 , wherein
 the polypeptide has the amino acid sequence of SEQ ID NO: 108, or is a first variant thereof, wherein the amino acid sequence of the first variant is at least 95% identical to the amino acid sequence of SEQ ID NO: 108;   the polypeptide has the amino acid sequence of SEQ ID NO: 109, or is a second variant thereof, wherein the amino acid sequence of the second variant is at least 95% identical to the amino acid sequence of SEQ ID NO: 109; or   the polypeptide has the amino acid sequence of SEQ ID NO: 110, or is a third variant thereof, wherein the amino acid sequence of the third variant is at least 95% to to the amino acid sequence of SEQ ID NO: 110.