Bioproduction of astaxanthin using mutant carotenoid ketolase and carotenoid hydroxylase genes
AssigneeE. I. Du Pont de Nemours and Company
InventorsXiao-Song Tang, Qiong Cheng, Joanne Shyr, Luan Tao
Life of this patent
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Abstract
Protein engineered nucleic acid fragments encoding a CrtO ketolase and a CrtZ hydroxylase are provided with increased astaxanthin synthesis activity. Methods using the present nucleic acid fragments are also provided for increasing or altering astaxanthin production in suitable production hosts.
Claims28
An isolated nucleic acid molecule encoding at least one carotenoid ketolase and at least one carotenoid hydroxylase, said nucleic acid molecule comprising:
- a) a nucleic acid fragment encoding a carotenoid ketolase having an amino acid sequence selected from the group consisting of SEQ ID NO:
- 17 and SEQ ID NO. 22;
- and
- b) a isolated nucleic acid fragment encoding a carotenoid hydroxylase having an amino acid sequence selected from the group consisting of SEQ ID NO:
- 19 and SEQ ID NO:
- 24:
- or
- an isolated nucleic acid molecule completely complementary to the nucleic acid molecule comprising the elements of (a) and (b).
An isolated nucleic acid molecule encoding at least one carotenoid ketolase and at least one carotenoid hydroxylase, said nucleic acid molecule comprising:
- a) a nucleic acid fragment encoding a carotenoid ketolase having a nucleic acid sequence selected from the group consisting of SEQ ID NO:
- 16 and SEQ ID NO:
- 21;
- and
- b) a nucleic acid fragment encoding a carotenoid hydroxylase having a nucleic acid sequence selected from the group consisting of SEQ ID NO:
- 18 and SEQ D NO:
- 23;
- or
- an isolated nucleic acid molecule completely complementary to the nucleic acid molecule comprising the elements of (a) and (b).
An isolated nucleic acid molecule encoding a carotenoid ketolase and a carotenoid hydroxylase, said isolated nucleic acid molecule comprising:
- a) a nucleic acid fragment encoding a carotenoid ketolase having the amino acid sequence as represented by SEQ ID NO:
- 17 and
- a nucleic acid fragment encoding a carotenoid hydroxylase enzyme having the amino acid sequence as represented by SEQ ID NO:
- 19;
- or
- (b) an isolated nucleic acid molecule completely complementary to the nucleic acid fragment of (a).
An isolated nucleic acid molecule encoding a carotenoid ketolase and a carotenoid hydroxylase, said isolated nucleic acid molecule comprising:
- (a) a nucleic acid fragment encoding a carotenoid ketolase having the amino acid sequence as represented by SEQ ID NO:
- 21;
- and
- a nucleic acid fragment encoding a carotenoid hydroxylase enzyme having the amino acid sequence as represented by SEQ ID NO:
- 23;
- or
- (b) an isolated nucleic acid molecule completely complementary to the nucleic acid fragment of (a).
(canceled)
(canceled)
A method to generate and identify nucleic acid molecules encoding a carotenoid ketolase and a carotenoid hydroxylase having improved astaxanthin biosynthesis activity comprising:
- a) providing a host cell capable of producing β-carotene;
- b) providing a starting pair of genes comprising a carotenoid ketolase gene and a carotenoid hydroxylase;
- c) exposing said starting pair of genes simultaneously to mutational conditions in vitro to form a mutated gene pair;
- wherein at least one nucleotide has been altered in either one or both of said carotenoid ketolase gene or said carotenoid hydroxylase gene;
- d) operably linking the mutated gene pair to a suitable regulatory sequence;
- e) transforming the host cell of step a) with the mutated gene pair from step d) to produce a recombinant host cell;
- f) growing the recombinant host cell under conditions whereby astaxanthin is produced;
- g) measuring the amount of astaxanthin produced in step f) and selecting those transformants having increased astaxanthin production relative to the level of astaxanthin produced by the starting pair of genes in the host cell;
- and
- h) identifying the mutated gene pair from the selected transformants which have increased astaxanthin biosynthesis activity.
Dependencies are inferred from the claim text.