Methods for determining ring number in carotenoids by lycopene epsilon-cyclasses and uses thereof

A method for identifying an enzyme-catalyzing domain in a eukaryotic or prokaryotic carotenoid-synthesizing enzyme, comprising

• a) providing a first polynucleotide encoding a full-length enzyme and a second polynucleotide encoding a full-length enzyme, each polynucleotide being subcloned in tandem into a vector;
• b) providing a first primer for hybridizing to the first polynucleotide and a second primer for hybridizing to the second polynucleotide;
• c) performing an inverse polymerase chain reaction using the first and the second primer and the vector to obtain a construct containing a chimeric polynucleotide containing a 5′ end of the first polynucleotide and a 3′ end of the second polynucleotide;
• d) repeating steps b) and c) with a plurality of different first primers and a plurality of different second primers for obtaining a plurality of constructs containing different chimeric polynucleotides for scanning along the encoded amino acid sequence one amino acid at a time;
• e) transfecting a host cell with one or more of the plurality of constructs and growing the host cell under conditions for expressing chimeric proteins encoded by the chimeric polynucleotides;
• f) performing enzyme catalysis with the chimeric proteins on an enzyme-specific substrate in the host cell, and
• g) identifying the enzyme-catalyzing domain encoded by the chimeric proteins by identification of at least one carotenoid compound from the enzyme catalysis of step f).

A method for identifying an enzyme-catalyzing domain in a eukaryotic or prokaryotic carotenoid-synthesizing enzyme, comprising

• a) providing a vector containing a polynucleotide encoding the full-length enzyme and a primer for hybridizing to the polynucleotide;
• b) performing site-directed mutagenesis using the vector and the primer for obtaining a construct containing a site-directed mutant of the polynucleotide encoding the enzyme;
• c) transfecting a host cell with the construct and growing the host cell under conditions for expressing a site-directed mutant of a protein encoded by the site-directed mutant of the polynucleotide;
• d) allowing enzyme catalysis with the site-directed mutant of the protein on an enzyme-specific substrate in the host cell;
• and
• e) identifying the enzyme-catalyzing domain encoded by the site-directed mutant of the protein by identification of a carotenoid compound from the enzyme catalysis of step d).

A method for identifying an enzyme-catalyzing domain in a eukaryotic or prokaryotic carotenoid-synthesizing enzyme, comprising

• a) providing a vector containing a polynucleotide encoding the full-length enzyme and a primer for hybridizing to the polynucleotide;
• b) performing site-directed mutagenesis using the primer and the vector for obtaining a construct containing a truncated polynucleotide encoding a fragment of the enzyme;
• c) transfecting a host cell with the construct and growing the host cell under conditions for expressing a truncated protein encoded by the truncated polynucleotide;
• d) allowing enzyme catalysis with the truncated protein on an enzyme-specific substrate in the host cell;
• and
• e) identifying the enzyme-catalyzing domain encoded by the truncated protein by identification of a carotenoid compound from the enzyme catalysis of step d).