Cloning and overexpression of the old yellow enzyme gene of Candida macedoniensis, and its application to the production of a chiral compound
The gene encoding old yellow enzyme (OYE), which catalyzes the conversion of ketoisophorone (KIP; 2,6,6-trimethyl-2-cyclohexen-1,4-dione) to (6 R)-levodione (2,2,6-trimethylcyclohexane-1,4-dione), of Candida macedoniensis was cloned and sequenced. A 1212 bp nucleotide fragment ( oye) was confirmed t...
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Veröffentlicht in: | Journal of biotechnology 2004-10, Vol.114 (1), p.1-9 |
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Zusammenfassung: | The gene encoding old yellow enzyme (OYE), which catalyzes the conversion of ketoisophorone (KIP; 2,6,6-trimethyl-2-cyclohexen-1,4-dione) to (6
R)-levodione (2,2,6-trimethylcyclohexane-1,4-dione), of
Candida macedoniensis was cloned and sequenced. A 1212
bp nucleotide fragment (
oye) was confirmed to be the gene encoding OYE based on the agreement of internal amino acid sequences.
Oye encodes a total 403 amino acid residues, and the deduced amino acid sequence shows a high degree of similarity to those of other microbial OYE family proteins. An expression vector, pETOYE, that contains the full length of
oye was constructed.
Escherichia coli harboring pETOYE exhibited an about six-fold increase in specific KIP-reducing activity under the control of the T7 promoter as compared with that of
C. macedoniensis. (6
R)-Levodione formed with washed cells of the transformant and a cofactor regeneration system amounted to 638
mM (98.2
mg
ml
−1), the a molar yield being 96.9%.
The asymmetric reduction of KIP to (6
R)-levodione with
E. coli cells, which co-expressed both
oye and the glucose dehydrogenase gene (
gdh), as a catalyst was investigated. The (6
R)-levodione formed amounted to 627
mM (96.6
mg
ml
−1), the a molar yield being 95.4%. Since the use of
E. coli BL21 (DE3) cells co-expressing
oye and
gdh as a catalyst is simple and does not require the addition of glucose dehydrogenase, it is highly advantageous for the practical synthesis of (6
R)-levodione. |
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ISSN: | 0168-1656 1873-4863 |
DOI: | 10.1016/j.jbiotec.2004.04.033 |