title: Molecular cloning and characterization of the ATP citrate lyase from carotenogenic yeast Phaffia rhodozyma.
abstract: ATP citrate lyase (ACL), is a key cytosolic source of acetyl-CoA for fatty acid and sterol biosynthesis and appear to be involved in carotenoid biosynthesis in yeasts. Three homologous DNA sequences encoding ACLs in Phaffia rhodozyma were isolated i.e two genes and one cDNA. The two genes were multi-intronic, with 3450-bp-coding sequences and both genes, as the cDNA, encoded identical 120.1-kDa polypeptides. Full-length amino acid sequences of these ACLs showed the two multidomains, PLN02235 and PLN02522, which are necessary for activity. The ACLs showed 82-87% similarity to putative ACLs from other basidiomycetes and 71% similarity to human ACL. The acl cDNA was used to express the heterologous ACL 6XHis-tagged which was identified using MALDI-TOF-MS. The sequenced peptides with 42.2% coverage showed 100% identity to the amino acid sequence generated in silico. The recombinant ACL purified to homogeneity showed an activity of 2 U. This is the first study to characterize a recombinant ACL from a carotenogenic yeast. The present study provides a key foundation for future studies to assess (a) the possible occurrence of alternative splicing, (b) identify the promoter(s) sequence(s) and (c) the involvement of ACL in the differential regulation of fatty acid and carotenoid biosynthesis in yeasts.
authors.affiliation: Department of Biotechnology and Bioengineering, Cinvestav-IPN, Av. Instituto Politécnico Nacional 2508, Col. San Pedro Zacatenco, México D.F. 07360, México.
authors.forename: Cipriano
authors.initials: C
authors.lastname: Chávez-Cabrera
authors.affiliation: Department of Biotechnology and Bioengineering, Cinvestav-IPN, Av. Instituto Politécnico Nacional 2508, Col. San Pedro Zacatenco, México D.F. 07360, México.
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Marsch
authors.affiliation: Department of Biotechnology and Bioengineering, Cinvestav-IPN, Av. Instituto Politécnico Nacional 2508, Col. San Pedro Zacatenco, México D.F. 07360, México.
authors.forename: Yaneth
authors.initials: Y
authors.lastname: Bartolo-Aguilar
authors.affiliation: Department of Biotechnology and Bioengineering, Cinvestav-IPN, Av. Instituto Politécnico Nacional 2508, Col. San Pedro Zacatenco, México D.F. 07360, México.
authors.forename: Zoila Rosa
authors.initials: ZR
authors.lastname: Flores-Bustamante
authors.affiliation: Department of Biotechnology and Bioengineering, Cinvestav-IPN, Av. Instituto Politécnico Nacional 2508, Col. San Pedro Zacatenco, México D.F. 07360, México.
authors.forename: María Eugenia
authors.initials: ME
authors.lastname: Hidalgo-Lara
authors.affiliation: Department of Biotechnology and Bioengineering, Cinvestav-IPN, Av. Instituto Politécnico Nacional 2508, Col. San Pedro Zacatenco, México D.F. 07360, México.
authors.forename: Anahí
authors.initials: A
authors.lastname: Martínez-Cárdenas
authors.affiliation: Department of Microbiology, National School of Biological Sciences -IPN, Prol. Carpio y Plan de Ayala S/N Col. Santo Tomás, México D.F. 11340, México.
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Cancino-Díaz
authors.affiliation: Department of Molecular Biology and Biotechnology, Biomedical Research Institute, National Autonomous University of Mexico (UNAM), México D.F. 04510, México.
authors.forename: Sergio
authors.initials: S
authors.lastname: Sánchez
authors.affiliation: Department of Biotechnology and Bioengineering, Cinvestav-IPN, Av. Instituto Politécnico Nacional 2508, Col. San Pedro Zacatenco, México D.F. 07360, México lfcotera@cinvestav.mx.
authors.forename: Luis Bernardo
authors.initials: LB
authors.lastname: Flores-Cotera
mesh_terms: D001275:ATP Citrate (pro-S)-Lyase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001487:Basidiomycota / Q000201:enzymology* / Q000235:genetics; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D016000:Cluster Analysis; D015870:Gene Expression; D010802:Phylogeny; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D011993:Recombinant Fusion Proteins; D001275:ATP Citrate (pro-S)-Lyase
keywords: Xanthophyllomyces dendrorhous; acetyl coenzyme A; astaxanthin; carotenoids; functional expression