Journal of Food Processing & Technology

Molecular cloning and functional characterization of the recombinant ATP:Citrate lyase of Phaffia rhodozyma

2^nd International Conference and Exhibition on Food Technology, Bioprocess & Cell Culture

October 28-30, 2013 Kansas City Marriott Country Club Plaza, USA

Cipriano Chavez-Cabrera, Zoila R. Flores-Bustamante and Luis B. Flores-Cotera

Posters: J Food Process Technol

Abstract:

Astaxanthin is a secondary metabolite and the main carotenoid produced in Phaffia rhodozyma. It is a powerful antioxidant commonly found in marine animals, and used in humans to prevent a number of degenerative diseases including cancer. Biosynthesis of astaxanthin requires acetyl-CoA as initial precursor. ATP-citrate lyase (ACL) is the key cytoplasmic enzyme which catalyses the formation of acetyl-CoA and oxaloacetate from citrate and CoA with a concomitant hydrolysis of ATP. ACL supplies acetyl-CoA for fatty acid biosynthesis in oleaginous yeasts. The biosynthesis of fatty acids and carotenoids had positive correlation with ACL activity in P. rhodozyma, consequently it has been suggested that both pathways have a common source of acetyl-CoA, although this has not been studied at the molecular level. Firstly, we isolated and cloned the ACL gene of P. rhodozyma in E. coli. An in silico analysis showed a 4,117 pb gene sequence, with 3,341bp open reading frame that encodes a 120.9 kDa protein. The ACL gene from P. rhodozyma had 82.2% identity with that of Postia placenta, and 85.6% similarity with that of Cryptococcus gattii. Additionally, the ACL enzyme from P. rhodozyma has the two multi domains PNL02235 and PLN02522, characteristic in all ACL enzymes. Secondly, the ACL ORF was subcloned in pPICZB, and the resultant construction used to transform the yeast Pichia pastoris X-33 for expression. The expressed enzyme shows ACL activity and has a tag-His fusion which serves to purify the enzyme by nickel attachment.

Biography :

Cipriano Chavez Cabrera is a 38 years old Ph.D. student at Research Center for Advanced Studies of the National Polytechnic Institute (CINVESTAVIPN), Mexico City. He has co-authored two papers on Phaffia rhodozyma and previously worked in the food industry and is a teacher at College of Science and Technology Studies of the Michoacan state (CECyTE Michoacan).