Tuesday, July 31, 2007 - 1:00 PM
S101
Exploitation of Cyanobacterial Genetics in the Development of an Escherichia coli Based Astaxanthin Production System
Mark A. Scaife1, Adam M. Burja2, Colin J. Barrow3, and Phillip C. Wright1. (1) Department of Chemical and Process Engineering, Biological and Environmental Systems Group, The University of Sheffield, Mappin Street, Sheffield, England, S1 3JD, United Kingdom, (2) Metabolic Engineering and Fermentation, Ocean Nutrition Canada, 101 Research Drive, Dartmouth, NS B2Y 4T6, Canada, (3) Research and Development, Ocean Nutrition Canada, 101 Research Drive, Dartmouth, NS B2Y 4T6, Canada
Carotenoids hold great promise due to their numerous health promoting properties. Of the plethora of compounds isolated to date, astaxanthin is the most intensively investigated. Astaxanthin can be produced by a number of organisms; however these are often slow growing, with complicated nutritional requirements, and low or impure carotenoid yields. Here we present the development of an Escherichia coli based expression system, able to rapidly accumulate astaxanthin. The genomes of six cyanobacterial strains were screened for the presence of putative b-carotene ketolase and hydroxylase genes. Results demonstrated the presence of multiple putative b-carotene ketolase genes present within individual cyanobacterial genomes. While putative b-carotene hydroxylase genes were found to be less abundant, being present as single copies in only four of the cyanobacteria investigated. Genes were cloned and functionally characterised, via complementation in E. coli. All b-carotene ketolase enzymes (CrtO and CrtW type) investigated were functional. Additionally, it was found that all CrtW type b-carotene ketolase enzymes were able to utilise the intermediate, zeaxanthin, in the biosynthesis of astaxanthin, while CrtO type enzymes were not. Of the CrtW type b-carotene ketolase enzymes, three with enhanced activity were identified from Anabaena variabilis ATCC 29413, Nostoc punctiforme PCC 73102 and Nostoc sp. PCC 7120. In contrast, of the four putative b-carotene hydroxylase enzymes investigated only two, derived from Synechocystis sp. PCC 6803 and Anabaena variabilis ATCC 29413, were found to be functional. Application of directed evolution methods to these CrtW and CrtZ genes resulted in enhancement of function with respect to astaxanthin biosynthesis.