Acyl-CoA elongase activity and gene from the marine microalga Pavlova lutheri (Haptophyceae)
ABSTRACT Microsomal elongases are proteins catalyzing the condensation of malonyl-CoA with acyl-CoA chains, the first and rate-limiting step in microsomal fatty acid elongation. Here we report the measurement of elongase activity of a microsomal enriched fraction from the marine microalga Pavlova lutheri (P. lutheri). By directly monitoring the production of C2 elongated acyl-CoA from a range of saturated and monounsaturated acyl-CoA substrates, we found that saturated 16:0-CoA is the preferred substrate for this elongase complex. Analysis of an EST database prepared from the exponential stage of growth of P. lutheri revealed the most abundant identifiable enzyme as a cDNA, Plelo1, encoding a protein similar to the plant β-ketoacyl-coenzyme A synthases (KCS, also known as elongases). Plelo1 is a single copy gene in the algal genome and gene expression analysis showed it to be highly expressed during the exponential phase of growth. It is suggested that microsomal elongation of 16:0-CoA represents a key intermediate step in the biosynthesis of the health beneficial very long chain polyunsaturated fatty acids eicosapentaenoic (20:5n3) and docosahexaenoic (22:6n3) acids.
- [show abstract] [hide abstract]
ABSTRACT: Gas chromatographic profiling of fatty acids was performed during the growth cycle of four marine microalgae in order to establish which, if any, of these could act as a reliable source of genes for the metabolic engineering of long chain polyunsaturated fatty acid (LC-PUFA) synthesis in alternative production systems. A high-throughput column based method for extraction of triacylglycerols (TAGs) was used to establish how much and at what stage in the growth phase LC-PUFAs partition to storage lipid in the different species. Differences in the time course of production and incorporation of docosahexaenoic acid (22:6n-3, DHA) and eicosapentaenoic acid (20:5n-3, EPA) into TAGs were found in the marine microalgae Nannochloropsis oculata (Eustigmatophyceae), Phaeodactylum tricornutum and Thalassiosira pseudonana (Bacillariophyceae), and the Haptophyte Pavlova lutheri. Differences were not only observed between species but also during the different phases of growth within a species. A much higher percentage of the total cellular EPA was partitioned to TAGs in stationary phase cells of N. oculata compared to P. tricornutum. Although P. tricornutum produces DHA it does not partition it to TAGs. Both T. pseudonana and P. lutheri produce EPA and DHA and partition these to TAGs during the stationary phase of growth. These two species are therefore good candidates for further biochemical and molecular analysis, in order to understand and manipulate the processes that are responsible for the incorporation of LC-PUFAs into storage oils.Phytochemistry 10/2002; 61(1):15-24. · 3.05 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: In studies of the outstanding salt tolerance of the unicellular green alga Dunaliella salina, we isolated a cDNA for a salt-inducible mRNA encoding a protein homologous to plant beta-ketoacyl-coenzyme A (CoA) synthases (Kcs). These microsomal enzymes catalyze the condensation of malonyl-CoA with acyl-CoA, the first and rate-limiting step in fatty acid elongation. Kcs activity, localized to a D. salina microsomal fraction, increased in cells transferred from 0.5 to 3.5 M NaCl, as did the level of the kcs mRNA. The function of the kcs gene product was directly demonstrated by the condensing activity exhibited by Escherichia coli cells expressing the kcs cDNA. The effect of salinity on kcs expression in D. salina suggested the possibility that salt adaptation entailed modifications in the fatty acid composition of algal membranes. Lipid analyses indicated that microsomes, but not plasma membranes or thylakoids, from cells grown in 3.5 M NaCl contained a considerably higher ratio of C18 (mostly unsaturated) to C16 (mostly saturated) fatty acids compared with cells grown in 0.5 M salt. Thus, the salt-inducible Kcs, jointly with fatty acid desaturases, may play a role in adapting intracellular membrane compartments to function in the high internal glycerol concentrations balancing the external osmotic pressure.Plant physiology 08/2002; 129(3):1320-9. · 6.56 Impact Factor
- Plant Journal - PLANT J. 01/2008; 25(1):115-125.