In algae, the biosynthesis of docosahexaenoic acid (22:6 3; DHA) proceeds via the elongation of eicosapentaenoic acid (20:5 3; EPA) to 22:5 3, which is required as a substrate for the final ⌬ 4 desaturation. To isolate the elongase specific for this step, we searched expressed sequence tag and genomic databases from the algae Ostreococcus tauri and Thalassiosira pseudonana , from the fish Oncorhynchus mykiss , from the frog Xenopus laevis , and from the sea squirt Ciona intestinalis using as a query the elongase sequence PpPSE1 from the moss Physcomitrella patens . The open reading frames of the identified elongase candidates were expressed in yeast for functional characterization. By this, we identified two types of elongases from O. tauri and T. pseudonana : one specific for the elongation of ( ⌬ 6-)C18-PUFAs and one specific for ( ⌬ 5-)C20-PUFAs, showing highest activity with EPA. The clones isolated from O. mykiss , X. laevis , and C. intestinalis accepted both C18-and C20-PUFAs. By coexpression of the ⌬ 6-and ⌬ 5-elongases from T. pseudonana and O. tauri , respectively, with the ⌬ 5-and ⌬ 4-desaturases from two other algae we successfully implemented DHA synthesis in stearidonic acid-fed yeast. This may be considered an encouraging first step in future efforts to implement this biosynthetic sequence into transgenic oilseed crops. -Meyer, A., H. Kirsch, F. Domergue, A. Abbadi, P. Sperling, J. Bauer, P. Cirpus, T. K. Zank, H. Moreau, T. J. Roscoe, U. Zähringer, and E. Heinz. Novel fatty acid elongases and their use for the reconstitution of docosahexaenoic acid biosynthesis.

Although Escherichia coli strain EC3132 possesses a chromosomally encoded sucrose metabolic pathway, its growth on low sucrose concentrations (5 mM) is unusually slow, with a doubling time of 20 h. In this report we describe the subcloning and further characterization of the corresponding csc genes and adjacent genes. The csc regulon comprises three genes for a sucrose permease, a fructokinase, and a sucrose hydrolase (genes cscB, cscK, and cscA, respectively). The genes are arranged in two operons and are negatively controlled at the transcriptional level by the repressor CscR. Furthermore, csc gene expression was found to be cyclic AMPCrpA dependent. A comparison of the genomic sequences of the E. coli strains EC3132, K-12, and O157:H7 in addition to Salmonella enterica serovar Typhimurium LT2 revealed that the csc genes are located in a hot spot region for chromosomal rearrangements in enteric bacteria. The comparison further indicated that the csc genes might have been transferred relatively recently to the E. coli wild-type EC3132 at around the time when the different strains of the enteric bacteria diverged. We found evidence that a mobile genetic element, which used the gene argW for site-specific integration into the chromosome, was probably involved in this horizontal gene transfer and that the csc genes are still in the process of optimal adaptation to the new host. Selection for such adaptational mutants growing faster on low sucrose concentrations gave three different classes of mutants. One class comprised cscR(Con) mutations that expressed all csc genes constitutively. The second class constituted a cscKo operator mutation, which became inducible for csc gene expression at low sucrose concentrations. The third class was found to be a mutation in the sucrose permease that caused an increase in transport activity.

Docosahexaenoic acid (DHA) can be synthesized via alternative routes from which only the omega3/omega6-pathways involve the action of a Delta4-fatty acid desaturase. We examined the suitability of Euglena gracilis, Thraustochytrium sp., Schizochytrium sp., and Crypthecodinium cohnii to serve as sources for cloning a cDNA encoding a Delta4-fatty acid desaturase. For this purpose we carried out in vivo labeling studies with radiolabeled C22 polyunsaturated fatty acid substrates. Schizochytrium sp. was unable to convert exogenously supplied [2-(14)C]-docosapentaenoic acid (DPA, 22:5(Delta)(7,10,13,16,19)) to DHA, while E. gracilis and Thraustochytrium sp. carried out this desaturation very efficiently. Hydrogenation and alpha-oxidation of the labeled DHA isolated from these two organisms showed that it was the result of direct Delta4-desaturation and not of substrate breakdown and resynthesis. To clone the desaturase gene, a cDNA library of E. gracilis was subjected to mass sequencing. A full-length clone with highest homology to the Delta4-desaturase of Thraustochytrium sp. was isolated, and its function was verified by heterologous expression in yeast. The desaturase efficiently converted DPA to DHA. Analysis of the substrate specificity demonstrated that the enzyme activity was not limited to C22 fatty acids, since it also efficiently desaturated C16 fatty acids. The enzyme showed strict Delta4-regioselectivity and required the presence of a Delta7-double bond in the substrate. Positional analysis of phosphatidylcholine revealed that the proportion of the Delta4-desaturated products was up to 20 times higher in the sn-2 position than in the sn-1 position.

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