A versatile transformation system for thraustochytrids, a promising producer for polyunsaturated fatty acids and fatty acid-derived fuels, was established. G418, hygromycin B, blasticidin, and zeocin inhibited the growth of thraustochytrids, indicating that multiple selectable marker genes could be used in the transformation system. A neomycin resistance gene (neo r ), driven with an ubiquitin or an EF-1␣ promoter-terminator from Thraustochytrium aureum ATCC 34304, was introduced into representatives of two thraustochytrid genera, Aurantiochytrium and Thraustochytrium. The neo r marker was integrated into the chromosomal DNA by random recombination and then functionally translated into neo r mRNA. Additionally, we confirmed that another two genera, Parietichytrium and Schizochytrium, could be transformed by the same method. By this method, the enhanced green fluorescent protein was functionally expressed in thraustochytrids. Meanwhile, T. aureum ATCC 34304 could be transformed by two 18S ribosomal DNA-targeting vectors, designed to cause single-or double-crossover homologous recombination. Finally, the fatty acid ⌬5 desaturase gene was disrupted by double-crossover homologous recombination in T. aureum ATCC 34304, resulting in an increase of dihomo-␥-linolenic acid (C 20:3n-6 ) and eicosatetraenoic acid (C 20:4n-3 ), substrates for ⌬5 desaturase, and a decrease of arachidonic acid (C 20:4n-6 ) and eicosapentaenoic acid (C 20:5n-3 ), products for the enzyme. These results clearly indicate that a versatile transformation system which could be applicable to both multiple transgene expression and gene targeting was established for thraustochytrids.

This article is available online at http://www.jlr.org Glycosphingolipids (GSLs), amphipathic compounds consisting of oligosaccharides and ceramide (Cer) moieties, are ubiquitous in the outer leafl et of the plasma membrane. More than 500 species of GSLs differing in oligosaccharides and Cer moieties have been characterized, some of which represent potential biomarkers for diseases and cell development. For instance, Forssman antigen is specifi cally detected in some gastric, colonic, and lung cancers ( 1 ), and stage-specifi c embryonic antigen (SSEA)-3 and SSEA-4, highly expressed at a stage of embryonic development, are utilized as a marker of embryonic stem cells ( 2 ) and induced pluripotent stem cells ( 3 ).The classifi cation of GSLs, into ganglio-series, globoseries and lacto-series GSLs, is entirely based on the structure of the saccharide moiety. However, the Cer moiety, composed of a sphingoid base and fatty acyl chain linked by an N -acyl linkage, shows heterogeneity in carbon chain length, and saturation and hydroxylation status, etc. The complexity of the Cer moiety prevents the comprehensive analysis of cellular GSLs by MS and/or LC-MS. Removal of the Cer moiety makes the analysis of GSLs much simpler and easier. For this purpose, chemical methods such as ozonolysis ( 4 ) and osmium-catalyzed periodate oxidation ( 5 ) have been developed. These chemical methods, however, require complicated and time-consuming procedures with relatively low yield ( 6 ). Alternatively, enzymes capable of detaching intact oligosaccharides from various GSLs Abstract Endoglycoceramidase (EGCase) is a glycosidase capable of hydrolyzing the ␤ -glycosidic linkage between the oligosaccharides and ceramides of glycosphingolipids (GSLs). Three molecular species of EGCase differing in specifi city were found in the culture fl uid of Rhodococcus equi (formerly Rhodococcus sp. M-750) and designated EGCase I, II, and III. This study describes the molecular cloning of EGCase I and characterization of the recombinant enzyme, which was highly expressed in a rhodococcal expression system using Rhodococcus erythropolis. Kinetic analysis revealed the turnover number (k cat ) ( k cat ) of the recombinant EGCase I to be 22-and 1,200-fold higher than that of EGCase II toward GM1a and Gb3Cer, respectively, although the K m of both enzymes was almost the same for these substrates.

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Enzymes capable of hydrolyzing the ␤-glycosidic linkage between oligosaccharides and ceramides in various glycosphingolipids has been found in microorganisms and invertebrates and designated endoglycoceramidase (EC 3.2.1.123) or ceramide glycanase. Here we report the molecular cloning, characterization, and homology modeling of a novel endoglycoceramidase that hydrolyzes oligogalactosylceramides to produce galactooligosaccharides and ceramides. The novel enzyme was purified from a culture supernatant of Rhodococcus equi, and the gene encoding 488 deduced amino acids was cloned using peptide sequences of the purified enzyme. Eight residues essential for the catalytic reaction in microbial and animal endoglycoceramidases were all conserved in the deduced amino acid sequence of the novel enzyme. Homology modeling of the enzyme using endocellulase E1 as a template revealed that the enzyme displays a (␤/␣) 8 barrel structure in which Glu 234 at the end of ␤-strand 4 and Glu 341 at the end of ␤-strand 7 could function as an acid/base catalyst and a nucleophile, respectively. Site-directed mutagenesis of these glutamates resulted in a complete loss of the activity without a change in their CD spectra. The recombinant enzyme hydrolyzed the ␤-galactosidic linkage between oligosaccharides and ceramides of 6-gala series glycosphingolipids that were completely resistant to hydrolysis by the enzymes reported so far. In contrast, the novel enzyme did not hydrolyze ganglio-, globo-, or lacto-series glycosphingolipids. The enzyme is therefore systematically named "oligogalactosyl-N-acylsphingosine 1,1-␤-galactohydrolase" or tentatively designated "endogalactosylceramidase." Glycosphingolipids (GSLs), 2 amphipathic compounds consisting of oligosaccharides and ceramides, are ubiquitous components of the plasma membrane (1). Recently, it was revealed that GSLs are enriched with other sphingolipids and cholesterol to form microdomains on ectoplasmic membranes. These lipid microdomains, known as detergent-insoluble sphingolipid-enriched domains, so-called DIMs or rafts (2), assemble receptors and signaling molecules such as glycosylphosphatidylinositol-anchored proteins, Src family kinases, and G-proteins (3).6-Gala series GSLs possessing the structure R-Gal␤1-6Gal␤1-1ЈCer have been found in the mollusk (Turbo cornutus) (4), the leech (Hirudo nipponia) (5), the earthworm (Pheretima sp.) (6), and some pathogenic cestode parasites (Echinococcus multilocularis (7), Taenia crassiceps (8), Spirometra erinacei (9), and Metroliasthes cotarunix (10). Recently, it was revealed that aureobasidin A-resistant Zygomycetes species synthesized 6-gala series GSLs (tentatively designated CDS, CTS, CTeS, and CPS) instead of inositol phosphorylceramide (11). Because aureobasidin A, a well known and widely used antifungal agent, was found to inhibit the synthesis of inositol phosphorylceramide, the resistance to the agent may stem from the lack of inositol phosphorylceramide in Zygomycetes. Thus, an inhibitor for the synthesis of 6-gala series GSLs see...

We cloned a novel beta-1,3-xylanase gene, consisting of a 1728-bp open reading frame encoding 576 amino acid residues, from a marine bacterium, Vibrio sp. strain AX-4. Sequence analysis revealed that the beta-1,3-xylanase is a modular enzyme composed of a putative catalytic module belonging to glycoside hydrolase family 26 and two putative carbohydrate-binding modules belonging to family 31. The recombinant enzyme hydrolysed beta-1,3-xylan to yield xylo-oligosaccharides with different numbers of xylose units, mainly xylobiose, xylotriose and xylotetraose. However, the enzyme did not hydrolyse beta-1,4-xylan, beta-1,4-mannan, beta-1,4-glucan, beta-1,3-xylobiose or p-nitrophenyl-beta-xyloside. When beta-1,3-xylo-oligosaccharides were used as the substrate, the kcat value of the enzyme for xylopentaose was found to be 40 times higher than that for xylotetraose, and xylotriose was extremely resistant to hydrolysis by the enzyme. A PSI-BLAST search revealed two possible catalytic Glu residues (Glu-138 as an acid/base catalyst and Glu-234 as a nucleophile), both of which are generally conserved in glycoside hydrolase superfamily A. Replacement of these two conserved Glu residues with Asp and Gln resulted in a significant decrease and complete loss of enzyme activity respectively, without a change in their CD spectra, suggesting that these Glu residues are the catalytic residues of beta-1,3-xylanase. The present study also clearly shows that the non-catalytic putative carbohydrate-binding modules play an important role in the hydrolysis of insoluble beta-1,3-xylan, but not that of soluble glycol-beta-1,3-xylan. Furthermore, repeating a putative carbohydrate-binding module strongly enhanced the hydrolysis of the insoluble substrate.

Thraustochytrids, marine protists known to accumulate polyunsaturated fatty acids (PUFAs) in lipid droplets, are considered an alternative to fish oils as a source of PUFAs. The major fatty acids produced in thraustochytrids are palmitic acid (C 16:0 ), n ؊ 6 docosapentaenoic acid (DPA) (C 22:5n ؊ 6 ), and docosahexaenoic acid (DHA) (C 22:6n ؊ 3 ), with eicosapentaenoic acid (EPA) (C 20:5n ؊ 3 ) and arachidonic acid (AA) (C 20:4n ؊ 6 ) as minor constituents. We attempted here to alter the fatty acid composition of thraustochytrids through the expression of a fatty acid ⌬5 desaturase gene driven by the thraustochytrid ubiquitin promoter. The gene was functionally expressed in Aurantiochytrium limacinum mh0186, increasing the amount of EPA converted from eicosatetraenoic acid (ETA) (C 20:4n ؊ 3 ) by the ⌬5 desaturase. The levels of EPA and AA were also increased by 4.6-and 13.2-fold in the transgenic thraustochytrids compared to levels in the mock transfectants when ETA and dihomo-␥-linolenic acid (DGLA) (C 20:3n ؊ 6 ) were added to the culture at 0.1 mM. Interestingly, the amount of EPA in the transgenic thraustochytrids increased in proportion to the amount of ETA added to the culture up to 0.4 mM. The rates of conversion and accumulation of EPA were much higher in the thraustochytrids than in baker's yeasts when the desaturase gene was expressed with the respective promoters. This report describes for the first time the finding that an increase of EPA could be accomplished by introducing the ⌬5 desaturase gene into thraustochytrids and indicates that molecular breeding of thraustochytrids is a promising strategy for generating beneficial PUFAs.

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