Naoya Takakuwa

Characterization and expression analysis of the exopolysaccharide gene cluster in Lactobacillus fermentum TDS030603.

Part of the exopolysaccharide gene cluster of Lactobacillus fermentum TDS030603 was characterized. It consists of 11,890 base pairs and is located in the chromosomal DNA, 13 open reading frames of which were encoded. Out of the 13 open reading frames, six were found to be involved in exopolysaccharide synthesis; however, five were similar to transposase genes of other lactobacilli, and two were functionally unrelated. Expression analysis revealed that the exopolysaccharide synthesis-related genes were expressed during cultivation. Southern analysis using specific primers for the exopolysaccharide genes indicated that duplication of the gene cluster did not occur. The plasmid-cured strain maintained its capacity for exopolysaccharide production, confirming that the exopolysaccharide gene cluster of this strain is located in the chromosomal DNA, similarly to thermophilic lactic acid bacteria. Our results indicate that this exopolysaccharide gene cluster is likely to be functional, although extensive gene rearrangement occurs.

Isolation and Characterization of the Genes Encoding Δ8-Sphingolipid Desaturase from Saccharomyces kluyveri and Kluyveromyces lactis

Saccharomyces kluyveri IFO 1685 and Kluyveromyces lactis IFO 1090 synthesize cerebroside containing 9-methyl-trans-4, trans-8-sphingadienine as a sphingoid base. From the genome of the two strains, the regions encompassing Δ8-sphingolipid desaturase were amplified and sequenced. The nucleotide sequences of these regions revealed single open reading frames of 1707 bp for S. kluyveri and 1722 bp for K. lactis, encoding polypeptides of 568 and 573 amino acids with molecular weights of 66.5 and 67.1 kDa, respectively. Conversion of 4-hydroxysphinganine to 4-hydroxy-trans-8-sphingenine in the cells of Saccharomyces cerevisiae was observed by the expressed gene from K. lactis and not by that from S. kluyveri. These findings may be explained by the difference in substrate specificity for the sphingoid base moiety between Δ8-sphingolipid desaturases of S. kluyveri and K. lactis.

Significance of the KlLAC1 gene in glucosylceramide production by Kluyveromyces lactis

Each of the 12 genes involved in the synthesis of glucosylceramide was overexpressed in cells of Kluyveromyces lactis to construct a strain accumulating a high quantity of glucosylceramide. Glucosylceramide was doubled by the KlLAC1 gene, which encodes ceramide synthase, and not by 11 other genes, including the KlLAG1 gene, a homologue of KlLAC1. Disruption of the KlLAC1 gene reduced the content below the detection level. Heterologous expression of the KlLAC1 gene in the cells of Saccharomyces cerevisiae caused the accumulation of ceramide, composed of C(18) fatty acid. The KlLAC1 protein preferred long-chain (C(18)) fatty acids to very-long-chain (C(26)) fatty acids for condensation with sphingoid bases and seemed to supply a ceramide moiety as the substrate for the formation of glucosylceramide. When the amino acid sequences of ceramide synthase derived from eight yeast species were compared, LAC1 proteins from five species producing glucosylceramide were clearly discriminated from those of the other three species and all LAG1 proteins. The LAC1 protein of K. lactis is the enzyme that plays a crucial role in the synthesis of glucosylceramide.

Production of Cerebroside from Beet Molasses by the Yeast Saccharomyces kluyveri

Twenty-two strains classified as Saccharomyces kluyveri were grown on beet molasses as the principal carbon source to select a strain for the production of a glycosphingolipid cerebroside. Six strains accumulated relatively high amounts of cerebroside (> 0.40 mg [g dry weight]−1) and grew well in an Erlenmeyer flask. Thirty strains were isolated from single spores of strain CBS 4800 after sporulation and further tested for cerebroside contents. Strain SP-25 accumulated greater amounts than the parental strain (0.59 mg [g dry weight]−1) and was finally selected and cultured in a 5 L jar fermentor. In a comparison with a conventional baking strain of S. cerevisiae, the selected strain SP-25 propagated more rapidly in conjunction with depressed synthesis of ethanol. The amount of cerebroside produced from 1 kg of sugar in molasses was estimated to be 0.15 g. The constituents of cerebroside accumulated in the yeast cells were principally 2-hydroxystearic acid as a fatty acid, 9-methyl-trans-4, trans-8-sphingadienine as a sphingoid base, and glucose as a hexose.

Distribution of Cerebroside in Genus Saccharomyces and Its Closely Related Yeasts, and Cloning of Cerebroside Metabolism-Related Genes

As sphingolipids, cerebroside (glucosylceramide) as well as free ceramide and inositolphosphate-containing ceramide, are widely distributed in fungi and plants. However, there has been no evidence of cerebroside or unsaturated sphingoid bases found in Saccharomyces yeasts. When thirty-one strains accepted in the genus Saccharomyces and its closely related yeasts were analyzed for sphingolipids, cerebrosides were found in Saccharomyces kluyveri, Zygosaccharomyces cidri, Z. fermentati, Kluyveromyces lactis, K. thermotolerans, and K. waltii. The cerebrosides of S. kluyveri and K. lactis included 9-methyl-trans-4, trans-8-sphingadienine and its putative metabolic intermediates; 4-unsaturated and 4,8-diunsaturated sphingoid bases. We further cloned genes encoding glucosylceramide synthase and Δ8-sphingolipid desaturase from S. kluyveri and K. lactis.