RESUMO
We describe the isolation and characterization of a gene (MAELO) that encodes a fatty acid elongase from arachidonic acid-producing fungus Mortierella alpina 1S-4. Although the homologous MAELO gene had already been isolated from M. alpina ATCC 32221, its function had not yet been identified. The MAELO gene from M. alpina 1S-4 was confirmed to encode a fatty acid elongase by its expression in yeast Saccharomyces cerevisiae. Analysis of the fatty acid composition of the yeast transformant revealed the accumulation of 22-, 24-, and 26-carbon saturated fatty acids. On the other hand, RNA interference of the MAELO gene in M. alpina 1S-4 was carried out. The gene-silenced strain obtained on RNA interference exhibited low contents of 20-, 22-, and 24-carbon saturated fatty acids and a high content of stearic acid (18 carbons), compared with those in the wild strain. The enzyme encoded by the MAELO gene was demonstrated to be involved in the biosynthesis of 20-, 22-, and 24-carbon saturated fatty acids in M. alpina 1S-4.
Assuntos
Acetiltransferases/metabolismo , Ácido Araquidônico/metabolismo , Ácidos Graxos/metabolismo , Proteínas Fúngicas/metabolismo , Mortierella/enzimologia , Acetiltransferases/química , Acetiltransferases/genética , Sequência de Aminoácidos , Elongases de Ácidos Graxos , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Expressão Gênica , Dados de Sequência Molecular , Mortierella/química , Mortierella/genética , Mortierella/metabolismo , Interferência de RNA , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de AminoácidosRESUMO
The oil-producing fungus Mortierella alpina 1S-4 is an industrial strain for arachidonic acid (AA) production. To determine its physiological properties and to clarify the biosynthetic pathways for PUFA, heterologous and homologous gene expression systems were established in this fungus. The first trial was performed with an enhanced green fluorescent protein gene to assess the transformation efficiency for heterologous gene expression. As a result, strong fluorescence was observed in the spores of the obtained transformant, suggesting that the foreign gene was inherited by the spores. The next trial was performed with a homologous PUFA elongase (GLELOp) gene, this enzyme having been reported to catalyze the elongation of GLA (18:3n-6) to dihomo-gamma-linolenic acid (20:3n-6), and to be the rate-limiting step of AA production. The FA composition of the transformant was different from that of the host strain: The GLA content was decreased whereas that of AA was increased. These data support the hypothesis that the GLELOp enzyme plays an important role in PUFA synthesis, and may indicate how to control PUFA biosynthesis.
Assuntos
Acetiltransferases/metabolismo , Ácido Araquidônico/biossíntese , Mortierella/enzimologia , Elongases de Ácidos Graxos , Ácidos Graxos/análise , Ácidos Graxos Insaturados/metabolismo , Proteínas Fúngicas , Cinética , Mortierella/metabolismo , Ácido gama-Linolênico/biossínteseRESUMO
Oil-producing fungus Mortierella alpina 1S-4 is an industrial strain. To determine its physiological properties and to clarify the biosynthetic pathways for polyunsaturated fatty acids, a transformation system for this fungus was established using a derivative of it, i.e., a ura5- mutant lacking orotate phosphoribosyl transferase (OPRTase, EC.2.4.2.10) activity. Transformation with a vector containing the homologous ura5 gene as a marker was successfully performed using microprojectile bombardment, other methods frequently used for transformation, such as the protoplasting, lithium acetate, or electroporation methods, not giving satisfactory results. As a result, two types of transformants were obtained: a few stable transformants overexpressing the ura5 gene, and many unstable transformants showing OPRTase activity comparable to that of the wild-type strain. The results of quantitative PCR indicated that the stable transformants could retain the ura5 genes originating from the transformation vector regardless of the culture conditions. On the other hand, unstable transformants easily lost the marker gene under uracil-containing conditions, as expected. In this paper, we report that an overall transformation system for this fungus was successfully established, and propose how to select useful transformants as experimental and industrial strains.
Assuntos
Mortierella/genética , Orotato Fosforribosiltransferase/genética , Transformação Genética , Acetatos/farmacologia , Meios de Cultura/química , Eletroporação , Genes Fúngicos , Vetores Genéticos , Mortierella/metabolismo , Mutação , Óleos/metabolismo , Orotato Fosforribosiltransferase/metabolismo , Protoplastos , Seleção GenéticaRESUMO
The oil-producing fungus Mortierella alpina 1S-4 is an industrial strain. In order to prepare host strains for a transformation system for this fungus, six uracil auxotrophs were obtained by means of random mutation with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). When the activities of orotate phosphoribosyl transferase (OPRTase, EC 2.4.2.10) and orotidine-5'-phosphate decarboxylase (OMPdecase, EC 4.1.1.23) were examined in the mutants and wild strain, OPRTase activity was found to be completely absent in all mutants, on the other hand, OMPdecase activity was intact. The genomic DNA and cDNA of the ura5 gene encoding OPRTase and the ura3 gene encoding OMPdecase were cloned and sequenced. The Ura5p deduced amino acid sequence of this fungus showed highest similarity to that of Vibrio cholerae classed among prokaryote. Furthermore, the mutational points in the ura5 genes of two selected mutants were identified; a base-replacement and a base-insertion.
