RESUMO
The gene encoding an aromatic and neutral aliphatic amino acid permease of Penicillium chrysogenum was cloned, functionally expressed and characterized in Saccharomyces cerevisiae M4276. The permease, designated PcMtr, is structurally and functionally homologous to Mtr of Neurospora crassa, and unrelated to the Amino Acid Permease (AAP) family which includes most amino acid permeases in fungi. Database searches of completed fungal genome sequences reveal that Mtr type permeases are not widely distributed among fungi, suggesting a specialized function.
Assuntos
Sistemas de Transporte de Aminoácidos/genética , Penicillium chrysogenum/enzimologia , Sistemas de Transporte de Aminoácidos/química , Sistemas de Transporte de Aminoácidos/metabolismo , Aminoácidos/química , Aminoácidos/metabolismo , Aminoácidos Aromáticos/metabolismo , Carbono/metabolismo , Radioisótopos de Carbono , Clonagem Molecular , Genes Fúngicos , Teste de Complementação Genética , Cinética , Dados de Sequência Molecular , Penicillium chrysogenum/genética , Penicillium chrysogenum/crescimento & desenvolvimento , Filogenia , Saccharomyces cerevisiae/genética , Especificidade por SubstratoRESUMO
External addition of the beta-lactam precursor alpha-aminoadipic acid to the filamentous fungus Penicillium chrysogenum leads to an increased intracellular alpha-aminoadipic acid concentration and an increase in penicillin production. The exact route for alpha-aminoadipic acid uptake is not known, although the general amino acid and acidic amino acid permeases have been implicated in this process. Their corresponding genes, PcGAP1 and PcDIP5, of P. chrysogenum were cloned and functionally expressed in a mutant of Saccharomyces cerevisiae (M4276) in which the acidic amino acid and general amino acid permease genes (DIP5 and GAP1, respectively) are disrupted. Transport assays show that both PcGap1 and PcDip5 mediated the uptake of alpha-aminoadipic acid, although PcGap1 showed a higher affinity for alpha-aminoadipic acid than PcDip5 (K(m) values, 230 and 800 microM, respectively). Leucine strongly inhibits alpha-aminoadipic acid transport via PcGap1 but not via PcDip5. This difference was exploited to estimate the relative contribution of each transport system to the alpha-aminoadipic acid flux in beta-lactam-producing P. chrysogenum. The transport measurements demonstrate that both PcGap1 and PcDip5 contribute to the alpha-aminoadipic acid flux.
Assuntos
Ácido 2-Aminoadípico/metabolismo , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Regulação Fúngica da Expressão Gênica , Penicillium chrysogenum/enzimologia , Sequência de Aminoácidos , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Dados de Sequência Molecular , Penicillium chrysogenum/genética , Penicillium chrysogenum/crescimento & desenvolvimento , Proteínas/genética , Proteínas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
The gene encoding the amino acid permease ArlP (Aromatic and leucine Permease) was isolated from the filamentous fungus Penicillium chrysogenum after PCR using degenerated oligonucleotides based on conserved regions of fungal amino acid permeases. The cDNA clone was used for expression of the permease in Saccharomyces cerevisiae M4054, which is defective in the general amino acid permease Gap1. Upon overexpression, an increase in the uptake of L-tyrosine, L-phenylalanine, L-tryptophan and L-leucine was observed. Further competition experiments indicate that ArlP recognizes neutral and aromatic amino acids with an unbranched beta-carbon atom.
