RESUMO
Phycomyces carRA gene encodes a protein with two domains. Domain R is characterized by red carR mutants that accumulate lycopene. Domain A is characterized by white carA mutants that do not accumulate significant amounts of carotenoids. The carRA-encoded protein was identified as the lycopene cyclase and phytoene synthase enzyme by sequence homology with other proteins. However, no direct data showing the function of this protein have been reported so far. Different Mucor circinelloides mutants altered at the phytoene synthase, the lycopene cyclase or both activities were transformed with the Phycomyces carRA gene. Fully transcribed carRA mRNA molecules were detected by Northern assays in the transformants and the correct processing of the carRA messenger was verified by RT-PCR. These results showed that Phycomyces carRA gene was correctly expressed in Mucor. Carotenoids analysis in these transformants showed the presence of ß-carotene, absent in the untransformed strains, providing functional evidence that the Phycomyces carRA gene complements the M. circinelloides mutations. Co-transformation of the carRA cDNA in E. coli with different combinations of the carotenoid structural genes from Erwinia uredovora was also performed. Newly formed carotenoids were accumulated showing that the Phycomyces CarRA protein does contain lycopene cyclase and phytoene synthase activities. The heterologous expression of the carRA gene and the functional complementation of the mentioned activities are not very efficient in E. coli. However, the simultaneous presence of both carRA and carB gene products from Phycomyces increases the efficiency of these enzymes, presumably due to an interaction mechanism.
Assuntos
Alquil e Aril Transferases/genética , Proteínas Fúngicas/genética , Liases Intramoleculares/genética , Phycomyces/genética , Alquil e Aril Transferases/metabolismo , Northern Blotting , Carotenoides/biossíntese , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Enzimológica da Expressão Gênica , Teste de Complementação Genética , Geranil-Geranildifosfato Geranil-Geraniltransferase , Liases Intramoleculares/metabolismo , Mucor/enzimologia , Mucor/genética , Mucor/metabolismo , Mutação , Phycomyces/enzimologia , Phycomyces/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , beta Caroteno/biossínteseRESUMO
Most Mucor species accumulate beta-carotene as the main carotenoid. The crtW and crtZ astaxanthin biosynthesis genes from Agrobacterium aurantiacum were placed under the control of Mucor circinelloides expression signals. Expression vectors containing the bacterial genes were constructed, and PEG-mediated transformations were performed on a selected M. circinelloides strain. Transformants that exhibited altered carotene production were isolated and analyzed. Southern analysis showed that all plasmids behave as autoreplicative elements. Northern analysis detected the actual heterologous transcription products, whereas thin layer chromatography and high-performance liquid chromatography studies revealed the presence of new carotenoid compounds and intermediates among the transformants.
Assuntos
Proteínas de Bactérias/genética , Genes Bacterianos , Mucor/genética , Oxigenases/genética , beta Caroteno/análogos & derivados , Carotenoides/análise , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Clonagem Molecular , Expressão Gênica , Vetores Genéticos , Mucor/metabolismo , Plasmídeos , Regiões Promotoras Genéticas , RNA Bacteriano/análise , RNA Mensageiro/análise , Proteínas Recombinantes , Rhizobium/genética , Rhizobium/metabolismo , Transformação Genética , Xantofilas , beta Caroteno/biossíntese , beta Caroteno/genéticaRESUMO
Two Mucor circinelloides structural genes involved in isoprenoid biosynthesis were isolated and characterised. The isoA gene encodes a typical eukaryotic farnesyl diphosphate synthase (EC 2.5.1.10), whereas the isoB gene deduced amino acid sequence shows similarity to fungal medium-chain prenyl diphosphate synthases. By functional complementation in Escherichia coli, the isoB gene product was shown to be a solanesyl diphosphate synthase (EC 2.5.1.11), which is the first fungal enzyme reported having this specificity. In addition, a M. circinelloides one-marker-per-chromosome map was completed by contour-clamped homogeneous electric field localisation of isoA, isoB and three other isoprenoid biosynthesis genes to individual chromosomes.
Assuntos
Alquil e Aril Transferases/genética , Cromossomos/genética , Proteínas Fúngicas/genética , Mucor/enzimologia , Alquil e Aril Transferases/isolamento & purificação , Alquil e Aril Transferases/metabolismo , Sequência de Bases , Clonagem Molecular , Difosfatos/metabolismo , Escherichia coli/genética , Regulação Fúngica da Expressão Gênica , Teste de Complementação Genética , Mucor/genética , Fosfatos de Poli-Isoprenil/biossíntese , Análise de Sequência de DNARESUMO
A new structural gene, carG, involved in the biosynthesis of carotenoids in the fungus Mucor circinelloides was isolated by heterologous hybridisation, using a probe derived from the Gibberella fujikuroi ggs1 gene. Functional analyses in Escherichia coli showed that the encoded protein has geranylgeranyl pyrophosphate (GGPP) synthase activity. A comparison of the deduced protein with other GGPP synthases suggested that the carG gene might have evolved from other larger genes present in some fungi. The analysis of carG mRNA accumulation after blue light irradiation showed that the expression of this gene is up-regulated by blue light, as happens with the other structural genes involved in carotenogenesis in M. circinelloides. Analysis of the promoter region revealed the presence of several APE-like sequences, which participate in the blue-light regulation of the expression of different fungal genes. These sequences are also present in the above-mentioned Mucor genes and strongly support the idea that this gene plays an important role in the regulation of carotenoid synthesis, despite belonging to a more general metabolic pathway.
Assuntos
Dimetilaliltranstransferase/genética , Regulação Fúngica da Expressão Gênica/fisiologia , Luz , Mucor/genética , Regulação para Cima , Alquil e Aril Transferases/genética , Sequência de Bases , Northern Blotting , Southern Blotting , Carotenoides/química , Primers do DNA , Farnesiltranstransferase , Teste de Complementação Genética , Dados de Sequência Molecular , Hibridização de Ácido Nucleico/genética , Plasmídeos/genética , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
The Phycomyces blakesleeanus wild-type is yellow, because it accumulates beta-carotene as the main carotenoid. A new carotenoid mutant of this fungus (A486) was isolated, after treatment with ethyl methane sulfonate (EMS), showing a whitish coloration. It accumulates large amounts of phytoene, small quantities of phytofluene, zeta-carotene and neurosporene, in decreasing amounts, and traces of beta-carotene. This phenotype indicates that it carries a leaky mutation affecting the enzyme phytoene dehydrogenase (EC 1.3.-.-), which is specified by the gene carB. Biochemical analysis of heterokaryons showed that mutant A486 complements two previously characterized carB mutants, C5 (carB10) and S442 (carB401). Sequence analysis of the carB gene genomic copy from these three strains revealed that they are all altered in the gene carB, giving information about the nature of the mutation in each carB mutant allele. The interallelic complementation provides evidence for the multimeric organization of the P. blakesleeanus phytoene dehydrogenase.
