RESUMO
The biotechnological potential of Yarrowia lipolytica, as a single cell oil-producing microorganism, is presented in this review. Although initially this yeast species was considered as a lipid-degrading, recently, it was reclassified as a lipid-producing microorganism, since it has been reported to be capable of accumulating diverse desirable fatty acids after metabolic pathway engineering. In the first part of the present document, a general revision of the oil metabolic pathways and the capacity of oil production in Y. lipolytica is presented. The single cell oil produced by these metabolic engineering strategies has been designed by optimization, introduction, or suppression of new pathways to increase yield on lipid production. Later on, the genetic regulation systems and the lipid composition generated by this yeast for industrial purposes are discussed. These lipids could be safely used in the chemical food and biofuel industries, due to their high proportion of oleic acid. This document emphasizes in the overviewing at Y. lipolytica as an ideal oil cell factory, and as an excellent model to produce single cell oil.
Assuntos
Metabolismo dos Lipídeos , Yarrowia/genética , Yarrowia/metabolismo , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Engenharia Metabólica , Redes e Vias MetabólicasRESUMO
Chitin is the second most abundant organic compound in nature and represents a rich carbon and nitrogen source that is primarily transformed by bacterial communities. Bacteria capable of gradually hydrolyzing chitin into N-acetylglucosamine monomers can have applications in the transformation of residues from shrimp and other crustaceans. The objective of the present study was to isolate, characterize and identify microorganisms with high chitinolytic activity. These microorganisms were isolated and characterized based on macro- and microscopic morphological traits. Strains were selected on colloidal chitin agar medium primarily based on a hydrolysis halo larger than 2 mm and a growing phase no longer than 6 days. Secondary selection consisted of semi-quantitative evaluation of chitinolytic activity with a drop dilution assay. From the above, ten strains were selected. Then, strain-specific activity was evaluated. The B4 strain showed the highest specific activity, which was 6,677.07 U/mg protein. Molecular identification indicated that the isolated strains belong to the species Stenotrophomonas maltophilia.
RESUMO
We isolated a gene encoding a histone acetyltransferase from Ustilago maydis (DC.) Cda., which is orthologous to the Saccharomyces cerevisiae GCN5 gene. The gene was isolated from genomic clones identified by their specific hybridization to a gene fragment obtained by the polymerase chain reaction (PCR). This gene (Umgcn5; um05168) contains an open reading frame (ORF) of 1421bp that encodes a putative protein of 473 amino acids with a Mr. of 52.6kDa. The protein exhibits a high degree of homology with histone acetyltransferases from different organisms. Null a2b2 ΔUmgcn5 mutants were constructed by substitution of the region encoding the catalytic site with a hygromycin B resistance cassette. Null a1b1 ΔUmgcn5 mutants were isolated from genetic crosses of a2b2 ΔUmgcn5 and a1b1 wild-type strains in maize. Mutants displayed a slight reduction in growth rate under different conditions, and were more sensitive than the wild type to stress conditions, but more important, they grew as long mycelial cells, and formed fuzz-like colonies under all conditions where wild-type strains grew in the yeast-like morphology and formed smooth colonies. This phenotype was not reverted by cAMP addition. Mutants were not virulent to maize plants, and were unable to form teliospores. These phenotypic alterations of the mutants were reverted by their transformation with the wild-type gene.
Assuntos
Proteínas Fúngicas/genética , Histona Acetiltransferases/genética , Ustilago/fisiologia , Ustilago/patogenicidade , Proteínas Fúngicas/metabolismo , Deleção de Genes , Histona Acetiltransferases/metabolismo , Mutação , Análise de Sequência , Estresse Fisiológico , Virulência , Zea mays/microbiologiaRESUMO
The whole MATA cassette from Yarrowia lipolytica, a dimorphic fungus, was replaced by the URA3 gene through a double homologous recombination. This MAT-less strain lost its mate capacity with A or B Y. lipolytica strains. Introduction of polymerase chain reaction-synthesized idiomorph MATB in a null strain of A locus by double homologous recombination gave rise to a "transsexual" B strain. Mating capacity of this engineered mutant was assayed using Y. lipolytica strains of either A or B mating type. Mating took place only with an A strain, demonstrating the MATB idiomorph functionality in a MATA phenotype. Our data suggest that specific downstream genes are responsible for the final A or B phenotypes present in all Y. lipolytica cells, independent of their MAT idiomorph phenotype.
Assuntos
Genes Fúngicos Tipo Acasalamento , Recombinação Genética , Yarrowia/fisiologia , DNA Fúngico/genética , Deleção de Genes , Genótipo , Fenótipo , Yarrowia/genéticaRESUMO
By use of the polymerase chain reaction and synthetic oligonucleotides designed from conserved regions, we amplified a fragment of a gene from Ustilago maydis encoding a putative histone deacetylase. With this probe we isolated the full gene from a minigenomic library. The gene (designated as Umhda2) contains an open reading frame (ORF) of 1701bp encoding a protein of 566 amino acids. Multiple comparison analysis with other histone deacetylases suggests that the Umhda2 gene product belongs to the Rpd3-related family of proteins. The highest degree of homology with histone deacetylases from other organisms corresponded to Hdalp of Schizosaccharomyces pombe and Rpd3p of Saccharomyces cerevisiae with 64.2 and 62.2% of sequence similarity, respectively. It displayed a substantially lower similarity with another histone deacetylase from U. maydis (Hdalp, 52.4%). Semi-quantitative RTPCR results indicate that the gene is transcriptionally up-regulated during the in vitro yeast-to-mycelium dimorphic transition.
Assuntos
Genes Fúngicos , Histona Desacetilases/genética , Ustilago/genética , Sequência de Aminoácidos , DNA Fúngico/genética , DNA Fúngico/isolamento & purificação , Proteínas Fúngicas/genética , Regulação Enzimológica da Expressão Gênica/genética , Regulação Fúngica da Expressão Gênica/genética , Biblioteca Genômica , Dados de Sequência Molecular , RNA Fúngico/genética , RNA Fúngico/isolamento & purificação , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
A gene encoding a sixth chitin synthase (Umchs6, sequence GenBank accession No. ) from the plant pathogenic hemibasidiomycete Ustilago maydis (DC.) Cda. was isolated and characterized. The predicted protein is 1103 amino acids in length with a calculated molecular mass of 123.5 kDa. a2b2 null mutants were obtained by substitution of a central fragment of the Umchs6 gene with the hygromycin resistance cassette, and a1b1 null mutants were obtained by genetic recombination in plants of an a2b2deltach6 and a wild-type a1b1 strain. The mutation had no effect on the dimorphic transition in vitro or on mating, and growth rate of the mutants was only slightly reduced. On the other hand, they displayed important alterations in cell morphology, particularly at the mycelial stage, and in the staining pattern with calcofluor white. Levels of chitin synthase activity in vitro and chitin content were reduced. The most noticeable characteristic of the mutants was their almost complete loss of virulence to maize (Zea mays L.). This was a recessive character. Microscopic observations during the infectious process suggest that chitin synthase 6 activity is very important for growth of the fungus into the plant. Transformation of a2b2deltach6 mutants with an autonomous replicating plasmid carrying the full Umchs6 gene restored their normal morphological phenotype and virulence. These results are evidence that the mutation in the Umchs6 gene was solely responsible for the phenotypic alterations observed.