RESUMO
BACKGROUND: Metabolic engineering is an attractive approach in order to improve the microbial production of drugs. Triterpenes is a chemically diverse class of compounds and many among them are of interest from a human health perspective. A systematic experimental or computational survey of all feasible gene modifications to determine the genotype yielding the optimal triterpene production phenotype is a laborious and time-consuming process. METHODOLOGY/PRINCIPAL FINDINGS: Based on the recent genome-wide sequencing of Saccharomyces cerevisiae CEN.PK 113-7D and its phenotypic differences with the S288C strain, we implemented a strategy for the construction of a ß-amyrin production platform. The genes Erg8, Erg9 and HFA1 contained non-silent SNPs that were computationally analyzed to evaluate the changes that cause in the respective protein structures. Subsequently, Erg8, Erg9 and HFA1 were correlated with the increased levels of ergosterol and fatty acids in CEN.PK 113-7D and single, double, and triple gene over-expression strains were constructed. CONCLUSIONS: The six out of seven gene over-expression constructs had a considerable impact on both ergosterol and ß-amyrin production. In the case of ß-amyrin formation the triple over-expression construct exhibited a nearly 500% increase over the control strain making our metabolic engineering strategy the most successful design of triterpene microbial producers.
Assuntos
Saccharomyces cerevisiae/genética , Triterpenos/metabolismo , Sítios de Ligação , Genótipo , Modelos Moleculares , Fenótipo , Fosfotransferases (Aceptor do Grupo Fosfato)/genética , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismo , Polimorfismo de Nucleotídeo Único , Saccharomyces cerevisiae/metabolismoRESUMO
Bowen-Conradi syndrome (BCS) is an autosomal-recessive disorder characterized by severely impaired prenatal and postnatal growth, profound psychomotor retardation, and death in early childhood. Nearly all reported BCS cases have been among Hutterites, with an estimated birth prevalence of 1/355. We previously localized the BCS gene to a 1.9 Mbp interval on human chromosome 12p13.3. The 59 genes in this interval were ranked as candidates for BCS, and 35 of these, including all of the best candidates, were sequenced. We identified variant NM_006331.6:c.400A-->G, p.D86G in the 18S ribosome assembly protein EMG1 as the probable cause of BCS. This mutation segregated with disease, was not found in 414 non-Hutterite alleles, and altered a highly conserved aspartic acid (D) residue. A structural model of human EMG1 suggested that the D86 residue formed a salt bridge with arginine 84 that would be disrupted by the glycine (G) substitution. EMG1 mRNA was detected in all human adult and fetal tissues tested. In BCS patient fibroblasts, EMG1 mRNA levels did not differ from those of normal cells, but EMG1 protein was dramatically reduced in comparison to that of normal controls. In mammalian cells, overexpression of EMG1 harboring the D86G mutation decreased the level of soluble EMG1 protein, and in yeast two-hybrid analysis, the D86G substitution increased interaction between EMG1 subunits. These findings suggested that the D-to-G mutation caused aggregation of EMG1, thereby reducing the level of the protein and causing BCS.
Assuntos
Cromossomos Humanos Par 12/genética , RNA Polimerases Dirigidas por DNA/genética , Retardo do Crescimento Fetal/genética , Mutação/genética , Transtornos Psicomotores/genética , Ribossomos/genética , Sequência de Aminoácidos , Animais , Estudos de Casos e Controles , Linhagem Celular , Cricetinae , RNA Polimerases Dirigidas por DNA/metabolismo , Feminino , Retardo do Crescimento Fetal/metabolismo , Retardo do Crescimento Fetal/patologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Genes Recessivos , Humanos , Immunoblotting , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Biogênese de Organelas , Linhagem , Conformação Proteica , Transtornos Psicomotores/metabolismo , Transtornos Psicomotores/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Síndrome , Técnicas do Sistema de Duplo-HíbridoRESUMO
The Snf1p/AMP-activated kinases are involved in transcriptional, metabolic, and developmental regulation in response to stress. In Saccharomyces cerevisiae, Snf1p (Cat1p) is one of the key regulators of carbohydrate metabolism, and cat1 (snf1) mutants fail to grow with non-fermentable carbon sources. In Candida albicans, Snf1p is an essential protein and cells depend on a functional Snf1 kinase even with glucose as carbon source. We investigated the CaSnf1p complex after tandem affinity purification and mass spectrometric analysis and show that the complex composition changes with the carbon source provided. Three subunits were identified, one of which was named CaSnf4p because of its homology to the ScSnf4 protein and the respective CaSNF4 gene could complement a S. cerevisiae snf4 mutant. The other two proteins revealed similarities to the S. cerevisiae kinase beta subunits ScGal83p, ScSip2p, and ScSip1p. Both genes complemented the scaffold function in a S. cerevisiae gal83,sip1,sip2 triple deletion mutant and were named according to their scaffold function as CaKIS1p and CaKIS2p. Matrix-assisted laser desorption ionization peptide mass fingerprint analysis indicated that CaKis2p is N-terminal myristoylated and the incorporation of CaKis2p in the Snf1p complex was reduced when compared with cells grown with glucose as a carbon source. To verify the different complex assemblies, a stable isotope labeling technique (iTraqtrade mark) was employed, confirming a 3-fold decrease of CaKis2p with ethanol. Yeast two-hybrid analysis confirmed the interaction partners, and these results showed an activator domain for the CaKis2 protein that has not been reported for S. cerevisiae scaffold subunits.
