RESUMO
Pds5p and the cohesin complex are required for sister chromatid cohesion and localize to the same chromosomal loci over the same cell cycle window. However, Pds5p and the cohesin complex likely have distinct roles in cohesion. We report that pds5 mutants establish cohesion, but during mitosis exhibit precocious sister dissociation. Thus, unlike the cohesin complex, which is required for cohesion establishment and maintenance, Pds5p is required only for maintenance. We identified SMT4, which encodes a SUMO isopeptidase, as a high copy suppressor of both the temperature sensitivity and precocious sister dissociation of pds5 mutants. In contrast, SMT4 does not suppress temperature sensitivity of cohesin complex mutants. Pds5p is SUMO conjugated, with sumoylation peaking during mitosis. SMT4 overexpression reduces Pds5p sumoylation, whereas smt4 mutants have increased Pds5p sumoylation. smt4 mutants were previously shown to be defective in cohesion maintenance during mitosis. These data provide the first link between a protein required for cohesion, Pds5p, and sumoylation, and suggest that Pds5p sumoylation promotes the dissolution of cohesion.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromátides/fisiologia , Segregação de Cromossomos/fisiologia , Proteínas Fúngicas/metabolismo , Mitose/fisiologia , Proteínas de Saccharomyces cerevisiae , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Proteínas de Ciclo Celular/genética , Sobrevivência Celular/genética , Células Cultivadas , Proteínas Cromossômicas não Histona , Endopeptidases/genética , Endopeptidases/metabolismo , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica/genética , Modelos Biológicos , Mutação/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Temperatura , Leveduras , CoesinasRESUMO
Methylomonas sp. strain 16a is an obligate methanotrophic bacterium that uses methane or methanol as the sole carbon source. An effort was made to engineer this organism for astaxanthin production. Upon expressing the canthaxanthin gene cluster under the control of the native hps promoter in the chromosome, canthaxanthin was produced as the main carotenoid. Further conversion to astaxanthin was carried out by expressing different combinations of crtW and crtZ genes encoding the beta-carotenoid ketolase and hydroxylase. The carotenoid intermediate profile was influenced by the copy number of these two genes under the control of the hps promoter. Expression of two copies of crtZ and one copy of crtW led to the accumulation of a large amount of the mono-ketolated product adonixanthin. On the other hand, expression of two copies of crtW and one copy of crtZ resulted in the presence of non-hydroxylated carotenoid canthaxanthin and the mono-hydroxylated adonirubin. Production of astaxanthin as the predominant carotenoid was obtained in a strain containing two complete sets of carotenoid biosynthetic genes. This strain had an astaxanthin titer ranging from 1 to 2.4 mg g(-1) of dry cell biomass depending on the growth conditions. More than 90% of the total carotenoid was astaxanthin, of which the majority was in the form of E-isomer. This result indicates that it is possible to produce astaxanthin with desirable properties in methanotrophs through genetic engineering.
Assuntos
Methylomonas/genética , Methylomonas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Cantaxantina/química , Cantaxantina/metabolismo , Carotenoides/química , Carotenoides/metabolismo , Cromatografia Líquida de Alta Pressão , Fermentação , Metano/metabolismo , Modelos Biológicos , Estrutura Molecular , Xantofilas/biossíntese , Xantofilas/químicaRESUMO
Biosynthesis of the commercial carotenoids canthaxanthin and astaxanthin requires beta-carotene ketolase. The functional importance of the conserved amino acid residues of this enzyme from Paracoccus sp. strain N81106 (formerly classified as Agrobacterium aurantiacum) was analyzed by alanine-scanning mutagenesis. Mutations in the three highly conserved histidine motifs involved in iron coordination abolished its ability to catalyze the formation of ketocarotenoids. This supports the hypothesis that the CrtW ketolase belongs to the family of iron-dependent integral membrane proteins. Most of the mutations generated at other highly conserved residues resulted in partial activity. All partially active mutants showed a higher amount of adonixanthin accumulation than did the wild type when expressed in Escherichia coli cells harboring the zeaxanthin biosynthetic gene cluster. Some of the partially active mutants also produced a significant amount of echinenone when expressed in cells producing beta-carotene. In fact, expression of a mutant carrying D117A resulted in the accumulation of echinenone as the predominant carotenoid. These observations indicate that partial inactivation of the CrtW ketolase can often lead to the production of monoketolated intermediates. In order to improve the conversion rate of astaxanthin catalyzed by the CrtW ketolase, a color screening system was developed. Three randomly generated mutants, carrying L175M, M99V, and M99I, were identified to have improved activity. These mutants are potentially useful in pathway engineering for the production of astaxanthin.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cantaxantina/biossíntese , Oxigenases/genética , Oxigenases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Sequência de Bases , Sequência Conservada , DNA Bacteriano/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Oxigenases/química , Paracoccus/genética , Paracoccus/metabolismo , Plasmídeos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Xantofilas/biossínteseRESUMO
Sister chromatid cohesion enables chromosomes to achieve bipolar attachment to the mitotic spindle and its dissolution is required for chromosome segregation. The cohesin complex serves as the primary molecular glue responsible for cohesion. Pds5p binds to the same chromosomal loci as the cohesin complex but plays a distinct role as a regulator of cohesion maintenance. Catenation between sister chromatids must also be removed by Topoisomerase II (Top2p) enzymatic activity to enable chromosome segregation. We identified TOP2 as a high-copy suppressor of the temperature sensitivity of pds5 mutants. TOP2 suppression is specific for pds5 mutants as it does not suppress mutants in the cohesin complex. TOP2 suppresses mini-chromosome loss in pds5 mutants indicating that it rescues a chromosome segregation defect. Surprisingly, TOP2 overexpression fails to suppress the cohesion defect of pds5 mutants, suggesting that it suppresses an additional and as yet uncharacterized defect in pds5 mutants that is essential for viability. A catalytically dead TOP2 allele suppresses pds5 temperature sensitivity, suggesting that suppression is unrelated to Top2p enzymatic function. Consistent with this idea, when the pds5 mutant is combined with the top2-4 mutant, which accumulates DNA catenanes due to defective enzymatic activity, the double mutants exhibit synthetic sickness indicating that increased catenation is toxic to pds5 cells. Our results suggest that Pds5p and Top2p cooperate to promote proper chromosome segregation by a mechanism unrelated to either cohesion or catenation/decatenation.