Advances in the production of chemicals by organelle compartmentalization in Saccharomyces cerevisiae / 生物工程学报

As a generally-recognized-as-safe microorganism, Saccharomyces cerevisiae is a widely studied chassis cell for the production of high-value or bulk chemicals in the field of synthetic biology. In recent years, a large number of synthesis pathways of chemicals have been established and optimized in S. cerevisiae by various metabolic engineering strategies, and the production of some chemicals have shown the potential of commercialization. As a eukaryote, S. cerevisiae has a complete inner membrane system and complex organelle compartments, and these compartments generally have higher concentrations of the precursor substrates (such as acetyl-CoA in mitochondria), or have sufficient enzymes, cofactors and energy which are required for the synthesis of some chemicals. These features may provide a more suitable physical and chemical environment for the biosynthesis of the targeted chemicals. However, the structural features of different organelles hinder the synthesis of specific chemicals. In order to ameliorate the efficiency of product biosynthesis, researchers have carried out a number of targeted modifications to the organelles grounded on an in-depth analysis of the characteristics of different organelles and the suitability of the production of target chemicals biosynthesis pathway to the organelles. In this review, the reconstruction and optimization of the biosynthesis pathways for production of chemicals by organelle mitochondria, peroxisome, golgi apparatus, endoplasmic reticulum, lipid droplets and vacuole compartmentalization in S. cerevisiae are reviewed in-depth. Current difficulties, challenges and future perspectives are highlighted.

Sortin2 enhances endocytic trafficking towards the vacuole in Saccharomyces cerevisiae

BACKGROUND: A highly regulated trafficking of cargo vesicles in eukaryotes performs protein delivery to a variety of cellular compartments of endomembrane system. The two main routes, the secretory and the endocytic pathways have pivotal functions in uni- and multi-cellular organisms. Protein delivery and targeting includes cargo recognition, vesicle formation and fusion. Developing new tools to modulate protein trafficking allows better understanding the endomembrane system mechanisms and their regulation. The compound Sortin2 has been described as a protein trafficking modulator affecting targeting of the vacuolar protein carboxypeptidase Y (CPY), triggering its secretion in Saccharomyces cerevisiae. RESULTS: A reverse chemical-genetics approach was used to identify key proteins for Sortin2 bioactivity. A genome-wide Sortin2 resistance screen revealed six yeast deletion mutants that do not secrete CPY when grown at Sortin2 condition where the parental strain does: met18, sla1, clc1, dfg10, dpl1 and yjl175w. Integrating mutant phenotype and gene ontology annotation of the corresponding genes and their interactome pointed towards a high representation of genes involved in the endocytic process. In wild type yeast endocytosis towards the vacuole was faster in presence of Sortin2, which further validates the data of the genome-wide screen. This effect of Sortin2 depends on structural features of the molecule, suggesting compound specificity. Sortin2 did not affect endocytic trafficking in Sortin2-resistant mutants, strongly suggesting that the Sortin2 effects on the secretory and endocytic pathways are linked. CONCLUSIONS: Overall, the results reveal that Sortin2 enhances the endocytic transport pathway in Saccharomyces cerevisiae. This cellular effect is most likely at the level where secretory and endocytic pathways are merged. Them Sortin2 specificity over the endomembrane system places it as a powerful biological modulator for cell biology.

GRA Proteins of Toxoplasma gondii: Maintenance of Host-Parasite Interactions across the Parasitophorous Vacuolar Membrane

The dense granule of Toxoplasma gondii is a secretory vesicular organelle of which the proteins participate in the modification of the parasitophorous vacuole (PV) and PV membrane for the maintenance of intracellular parasitism in almost all nucleated host cells. In this review, the archives on the research of GRA proteins are reviewed on the foci of finding GRA proteins, characterizing molecular aspects, usefulness in diagnostic antigen, and vaccine trials in addition to some functions in host-parasite interactions.

Marginally low copper causes lesions of the midbrain in animal models: the implications for man

Serum copper levels must be maintained between very strict limits for the maintenance of good health. High levels have recently been linked to Alzheimer's disease while low levels during pregnancy cause enzootic ataxia (swayback disease) in offspring. In this study, we investigated the prolonged effect of serum copper that was maintained at and around 0.5 ppm, the level presently regarded as safe. Pregnant sheep and rabbits in the last trimester (1-4 weeks) of pregnancy were treated with the copper chelator ammonium tetrathiomolybdate (ATM). Treatment was continued until the young were one month old at which time the animals were sacrificed Serum copper levels of the parents and offspring were monitored by atomic absorption. The difference spectra (400-630 nm) was examined and SDS PAGE was used to evaluate the protein composition of the brain mitochondria. The anatomy of the midbrain was also studied. Although the young sheep and rabbits from the ATM-treated mothers showed no visible signs of disability or swayback disease, the midbrain of those young animals with serum copper between 0.3-0.9 ppm showed evidence of vacuolation, cavitation and chromatolysis. In contrast, the difference spectra and the protein composition of the brain mitochondria from these animals were all normal. These results suggest that although animals may appear normal and exhibit some normal biochemical markers, serum copper in the region of 0.5 ppm may not be safe for some breeds of sheep or rabbits. It is possible that a similar situation applies to man.

Los niveles séricos cúpricos tienen que ser mantenidos dentro de límites muy estrictos, si se quiere tener una buena salud. Los altos niveles de cobre han sido asociados recientemente con la enfermedad de Alzheimer, mientras que los niveles bajos durante el embarazo causan ataxia enzoótica (swayback) enla descendencia. En este estudio investigamos el efecto prolongado del cobre sérico mantenido a 0.5 ppm ó alrededor de 0.5 ppm – el nivel considerado seguro actualmente. Ovejas y conejas preñadas, en el último trimestre (1-4 semanas) de gestación, fueron tratadas con el quelante del cobre conocido como tetratiomolibdato de amonio (TM). El tratamiento continuó hasta que las crías tuvieron un mes,momento en el que los animales fueron sacrificados. Los niveles séricos cúpricos de los progenitores y la progenie fueron monitoreados mediante absorción atómica. Se examinaron los espectros de diferencia (400– 630 nm). Se usó la técnica de SDS-PAGE para evaluar la composición proteica de lasmitocondrias cerebrales. También se estudió la anatomía del mesencéfalo. Aunque la anatomía de las crías de ovejas y conejas madres tratadas con TM no mostraron señales visibles de discapacidad o enfermedad swayback, el mesencéfalo de estas crías con cobre sérico entre 0.3–0.9 ppm, mostróevidencias de vacuolación, cavitación y cromatolisis. En contraste con ello, los espectros de diferencia y la composición proteica de las mitocondrias del cerebro de estos animales, fueron todos normales. Estos resultados sugieren que aunque los animales puedan parecer normales y presentar marcas bioquímicas normales, el cobre sérico en el rango de 0.5 ppm, puede no ser seguro para algunas crías de ovejas y conejos. Es posible que una situación similar se aplique al ser humano.

Dorfman-Chanarin syndrome.

A four-year-old girl was brought to the dermatology outpatient department with scaling all over the body since birth. She had history of episodic vomiting and abdominal distension. A dermatological diagnosis of lamellar ichthyosis was made. Abdominal examination revealed a nontender hepatomegaly, fatty liver on ultrasonography and deranged liver function tests. Peripheral blood smear showed lipid vacuoles in the granulocytes consistent with Jordans' anomaly. Similar lipid vacuoles were seen in the basal layer in skin biopsy. An inflammatory infiltrate, moderate fibrosis in the portal tract and diffuse severe fatty change in hepatocytes were seen in liver biopsy. The patient was diagnosed as a case of Dorfman-Chanarin syndrome.

Interactions between secreted GRA proteins and host cell proteins across the paratitophorous vacuolar membrane in the parasitism of Toxoplasma gondii

Interactions between GRA proteins of dense granules in Toxoplasma gondii and host cell proteins were analyzed by yeast two-hybrid technique. The cMyc-GRA fusion proteins expressed from pGBKT7 plasmid in Y187 yeast were bound to host cell proteins from pGADT7-Rec-HeLa cDNA library transformed to AH109 yeast by mating method. By the selection procedures, a total of 939 colonies of the SD/-AHLT culture, 348 colonies of the X-alpha-gal positive and PCR, 157 colonies of the X-beta-gal assay were chosen for sequencing the cDNA and finally 90 colonies containing ORF were selected to analyze the interactions. GRA proteins interacted with a variety of host cell proteins such as enzymes, structural and functional proteins of organellar proteins of broad spectrum. Several specific bindings of each GRA protein to host proteins were discussed presumptively the role of GRA proteins after secreting into the parasitophorous vacuoles (PV) and the PV membrane in the parasitism of this parasite.

Acid phosphatase activity in ungerminated conidia from Colletotrichum graminicola as determined by spectrophotometric and cytochemical methods

The acid phosphatase in ungerminated conidia from Colletotrichum graminicola, a corn pathogen, was investigated using spectrophotometric and cytochemical methods. Acid phosphatase activity was studied in a homogenate obtained by fragmentation of ungerminated conidia. With p-nitrophenylphosphate as substrate, the apparent Vmax and Km were 1.000 nmol p-nitrophenol/mg of protein/min and 0.631 mM, respectively. The pH and temperature optima were 5.5 and 60 graus Celsius, respectively. A cytochemical ultrastructural assay showed deposition of the reaction product inside vacuoles but not extracellularly on the cell surface. The permeabilization of conidia with Triton X-100 increased acid phosphatase activity eight fold. Compared to other procedures, our method was fast, easy to perform and gave consistent results.

Envefalitis equina venezolana: leucocitos vacuolados / Venezuelan equine encephalitis: vacuolized leukocytes

Se estudia, mediante frotis obtenidos por punción digital, la sangre periférica (capilar) de 50 enfermos de encefalitis equina venezolana, en el período de estado de la enfermedad. Se encuentra que casi todos (49 de 50: 98 por ciento), presentaron vacuolas en los leucocitos. Se piensa en la posibilidad de que las vacuolas sean consecuencia de acción viral citopatológica