Parallel laboratory evolution and rational debugging reveal genomic plasticity to S. cerevisiae synthetic chromosome XIV defects

Synthetic chromosome engineering is a complex process due to the need to identify and repair growth defects and deal with combinatorial gene essentiality when rearranging chromosomes. To alleviate these issues, we have demonstrated novel approaches for repairing and rearranging synthetic Saccharomyces cerevisiae genomes. We have designed, constructed, and restored wild-type fitness to a synthetic 753,096-bp version of S. cerevisiae chromosome XIV as part of the Synthetic Yeast Genome project. In parallel to the use of rational engineering approaches to restore wild-type fitness, we used adaptive laboratory evolution to generate a general growth-defect-suppressor rearrangement in the form of increased TAR1 copy number. We also extended the utility of the synthetic chromosome recombination and modification by loxPsym-mediated evolution (SCRaMbLE) system by engineering synthetic-wild-type tetraploid hybrid strains that buffer against essential gene loss, highlighting the plasticity of the S. cerevisiae genome in the presence of rational and non-rational modifications. </p

Comparative genomic analysis of glycoylation pathways in yeast, plants and higher eukaryotes

N-linked glycosylation is an essential modification of secretory and membrane proteins in all eukaryotic cells. Here, we review the current metabolic pathways of N-linked oligosaccharide biosynthesis in the endoplasmic reticulum and in the Golgi apparatus for yeasts: Saccharomyces cerevisiae and Schizosaccharomyces pombe, and higher eukaryotes: plants and human. The evolutionarily conserved proteins, processed in the cytosolic and the luminal side of the ER membrane, and the unique genes and their specific functions, occurring in the Golgi complex, for each selected organism, will be collated and discussed. This precise knowledge of the glycosylation pathway contributes to better understanding of the N-linked glycoprotein biosynthesis among different species, resulting in the recently successfully engineered strains for heterologous gene expression systems for industrial and therapeutic protein production.22 page(s

Control of plant-parasitic nematodes by Paecilomyces lilacinus and Monacrosporium lysipagum in pot trials

The common soil inhabiting nematophagous fungus Paecilomyces lilacinus (Thom) Samson and the nematode trapping fungus Monacrosporium lysipagum (Drechsler) Subram were assayed for their ability to reduce the populations of three economically important plant-parasitic nematodes in pot trials. The fungi were tested individually and in combination against the root-knot nematode Meloidogyne javanica (Treub) Chitwood, cereal cyst nematode Heterodera avenae Wollenweber, or burrowing nematode Radopholus similis (Cobb) Thorne on tomato, barley and tissue cultured banana plants, respectively. In all cases, nematode populations were controlled substantially by both individual and combined applications of the fungi. Combined application of P. lilacinus and M. lysipagum reduced 62% of galls and 94% of M.javanica juveniles on tomato when compared to the experiment with no fungi added. Sixty five percent of H. avenae cysts were reduced on barley by combined application of fungi. Control of R. similis on banana, both in the roots and in the soil, was greatest when M. lysipagum was applied alone (86%) or in combination with P. lilacinus (96%), using a strategy where the fungi were inoculated twice in 18 weeks growth period. Overall, combined application of P. lilacinus and M. lysipagum was the most effective treatment in controlling nematode populations, although in some cases M. lysipagum alone was as effective as the combined application of fungi, particularly against M. javanica.16 page(s

Infection of plant-parasitic nematodes by Paecilomyces lilacinus and Monacrosporium lysipagum

Studying the mode of infection of a biocontrol agent is important in order to assess its efficiency. The mode and severity of infection of nematodes by a soil saprophyte Paecilomyces lilacinus (Thom) Samson and a knob-producing nematode trapping fungus Monacrosporium lysipagum (Drechsler) Subram were studied under laboratory conditions using microscopy. Infection of stationary stages of nematodes by P. lilacinus was studied with three plant-parasitic nematodes Meloidogyne javanica (Treub) Chitwood, Heterodera avenae Wollenweber and Radopholus similis (Cobb) Thorne. Paecilomyces lilacinus infected eggs, juveniles and females of M. javanica by direct hyphal penetration. The early developed eggs were more susceptible than the eggs containing fully developed juveniles. As observed by transmission electron microscopy, fungal hypha penetrated the M. javanica female cuticle directly. Paecilomyces lilacinus also infected immature cysts of H. avenae including eggs in the cysts and the eggs of R. similis. Trapping and subsequent killing of mobile stages of nematodes by M. lysipagum were studied with the above three nematodes. In addition, plant-parasitic nematodes Pratylenchus neglectus (Rensch) Chitwood and Oteifa and Ditylenchus dipsaci (Kuhn) Filipjev were tested with M. lysipagum. This fungus was shown to infect mobile stages of all the plant-parasitic nematodes. In general, juveniles except those of P. neglectus, were more susceptible to the attack than adults.20 page(s

Activity-based identification of secreted serine proteases of the filamentous fungus, Ophiostoma

A general activity probe was synthesized and applied to the supernatant of a filamentous fungus, Ophiostoma, culture to identify directly the secreted serine proteases by covalent enzyme labeling. The activity probe contained a chemically reactive group that reacted with, and thus covalently labeled, the serine residues of only active proteases and not heat-inactivated proteases. The activity probe also contained a fluorescent group that allowed for the subsequent visualization of the captured proteases in 1-D gels and their identification by N-terminal sequencing. This use of a chemical probe led to the rapid discovery of subtilisin-like serine protease of Ophiostoma. Two hypothetical proteins were also captured, with one being a probable endopeptidase K type of protease.7 page(s

Heterologous protein expression in filamentous fungi

Filamentous fungi are commonly used in the fermentation industry for the large-scale production of proteins – mainly industrial enzymes. Recent advances in fungal genomics and related experimental technologies such as gene arrays and proteomics are rapidly changing the approaches to the development and use of filamentous fungi as hosts for the production of both homologous and heterologous gene products. The emphasis is moving towards sourcing new genes of interest through database mining and unravelling the circuits related to fungal gene regulation, applying, for example, transcriptomics. However, although heterologous fungal proteins are efficiently expressed, expression of gene products from other organisms is subject to several bottlenecks that reduce yield. Current approaches emphasize the study of pathways involved in protein modification and degradation in general rather than gene-by-gene approaches.7 page(s

Directed evolution of thermophilic enzymes and their high level expression in Trichoderma reesei

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α-amylase from a filamentous fungus - Ophiostoma floccosum

Ophiostoma floccosum, an ascomycete, is being developed as a new expression system for the production of foreign proteins. Enzymes for starch degradation and several proteases are amongst the most efficiently secreted proteins of Ophiostoma. The organism secretes only a few proteins into the culture medium, which provides a considerable advantage for the purification of any recombinant gene product. Several mutants of O. floccosum derived by UV mutagenesis have been isolated and the total the amount of secreted protein was increased by 4 to 6 times. The amylase activity of the best mutant was improved 240-fold compared to the parental strain. The proteinase profiles in the culture supernatants of several key mutants have been characterised for the selection of a suitable expression host for a particular gene product. The regulatory sequences and the protein encoding region of α-amylase, one of the dominant secreted proteins, have been isolated. A series of expression vectors containing the α-amylase regulatory sequences and sequences encoding the mature α-amylase enzyme gene have been constructed. The expression system is being tested using dsRed as a reporter gene.1 page(s

The Trichoderma reesei proteasome

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