CRABTREE POZİTİF VE CRABTREE NEGATİF MAYA TÜRLERİNDE GCR1 GENİNİN IN SILICO ANALİZİ

The use of fermentation in the presence of oxygen and at high glucose concentrations is referred to as the Crabtree effect. Yeast species that have the Crabtree effect are called Crabtree positive, and yeast species that do not have the Crabtree effect are called Crabtree negative. While Crabtree negative yeast strains are mostly used for heterologous protein production in the industrial field, Crabtree positive yeast strains are used to understand metabolic events in cancer cells. The genes encoding the enzymes involved in the glycolytic pathway in S. cerevisiae yeast cells are controlled by Gcr1p. Gcr1p binds to CT elements located in the promoter regions of glycolytic genes and activates their transcription. In our study, Crabtree positive and negative yeast strains containing Sc-Gcr1p similar proteins were determined, and protein similarity analyzes and promoter analyzes of genes encoding the relevant proteins in these yeast strains were compared in silico using different databases and analysis programs. For this purpose, SGD, UNIPROT, NCBI-Genome and Yeastract databases and BLASTp-NCBI, MEGA-X and Chromatin Folding V2 programs were used. Using the SGD database, 32 different yeast strains were identified that matched with Sc-Gcr1p. Five different Crabtree positive and 5 different Crabtree negative yeast strains were selected from these yeast strains and in silico analyzes were performed using these yeast strains. After protein analysis and promoter analysis, it was determined that the similarities and differences between yeast species were not specific for Crabtree positive and Crabtree negative yeast species, but varied between species.

Download Full-text

Controlling Unconventional Secretion for Production of Heterologous Proteins in Ustilago maydis through Transcriptional Regulation and Chemical Inhibition of the Kinase Don3

Journal of Fungi ◽

10.3390/jof7030179 ◽

2021 ◽

Vol 7 (3) ◽

pp. 179

Author(s):

Kai P. Hussnaetter ◽

Magnus Philipp ◽

Kira Müntjes ◽

Michael Feldbrügge ◽

Kerstin Schipper

Keyword(s):

Protein Production ◽

Heterologous Protein ◽

Ustilago Maydis ◽

Downstream Processing ◽

Culture Broth ◽

Yeast Cells ◽

Heterologous Protein Expression ◽

Heterologous Proteins ◽

Regulatory Systems ◽

Unconventional Secretion

Heterologous protein production is a highly demanded biotechnological process. Secretion of the product to the culture broth is advantageous because it drastically reduces downstream processing costs. We exploit unconventional secretion for heterologous protein expression in the fungal model microorganism Ustilago maydis. Proteins of interest are fused to carrier chitinase Cts1 for export via the fragmentation zone of dividing yeast cells in a lock-type mechanism. The kinase Don3 is essential for functional assembly of the fragmentation zone and hence, for release of Cts1-fusion proteins. Here, we are first to develop regulatory systems for unconventional protein secretion using Don3 as a gatekeeper to control when export occurs. This enables uncoupling the accumulation of biomass and protein synthesis of a product of choice from its export. Regulation was successfully established at two different levels using transcriptional and post-translational induction strategies. As a proof-of-principle, we applied autoinduction based on transcriptional don3 regulation for the production and secretion of functional anti-Gfp nanobodies. The presented developments comprise tailored solutions for differentially prized products and thus constitute another important step towards a competitive protein production platform.

Download Full-text

Secretion of Human Serum Albumin by Kluyveromyces lactis Overexpressing KlPDI1 and KlERO1

Applied and Environmental Microbiology ◽

10.1128/aem.71.8.4359-4363.2005 ◽

2005 ◽

Vol 71 (8) ◽

pp. 4359-4363 ◽

Cited By ~ 29

Author(s):

Tiziana Lodi ◽

Barbara Neglia ◽

Claudia Donnini

Keyword(s):

Human Serum Albumin ◽

Serum Albumin ◽

Human Serum ◽

Protein Conformation ◽

Protein Production ◽

Heterologous Protein ◽

Kluyveromyces Lactis ◽

Heterologous Proteins ◽

Disulfide Bridges ◽

Genes Encoding

ABSTRACT The control of protein conformation during translocation through the endoplasmic reticulum is often a bottleneck for heterologous protein production. The core pathway of the oxidative folding machinery includes two conserved proteins: Pdi1p and Ero1p. We increased the dosage of the genes encoding these proteins in the yeast Kluyveromyces lactis and evaluated the secretion of heterologous proteins. KlERO1, an orthologue of Saccharomyces cerevisiae ERO1, was cloned by functional complementation of the ts phenotype of an Scero1 mutant. The expression of KlERO1 was induced by treatment of the cells with dithiothreitol and by overexpression of human serum albumin (HSA), a disulfide bond-rich protein. Duplication of either PDI1 or ERO1 led to a similar increase in HSA yield. Duplication of both genes accelerated the secretion of HSA and improved cell growth rate and yield. Increasing the dosage of KlERO1 did not affect the production of human interleukin 1β, a protein that has no disulfide bridges. The results confirm that the ERO1 genes of S. cerevisiae and K. lactis are functionally similar even though portions of their coding sequence are quite different and the phenotypes of mutants overexpressing the genes differ. The marked effects of KlERO1 copy number on the expression of heterologous proteins with a high number of disulfide bridges suggests that control of KlERO1 and KlPDI1 is important for the production of high levels of heterologous proteins of this type.

Download Full-text

Establishing regulated unconventional secretion for production of heterologous proteins

10.1101/2021.02.02.429340 ◽

2021 ◽

Author(s):

Kai P. Hussnaetter ◽

Magnus Philipp ◽

Kira Müntjes ◽

Michael Feldbrügge ◽

Kerstin Schipper

Keyword(s):

Protein Production ◽

Heterologous Protein ◽

Culture Broth ◽

Yeast Cells ◽

Heterologous Protein Expression ◽

Heterologous Proteins ◽

Regulatory Systems ◽

Production Platform ◽

Unconventional Secretion ◽

Different Levels

AbstractHeterologous protein production is a highly demanded biotechnological process. Secretion of the product to the culture broth is advantageous because it drastically reduces downstream pro-cessing costs. We exploit unconventional secretion for heterologous protein expression in the fungal model microorganism Ustilago maydis. Proteins of interest are fused to carrier chitinase Cts1 for export via the fragmentation zone of dividing yeast cells in a lock-type mechanism. Here, we are first to develop regulatory systems for unconventional protein secretion. This enables uncoupling the accumulation of biomass and protein synthesis of a product of choice from its export. Regulation was successfully established at two different levels using transcriptional and post-translational induction strategies. As a proof-of-principle, we applied autoinduction based on transcriptional regulation for the production and secretion of functional anti-Gfp nanobodies. The presented developments comprise tailored solutions for differentially prized products and thus constitute another important step towards a competitive protein production platform.

Download Full-text

In Silico Modeling of Crabtree Effect

Endocrine Metabolic & Immune Disorders - Drug Targets ◽

10.2174/1871530317666170828111113 ◽

2017 ◽

Vol 17 (3) ◽

Cited By ~ 1

Author(s):

Debraj Ghosh ◽

Rajat K. De

Keyword(s):

In Silico ◽

Crabtree Effect ◽

In Silico Modeling

Download Full-text

New Promoters to Improve Heterologous Protein Production in Aspergillus vadensis

Current Biotechnology ◽

10.2174/2211550103666140529115755 ◽

2014 ◽

Vol 3 (3) ◽

pp. 244-251 ◽

Cited By ~ 6

Author(s):

Helena Culleton ◽

Ourdia Bouzid ◽

Vincent McKie ◽

Ronald Vries

Keyword(s):

Protein Production ◽

Heterologous Protein ◽

Heterologous Protein Production

Download Full-text

Genes encoding components of the olfactory signal transduction cascade contain a DNA binding site that may direct neuronal expression.

Molecular and Cellular Biology ◽

10.1128/mcb.13.9.5805 ◽

1993 ◽

Vol 13 (9) ◽

pp. 5805-5813 ◽

Cited By ~ 52

Author(s):

M M Wang ◽

R Y Tsai ◽

K A Schrader ◽

R R Reed

Keyword(s):

Signal Transduction ◽

Dna Binding ◽

Binding Site ◽

Binding Sites ◽

Consensus Sequence ◽

Initiation Site ◽

Olfactory Neuron ◽

Promoter Regions ◽

Transcriptional Initiation ◽

Genes Encoding

Genes which mediate odorant signal transduction are expressed at high levels in neurons of the olfactory epithelium. The molecular mechanism governing the restricted expression of these genes likely involves tissue-specific DNA binding proteins which coordinately activate transcription through sequence-specific interactions with olfactory promoter regions. We have identified binding sites for the olfactory neuron-specific transcription factor, Olf-1, in the sequences surrounding the transcriptional initiation site of five olfactory neuron-specific genes. The Olf-1 binding sites described define the consensus sequence YTCCCYRGGGAR. In addition, we have identified a second binding site, the U site, in the olfactory cyclic nucleotide gated channel and type III cyclase promoters, which binds factors present in all tissue examined. These experiments support a model in which expression of Olf-1 in the sensory neurons coordinately activates a set of olfactory neuron-specific genes. Furthermore, expression of a subset of these genes may be modulated by additional binding factors.

Download Full-text

Lupeol Accumulation Correlates with Auxin in the Epidermis of Castor

Molecules ◽

10.3390/molecules26102978 ◽

2021 ◽

Vol 26 (10) ◽

pp. 2978

Author(s):

Donghai Li ◽

Cheng Pan ◽

Jianjun Lu ◽

Wajid Zaman ◽

Huayan Zhao ◽

...

Keyword(s):

Auxin Transport ◽

Biosynthesis Pathway ◽

Comparative Transcriptome ◽

Hormone Activity ◽

Promoter Regions ◽

Pentacyclic Triterpene ◽

Large Numbers ◽

Genes Encoding ◽

Low Levels ◽

Underlying Mechanisms

Lupeol, a natural lupane-type pentacyclic triterpene, possesses various pharmacological properties, and its production attracts attention. Significant quantities of lupeol are deposited on the castor aerial organ surface and are easily extractable as a predominant wax constituent. Thus, castor might be considered as a potential bioreactor for the production of lupeol. The lupeol biosynthesis pathway is well known, but how it is regulated remains largely unknown. Among large numbers of castor cultivars, we targeted one accession line (337) with high levels of lupeol on its stem surface and low levels thereof on its hypocotyl surface, implicating that lupeol synthesis is differentially regulated in the two organs. To explore the underlying mechanisms, we did comparative transcriptome analysis of the first internode of 337 stem and the upper hypocotyl. Our results show that large amounts of auxin-related genes are differentially expressed in both parts, implying some possible interactions between auxin and lupeol production. We also found that several auxin-responsive cis-elements are present in promoter regions of HMGR and LUS genes encoding two key enzymes involved in lupeol production. Furthermore, auxin treatments apparently induced the expression levels of RcHMGR and RcLUS. Furthermore, we observed that auxin treatment significantly increased lupeol contents, whereas inhibiting auxin transport led to an opposite phenotype. Our study reveals some relationships between hormone activity and lupeol synthesis and might provide a promising way for improving lupeol yields in castor.

Download Full-text

RegA, an AraC-Like Protein, Is a Global Transcriptional Regulator That Controls Virulence Gene Expression in Citrobacter rodentium

Infection and Immunity ◽

10.1128/iai.00770-08 ◽

2008 ◽

Vol 76 (11) ◽

pp. 5247-5256 ◽

Cited By ~ 44

Author(s):

Emily Hart ◽

Ji Yang ◽

Marija Tauschek ◽

Michelle Kelly ◽

Matthew J. Wakefield ◽

...

Keyword(s):

Mammalian Cells ◽

Virulence Gene ◽

Virulence Determinants ◽

Intestinal Colonization ◽

Citrobacter Rodentium ◽

Promoter Regions ◽

Reading Frame ◽

Virulence Gene Expression ◽

Genes Encoding ◽

Colonization Factors

ABSTRACT Citrobacter rodentium is an attaching and effacing pathogen which causes transmissible colonic hyperplasia in mice. Infection with C. rodentium serves as a model for infection of humans with enteropathogenic and enterohemorrhagic Escherichia coli. To identify novel colonization factors of C. rodentium, we screened a signature-tagged mutant library of C. rodentium in mice. One noncolonizing mutant had a single transposon insertion in an open reading frame (ORF) which we designated regA because of its homology to genes encoding members of the AraC family of transcriptional regulators. Deletion of regA in C. rodentium resulted in markedly reduced colonization of the mouse intestine. Examination of lacZ transcriptional fusions using promoter regions of known and putative virulence-associated genes of C. rodentium revealed that RegA strongly stimulated transcription of two newly identified genes located close to regA, which we designated adcA and kfcC. The cloned adcA gene conferred autoaggregation and adherence to mammalian cells to E. coli strain DH5α, and a kfc mutation led to a reduction in the duration of intestinal colonization, but the kfc mutant was far less attenuated than the regA mutant. These results indicated that other genes of C. rodentium whose expression required activation by RegA were required for colonization. Microarray analysis revealed a number of RegA-regulated ORFs encoding proteins homologous to known colonization factors. Transcription of these putative virulence determinants was activated by RegA only in the presence of sodium bicarbonate. Taken together, these results show that RegA is a global regulator of virulence in C. rodentium which activates factors that are required for intestinal colonization.

Download Full-text