Conditions Of Derepression Research Articles

Improving phytase production in Pichia pastoris fermentations through de-repression and methanol induction optimization.

Pichia pastoris (Komagataella phaffii) is a fast-growing methylotrophic yeast with the ability to assimilate several carbon sources such as methanol, glucose, or glycerol. It has been shown to have outstanding secretion capability with a variety of heterologous proteins. In previous studies, we engineered P. pastoris to co-express Escherichia coli AppA phytase and the HAC1 transcriptional activator using a bidirectional promoter. Phytase production was characterized in shake flasks and did not reflect industrial conditions. In the present study, phytase expression was explored and optimized using instrumented fermenters in continuous and fed-batch modes. First, the production of phytase was investigated under glucose de-repression in continuous culture at three dilution factors, 0.5 d-1 , 1 d-1 , and 1.5 d-1 . The fermenter parameters of these cultures were used to inform a kinetic model in batch and fed-batch modes for growth and phytase production. The kinetic model developed aided to design the glucose-feeding profile of a fed-batch culture. Kinetic model simulations under glucose de-repression and fed-batch conditions identified optimal phytase productivity at the specific growth rate of 0.041 h-1 . Validation of the model simulation with experimental data confirmed the feasibility of the model to predict phytase production in our newly engineered strain. Methanol was used only to induce the expression of phytase at high cell densities. Our results showed that high phytase production required two stages, the first stage used glucose under de-repression conditions to generate biomass while expressing phytase, and stage two used methanol to induce phytase expression. The production of phytase was improved 3.5-fold by methanol induction compared to the expression with glucose alone under de-repression conditions to a final phytase activity of 12.65 MU/L. This final volumetric phytase production represented an approximate 36-fold change compared to the flask fermentations. Finally, the phytase protein produced was assayed to confirm its molecular weight, and pH and temperature profiles. This study highlights the importance of optimizing protein production in P. pastoris when using novel promoters and presents a general approach to performing bioprocess optimization in this important production host.

Differing SAGA module requirements for NCR-sensitive gene transcription in yeast.

Nitrogen catabolite repression (NCR) is a means for yeast to adapt its transcriptome to changing nitrogen sources in its environment. In conditions of derepression (under poor nitrogen conditions, upon rapamycin treatment, or when glutamine production is inhibited), two transcriptional activators of the GATA family are recruited to NCR-sensitive promoters and activate transcription of NCR-sensitive genes. Earlier observations have involved the Spt-Ada-Gcn5 acetyltransferase(SAGA) chromatin remodeling complex in these transcriptional regulations. In this report, we provide an illustration of the varying NCR-sensitive responses and question whether differing SAGA recruitment could explain this diversity of responses.

Iron effects localization of Ght5 in fission yeast.

Iron is an essential cofactor for eukaryotic cells, as well as a toxic metal under certain conditions. On the other hand, glucose is the preferred energy and carbon source by most organisms and is an important signaling molecule in the regulation of biological processes. In Schizosaccharomyces pombe, the Ght5 hexose transporter, known as a high affinity glucose transporter, is required for cell proliferation in the low glucose condition. In this study, it is aimed to investigate the effects of iron stress on the Ght5 hexose transporter under glucose repression and derepression conditions. The effect of iron stress on the expression profile of the ght5 gene was analyzed by RT-qPCR and western blot. The localization of the Ght5-mNeongreen fusion protein examined with confocal microscopy. As a result, it was revealed that iron stress had an inhibitory effect on ght5 expression, and it altered Ght5 localization on the cell surface causing accumulation in the cytoplasm.

Ubiquitin-Conjugating Enzymes Ubc1 and Ubc4 Mediate the Turnover of Hap4, a Master Regulator of Mitochondrial Biogenesis in Saccharomyces cerevisiae.

Mitochondrial biogenesis is tightly regulated in response to extracellular and intracellular signals, thereby adapting yeast cells to changes in their environment. The Hap2/3/4/5 complex is a master transcriptional regulator of mitochondrial biogenesis in yeast. Hap4 is the regulatory subunit of the complex and exhibits increased expression when the Hap2/3/4/5 complex is activated. In cells grown under glucose derepression conditions, both the HAP4 transcript level and Hap4 protein level are increased. As part of an inter-organellar signaling mechanism coordinating gene expression between the mitochondrial and nuclear genomes, the activity of the Hap2/3/4/5 complex is reduced in respiratory-deficient cells, such as ρ0 cells lacking mitochondrial DNA, as a result of reduced Hap4 protein levels. However, the underlying mechanism is unclear. Here, we show that reduced HAP4 expression in ρ0 cells is mediated through both transcriptional and post-transcriptional mechanisms. We show that loss of mitochondrial DNA increases the turnover of Hap4, which requires the 26S proteasome and ubiquitin-conjugating enzymes Ubc1 and Ubc4. Stabilization of Hap4 in the ubc1 ubc4 double mutant leads to increased expression of Hap2/3/4/5-target genes. Our results indicate that mitochondrial biogenesis in yeast is regulated by the functional state of mitochondria partly through ubiquitin/proteasome-dependent turnover of Hap4.

On the Evidence Supporting That AN11127 Encodes an Aspergillus Nidulans Sec12 Orthologous Protein. Reply to Bravo-Plaza et al. Comment on "Dimou et al. Profile of Membrane Cargo Trafficking Proteins and Transporters Expressed under N Source Derepressing Conditions in Aspergillus nidulans. J. Fungi 2021, 7, 560".

Prof. Peñalva and co-workers provided evidence that AN11127 is related by sequence and function to Sec12 [...].

Comment on Dimou et al. Profile of Membrane Cargo Trafficking Proteins and Transporters Expressed under N Source Derepressing Conditions in Aspergillus nidulans. J. Fungi 2021, 7, 560.

Contrary to the opinion recently offered by Dimou et al., our previously published biochemical, subcellular and genetic data supported our contention that AN11127 corresponds to the A. nidulans gene encoding Sec12, which is the guanine nucleotide exchange factor (GEF) specific for SAR1. We add here additional bioinformatics evidence that fully disprove the otherwise negative evidence reported by Dimou et al., highlighting the dangers associated with the lax interpretation of genomic data. On the positive side, we establish guidelines for the identification of this key secretory gene in other species of Ascomycota and Basidiomycota, including species of medical and applied interest.

Profile of Membrane Cargo Trafficking Proteins and Transporters Expressed under N Source Derepressing Conditions in Aspergillus nidulans.

Solute and ion transporters are proteins essential for cell nutrition, detoxification, signaling, homeostasis and drug resistance. Being polytopic transmembrane proteins, they are co-translationally inserted and folded into the endoplasmic reticulum (ER) of eukaryotic cells and subsequently sorted to their final membrane destination via vesicular secretion. During their trafficking and in response to physiological/stress signals or prolonged activity, transporters undergo multiple quality control processes and regulated turnover. Consequently, transporters interact dynamically and transiently with multiple proteins. To further dissect the trafficking and turnover mechanisms underlying transporter subcellular biology, we herein describe a novel mass spectrometry-based proteomic protocol adapted to conditions allowing for maximal identification of proteins related to N source uptake in A. nidulans. Our analysis led to identification of 5690 proteins, which to our knowledge constitutes the largest protein dataset identified by omics-based approaches in Aspergilli. Importantly, we detected possibly all major proteins involved in basic cellular functions, giving particular emphasis to factors essential for membrane cargo trafficking and turnover. Our protocol is easily reproducible and highly efficient for unearthing the full A. nidulans proteome. The protein list delivered herein will form the basis for downstream systematic approaches and identification of protein–protein interactions in living fungal cells.

Apoptosis, Induced by Human α-Synuclein in Yeast, Can Occur Independent of Functional Mitochondria.

Human α-synuclein expression in baker’s yeast reportedly induces mitochondria-dependent apoptosis. Surprisingly, we find that, under de-repressing conditions of the inducible MET25/GAL1 promoters, yeast cells expressing chromosomally-integrated copies of the human α-synuclein gene are not killed, but spontaneously form respiration-deficient rho-minus (ρ−) petites. Although yeast cells can undergo cell death (apoptosis) from loss of mitochondrial function, they can also survive without functional mitochondria. Such cells are referred to as ρ0 or ρ− petites. This study reports that minimal expression of human α-synuclein in yeast, from MET25/GAL1 promoter, gives rise to ρ− petites. Interestingly, the full expression of α-synuclein, from the same promoters, in α-synuclein-triggered ρ− petites and also in ρ0 petites (produced by treating ρ+ cells with the mutagen ethidium bromide) initiates apoptosis. The percentages of petites increase with increasing α-synuclein gene copy-number. ρ− petites expressing α-synuclein from fully-induced MET25/GAL1 promoters exhibit increased ROS levels, loss of mitochondrial membrane potential, and nuclear DNA fragmentation, with increasing copies of α-synuclein. Our results indicate that, for the first time in yeast, α-synuclein-triggered apoptosis can occur independently of functional mitochondria. The observation that α-synuclein naturally forms petites and that they can undergo apoptosis may have important implications in understanding the pathogenesis of Parkinson’s disease.

Musashi interaction with poly(A)-binding protein is required for activation of target mRNA translation

The Musashi family of mRNA translational regulators controls both physiological and pathological stem cell self-renewal primarily by repressing target mRNAs that promote differentiation. In response to differentiation cues, Musashi can switch from a repressor to an activator of target mRNA translation. However, the molecular events that distinguish Musashi-mediated translational activation from repression are not understood. We have previously reported that Musashi function is required for the maturation of Xenopus oocytes and specifically for translational activation of specific dormant maternal mRNAs. Here, we employed MS to identify cellular factors necessary for Musashi-dependent mRNA translational activation. We report that Musashi1 needs to associate with the embryonic poly(A)-binding protein (ePABP) or the canonical somatic cell poly(A)-binding protein PABPC1 for activation of Musashi target mRNA translation. Co-immunoprecipitation studies demonstrated an increased Musashi1 interaction with ePABP during oocyte maturation. Attenuation of endogenous ePABP activity severely compromised Musashi function, preventing downstream signaling and blocking oocyte maturation. Ectopic expression of either ePABP or PABPC1 restored Musashi-dependent mRNA translational activation and maturation of ePABP-attenuated oocytes. Consistent with these Xenopus findings, PABPC1 remained associated with Musashi under conditions of Musashi target mRNA de-repression and translation during mammalian stem cell differentiation. Because association of Musashi1 with poly(A)-binding proteins has previously been implicated only in repression of Musashi target mRNAs, our findings reveal novel context-dependent roles for the interaction of Musashi with poly(A)-binding protein family members in response to extracellular cues that control cell fate.

Regulation of Aspergillus nidulans CreA-Mediated Catabolite Repression by the F-Box Proteins Fbx23 and Fbx47.

ABSTRACTThe attachment of one or more ubiquitin molecules by SCF (Skp–Cullin–F-box) complexes to protein substrates targets them for subsequent degradation by the 26S proteasome, allowing the control of numerous cellular processes. Glucose-mediated signaling and subsequent carbon catabolite repression (CCR) are processes relying on the functional regulation of target proteins, ultimately controlling the utilization of this carbon source. In the filamentous fungus Aspergillus nidulans, CCR is mediated by the transcription factor CreA, which modulates the expression of genes encoding biotechnologically relevant enzymes. Although CreA-mediated repression of target genes has been extensively studied, less is known about the regulatory pathways governing CCR and this work aimed at further unravelling these events. The Fbx23 F-box protein was identified as being involved in CCR and the Δfbx23 mutant presented impaired xylanase production under repressing (glucose) and derepressing (xylan) conditions. Mass spectrometry showed that Fbx23 is part of an SCF ubiquitin ligase complex that is bridged via the GskA protein kinase to the CreA-SsnF-RcoA repressor complex, resulting in the degradation of the latter under derepressing conditions. Upon the addition of glucose, CreA dissociates from the ubiquitin ligase complex and is transported into the nucleus. Furthermore, casein kinase is important for CreA function during glucose signaling, although the exact role of phosphorylation in CCR remains to be determined. In summary, this study unraveled novel mechanistic details underlying CreA-mediated CCR and provided a solid basis for studying additional factors involved in carbon source utilization which could prove useful for biotechnological applications.

The transcription factors ADR1 or CAT8 are required for RTG pathway activation and evasion from yeast acetic acid-induced programmed cell death in raffinose.

Yeast Saccharomyces cerevisiae grown on glucose undergoes programmed cell death (PCD) induced by acetic acid (AA-PCD), but evades PCD when grown in raffinose. This is due to concomitant relief of carbon catabolite repression (CCR) and activation of mitochondrial retrograde signaling, a mitochondria-to-nucleus communication pathway causing up-regulation of various nuclear target genes, such as CIT2, encoding peroxisomal citrate synthase, dependent on the positive regulator RTG2 in response to mitochondrial dysfunction. CCR down-regulates genes mainly involved in mitochondrial respiratory metabolism. In this work, we investigated the relationships between the RTG and CCR pathways in the modulation of AA-PCD sensitivity under glucose repression or de-repression conditions. Yeast single and double mutants lacking RTG2 and/or certain factors regulating carbon source utilization, including MIG1, HXK2, ADR1, CAT8, and HAP4, have been analyzed for their survival and CIT2 expression after acetic acid treatment. ADR1 and CAT8 were identified as positive regulators of RTG-dependent gene transcription. ADR1 and CAT8 interact with RTG2 and with each other in inducing cell resistance to AA-PCD in raffinose and controlling the nature of cell death. In the absence of ADR1 and CAT8, AA-PCD evasion is acquired through activation of an alternative factor/pathway repressed by RTG2, suggesting that RTG2 may play a function in promoting necrotic cell death in repressing conditions when RTG pathway is inactive. Moreover, our data show that simultaneous mitochondrial retrograde pathway activation and SNF1-dependent relief of CCR have a key role in central carbon metabolism reprogramming which modulates the yeast acetic acid-stress response.

Role of Base Excision Repair (BER) in Transcription-associated Mutagenesis of Nutritionally Stressed Nongrowing Bacillus subtilis Cell Subpopulations.

Compelling evidence points to transcriptional processes as important factors contributing to stationary-phase associated mutagenesis. However, it has not been documented whether or not base excision repair mechanisms play a role in modulating mutagenesis under conditions of transcriptional derepression. Here, we report on a flow cytometry-based methodology that employs a fluorescent reporter system to measure at single-cell level, the occurrence of transcription-associated mutations in nutritionally stressed B. subtilis cultures. Using this approach, we demonstrate that (i) high levels of transcription correlates with augmented mutation frequency, and (ii) mutation frequency is enhanced in nongrowing population cells deficient for deaminated (Ung, YwqL) and oxidized guanine (GO) excision repair, strongly suggesting that accumulation of spontaneous DNA lesions enhance transcription-associated mutagenesis.

Construction and use of a Cupriavidus necator H16 soluble hydrogenase promoter (PSH) fusion to gfp (green fluorescent protein).

Hydrogenases are metalloenzymes that reversibly catalyse the oxidation or production of molecular hydrogen (H2). Amongst a number of promising candidates for application in the oxidation of H2 is a soluble [Ni–Fe] uptake hydrogenase (SH) produced by Cupriavidus necator H16. In the present study, molecular characterisation of the SH operon, responsible for functional SH synthesis, was investigated by developing a green fluorescent protein (GFP) reporter system to characterise PSH promoter activity using several gene cloning approaches. A PSH promoter-gfp fusion was successfully constructed and inducible GFP expression driven by the PSH promoter under de-repressing conditions in heterotrophic growth media was demonstrated in the recombinant C. necator H16 cells. Here we report the first successful fluorescent reporter system to study PSH promoter activity in C. necator H16. The fusion construct allowed for the design of a simple screening assay to evaluate PSH activity. Furthermore, the constructed reporter system can serve as a model to develop a rapid fluorescent based reporter for subsequent small-scale process optimisation experiments for SH expression.

Combinatorial function of velvet and AreA in transcriptional regulation of nitrate utilization and secondary metabolism

Velvet is a conserved protein complex that functions as a regulator of fungal development and secondary metabolism. In the soil-inhabiting pathogen Fusarium oxysporum, velvet governs mycotoxin production and virulence on plant and mammalian hosts. Here we report a previously unrecognized role of the velvet complex in regulation of nitrate metabolism. F. oxysporum mutants lacking VeA or LaeA, two key components of the complex, were impaired in growth on the non-preferred nitrogen sources nitrate and nitrite. Both velvet and the general nitrogen response GATA factor AreA were required for transcriptional activation of nitrate (nit1) and nitrite (nii1) reductase genes under de-repressing conditions, as well as for the nitrate-triggered increase in chromatin accessibility at the nit1 locus. AreA also contributed to chromatin accessibility and expression of two velvet-regulated gene clusters, encoding biosynthesis of the mycotoxin beauvericin and of the siderophore ferricrocin. Thus, velvet and AreA coordinately orchestrate primary and secondary metabolism as well as virulence functions in F. oxysporum.

Gln3–Gcn4 hybrid transcriptional activator determines catabolic and biosynthetic gene expression in the yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is able to sense the availability and quality of nitrogen sources and the intrinsic variation of amino acid disponibility for protein synthesis. When this yeast is provided with secondary nitrogen sources, transcription of genes encoding enzymes involved in their catabolism is elicited through the action of Gln3, which constitutes the main activator of the Nitrogen Catabolite Repression network (NCR). Activation of genes encoding enzymes involved in the amino acid biosynthetic pathways is achieved through the action of the GCN4-encoded transcriptional modulator whose transcriptional activation is induced at the translational level by limitation for any amino acid. Thus the role of each one of these activators had been secluded to either catabolic or biosynthetic pathways. However, some observations have suggested that under peculiar physiological conditions, Gln3 and Gcn4 could act simultaneously in order to contemporaneously increase expression of both sets of genes. This paper addresses the question of whether Gln3 and Gcn4 cooperatively determine expression of their target genes. Results presented herein show that induced expression of catabolic and biosynthetic genes when cells are grown under nitrogen derepressive conditions and amino acid deprivation is dependent on the concurrent action of Gln3 and Gcn4, which form part of a unique transcriptional complex. We propose that the combination of Gln3 and Gcn4 results in the constitution of a hybrid modulator which elicits a novel transcriptional response, not evoked when these modulators act in a non-combinatorial fashion.

The snf1 Gene of Ustilago maydis Acts as a Dual Regulator of Cell Wall Degrading Enzymes

Many fungal plant pathogens are known to produce extracellular enzymes that degrade cell wall elements required for host penetration and infection. Due to gene redundancy, single gene deletions generally do not address the importance of these enzymes in pathogenicity. Cell wall degrading enzymes (CWDEs) in fungi are often subject to carbon catabolite repression at the transcriptional level such that, when glucose is available, CWDE-encoding genes, along with many other genes, are repressed. In Saccharomyces cerevisiae, one of the main players controlling this process is SNF1, which encodes a protein kinase. In this yeast, Snf1p is required to release glucose repression when this sugar is depleted from the growth medium. We have employed a reverse genetic approach to explore the role of the SNF1 ortholog as a potential regulator of CWDE gene expression in Ustilago maydis. We identified U. maydis snf1 and deleted it from the fungal genome. Consistent with our hypothesis, the relative expression of an endoglucanase and a pectinase was higher in the wild type than in the Δsnf1 mutant strain when glucose was depleted from the growth medium. However, when cells were grown in derepressive conditions, the relative expression of two xylanase genes was unexpectedly higher in the Δsnf1 strain than in the wild type, indicating that, in this case, snf1 negatively regulated the expression of these genes. Additionally, we found that, contrary to several other fungal species, U. maydis Snf1 was not required for utilization of alternative carbon sources. Also, unlike in ascomycete plant pathogens, deletion of snf1 did not profoundly affect virulence in U. maydis.

Metabolic Impact of Increased NADH Availability in Saccharomyces cerevisiae

Engineering the level of metabolic cofactors to manipulate metabolic flux is emerging as an attractive strategy for bioprocess applications. We present the metabolic consequences of increasing NADH in the cytosol and the mitochondria of Saccharomyces cerevisiae. In a strain that was disabled in formate metabolism, we either overexpressed the native NAD(+)-dependent formate dehydrogenase in the cytosol or directed it into the mitochondria by fusing it with the mitochondrial signal sequence encoded by the CYB2 gene. Upon exposure to formate, the mutant strains readily consumed formate and induced fermentative metabolism even under conditions of glucose derepression. Cytosolic overexpression of formate dehydrogenase resulted in the production of glycerol, while when this enzyme was directed into the mitochondria, we observed glycerol and ethanol production. Clearly, these results point toward different patterns of compartmental regulation of redox homeostasis. When pulsed with formate, S. cerevisiae cells growing in a steady state on glucose immediately consumed formate. However, formate consumption ceased after 20 min. Our analysis revealed that metabolites at key branch points of metabolic pathways were affected the most by the genetic perturbations and that the intracellular concentrations of sugar phosphates were specifically affected by time. In conclusion, the results have implications for the design of metabolic networks in yeast for industrial applications.

Quantitative transcriptional analysis of the genes encoding glycoside hydrolase family 7 cellulase isozymes in the basidiomycetePhanerochaete chrysosporium

Cellulolytic fungi generally secrete a cellulase mixture consisting mainly of glycoside hydrolase family 7 cellulases (Cel7s) during degradation of crystalline cellulose. Although several Cel7s have been investigated so far, the marked similarity in their amino acid and nucleotide sequences makes independent quantitative analysis difficult. Here, we present a real-time PCR method for the detection and quantification of Cel7 genes (cel7A-F/G) in the basidiomycete Phanerochaete chrysosporium using PCR primer sets designed based on the 3' untranslated region sequences. It was confirmed by agarose gel electrophoresis, sequencing, and dissociation curve analysis of the PCR products that each cel7 transcript was specifically amplified by the corresponding primers. We applied this real-time reverse-transcription PCR method using the presented primer sets to evaluate quantitatively the expression changes of cel7 genes in P. chrysosporium under conditions of carbon catabolite derepression.

Real-time quantitative analysis of carbon catabolite derepression of cellulolytic genes expressed in the basidiomycete Phanerochaete chrysosporium

Production of cellulolytic enzymes, such as cellobiohydrolases (CBH) and cellobiose dehydrogenase (CDH), by the basidiomycete Phanerochaete chrysosporium is significantly repressed in glucose-containing media; this is known as carbon catabolite repression. We have analyzed the glucose concentration dependence of transcript numbers of the cellulolytic genes (cel6A, cel7D, and cdh) and beta-glucosidase gene (bgl3A) by means of real-time quantitative reverse transcriptase polymerase chain reaction to investigate the roll of carbon catabolite derepression in these gene expression. When the mycelium of P. chrysosporium grown in glucose culture was transferred to media containing various concentrations of glucose (0-5,000 microM), the expression levels of cel6A, cel7D, and cdh were drastically influenced by glucose, whereas no significant change was observed in bgl3A. The numbers of transcripts of cel6A, cel7D, and cdh increased exponentially during incubation for 6 h in the culture without glucose, and the rates of increase were 2.1 times per hour for cel6A transcripts and 2.7 times per hour for cel7D transcripts. Moreover, derepression of cel6A and cel7D was delayed (by 1.6 and 0.6 h, respectively) when the culture contained 50 microM glucose compared with that in the absence of glucose, suggesting that the promoter activities of cel7D and cel6A are distinct under conditions of carbon catabolite derepression.

Dimerization of yeast transcription factors Ino2 and Ino4 is regulated by precursors of phospholipid biosynthesis mediated by Opi1 repressor

Structural genes of phospholipid biosynthesis in the yeast S. cerevisiae are activated by the heterodimeric transcription factor Ino2 + Ino4, binding to ICRE (inositol/choline-responsive element) promoter motifs. In the presence of phospholipid precursors inositol and choline, Ino2-dependent activation is inhibited by the Opi1 repressor which interacts with Ino2. In this work, we systematically investigated the importance of regulatory mechanisms possibly affecting ICRE-dependent gene expression. Autoregulatory expression of INO2, INO4 and OPI1 was abolished by promoter exchange experiments, showing that autoregulation of regulators contributes to the degree of differential gene expression but is not responsible for it. Using GFP fusion proteins, Ino2 and Ino4 were found to localize to the nucleus under conditions of repression and derepression. Interestingly, nuclear localization of Ino2 required a functional INO4 gene. Targeting of a lexA-Ino2 fusion to a heterologous promoter containing lexA operator motifs revealed a constitutive gene activation which was not influenced by phospholipid precursors. We could show that Ino2-dependent activation of a lexA-Ino4 fusion is affected by inositol and choline. Since gene activation required interaction of Ino2 and Ino4 mediated by their helix-loop-helix domains, formation/dissociation of the heterodimer must be considered as an important step of target gene regulation.