Implications of carbon catabolite repression for Aspergillus-based cell factories: A review.

Carbon catabolite repression (CCR) is a global regulatory mechanism that allows organisms to preferentially utilize a preferred carbon source (usually glucose) by suppressing the expression of genes associated with the utilization of nonpreferred carbon sources. Aspergillus is a large genus of filamentous fungi, some species of which have been used as microbial cell factories for the production of organic acids, industrial enzymes, pharmaceuticals, and other fermented products due to their safety, substrate convenience, and well-established post-translational modifications. Many recent studies have verified that CCR-related genetic alterations can boost the yield of various carbohydrate-active enzymes (CAZymes), even under CCR conditions. Based on these findings, we emphasize that appropriate regulation of the CCR pathway, especially the expression of the key transcription factor CreA gene, has great potential for further expanding the application of Aspergillus cell factories to develop strains for industrial CAZymes production. Further, the genetically modified CCR strains (chassis hosts) can also be used for the production of other useful natural products and recombinant proteins, among others. We here review the regulatory mechanisms of CCR in Aspergillus and its direct application in enzyme production, as well as its potential application in organic acid and pharmaceutical production to illustrate the effects of CCR on Aspergillus cell factories.

The molecular mechanism for carbon catabolite repression of the chitin response in Vibrio cholerae.

Vibrio cholerae is a facultative pathogen that primarily occupies marine environments. In this niche, V. cholerae commonly interacts with the chitinous shells of crustacean zooplankton. As a chitinolytic microbe, V. cholerae degrades insoluble chitin into soluble oligosaccharides. Chitin oligosaccharides serve as both a nutrient source and an environmental cue that induces a strong transcriptional response in V. cholerae. Namely, these oligosaccharides induce the chitin sensor, ChiS, to activate the genes required for chitin utilization and horizontal gene transfer by natural transformation. Thus, interactions with chitin impact the survival of V. cholerae in marine environments. Chitin is a complex carbon source for V. cholerae to degrade and consume, and the presence of more energetically favorable carbon sources can inhibit chitin utilization. This phenomenon, known as carbon catabolite repression (CCR), is mediated by the glucose-specific Enzyme IIA (EIIAGlc) of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). In the presence of glucose, EIIAGlc becomes dephosphorylated, which inhibits ChiS transcriptional activity by an unknown mechanism. Here, we show that dephosphorylated EIIAGlc interacts with ChiS. We also isolate ChiS suppressor mutants that evade EIIAGlc-dependent repression and demonstrate that these alleles no longer interact with EIIAGlc. These findings suggest that EIIAGlc must interact with ChiS to exert its repressive effect. Importantly, the ChiS suppressor mutations we isolated also relieve repression of chitin utilization and natural transformation by EIIAGlc, suggesting that CCR of these behaviors is primarily regulated through ChiS. Together, our results reveal how nutrient conditions impact the fitness of an important human pathogen in its environmental reservoir.

Preliminary study on the effect of catabolite repression gene knockout on p-nitrophenol degradation in Pseudomonas putida DLL-E4.

P-nitrophenol (PNP) is a carcinogenic, teratogenic, and mutagenic compound that can cause serious harm to the environment. A strain of Pseudomonas putida DLL-E4, can efficiently degrade PNP in a complex process that is influenced by many factors. Previous studies showed that the expression level of pnpA, a key gene involved in PNP degradation, was upregulated significantly and the degradation of PNP was obviously accelerated in the presence of glucose. In addition, the expression of crc, crcY, and crcZ, key genes involved in catabolite repression, was downregulated, upregulated, and upregulated, respectively. To investigate the effect of the carbon catabolite repression (CCR) system on PNP degradation, the crc, crcY, and crcZ genes were successfully knocked out by conjugation experiments. Our results showed that the knockout of crc accelerated PNP degradation but slowed down the cell growth. However, the knockout of crcY or crcZ alone accelerated PNP degradation when PNP as the sole carbon source, but that knockout slowed down PNP degradation when glucose was added. The results indicate that the CCR system is involved in the regulation of PNP degradation, and further work is required to determine the details of the specific regulatory mechanism.

Implications of carbon catabolite repression for plant-microbe interactions.

Carbon catabolite repression (CCR) plays a key role in many physiological and adaptive responses in a broad range of microorganisms that are commonly associated with eukaryotic hosts. When a mixture of different carbon sources is available, CCR, a global regulatory mechanism, inhibits the expression and activity of cellular processes associated with utilization of secondary carbon sources in the presence of the preferred carbon source. CCR is known to be executed by completely different mechanisms in different bacteria, yeast, and fungi. In addition to regulating catabolic genes, CCR also appears to play a key role in the expression of genes involved in plant-microbe interactions. Here, we present a detailed overview of CCR mechanisms in various bacteria. We highlight the role of CCR in beneficial as well as deleterious plant-microbe interactions based on the available literature. In addition, we explore the global distribution of known regulatory mechanisms within bacterial genomes retrieved from public repositories and within metatranscriptomes obtained from different plant rhizospheres. By integrating the available literature and performing targeted meta-analyses, we argue that CCR-regulated substrate use preferences of microorganisms should be considered an important trait involved in prevailing plant-microbe interactions.

DNA affinity purification sequencing and transcriptional profiling reveal new aspects of nitrogen regulation in a filamentous fungus.

Sensing available nutrients and efficiently utilizing them is a challenge common to all organisms. The model filamentous fungus Neurospora crassa is capable of utilizing a variety of inorganic and organic nitrogen sources. Nitrogen utilization in N. crassa is regulated by a network of pathway-specific transcription factors that activate genes necessary to utilize specific nitrogen sources in combination with nitrogen catabolite repression regulatory proteins. We identified an uncharacterized pathway-specific transcription factor, amn-1, that is required for utilization of the nonpreferred nitrogen sources proline, branched-chain amino acids, and aromatic amino acids. AMN-1 also plays a role in regulating genes involved in responding to the simple sugar mannose, suggesting an integration of nitrogen and carbon metabolism. The utilization of nonpreferred nitrogen sources, which require metabolic processing before being used as a nitrogen source, is also regulated by the nitrogen catabolite regulator NIT-2. Using RNA sequencing combined with DNA affinity purification sequencing, we performed a survey of the role of NIT-2 and the pathway-specific transcription factors NIT-4 and AMN-1 in directly regulating genes involved in nitrogen utilization. Although previous studies suggested promoter binding by both a pathway-specific transcription factor and NIT-2 may be necessary for activation of nitrogen-responsive genes, our data show that pathway-specific transcription factors regulate genes involved in the catabolism of specific nitrogen sources, while NIT-2 regulates genes involved in utilization of all nonpreferred nitrogen sources, such as nitrogen transporters. Together, these transcription factors form a nutrient sensing network that allows N. crassa cells to regulate nitrogen utilization.

Molecular mechanisms controlling fructose-specific memory and catabolite repression in lactose metabolism by Streptococcus mutans.

Lactose is an abundant dietary carbohydrate metabolized by the dental pathogen Streptococcus mutans. Lactose metabolism presents both classic diauxic behaviors and long-term memory, where the bacteria can pause for >11 h before initiating growth on lactose. Here, we explored mechanisms contributing to unusual aspects of regulation of the lac operon. The fructose-phosphate metabolites, F-1-P and F-6-P, could modulate the DNA-binding activities of the lactose repressor. Recombinant LacR proteins bound upstream of lacA and Gal-6-P induced the formation of different LacR-DNA complexes. Deletion of lacR resulted in strain-specific growth phenotypes on lactose, but also on a number of mono- and di-saccharides that involve the glucose-PTS or glucokinase in their catabolism. The phenotypes were consistent with the novel findings that loss of LacR altered glucose-PTS activity and expression of the gene for glucokinase. CcpA was also shown to affect lactose metabolism in vivo and to bind to the lacA promoter region in vitro. Collectively, our study reveals complex molecular circuits controlling lactose metabolism in S. mutans, where LacR and CcpA integrate cellular and environmental cues to regulate metabolism of a variety of carbohydrates that are critical to persistence and pathogenicity of S. mutans.

Carbon catabolite repressor MoCreA is required for the asexual development and pathogenicity of the rice blast fungus.

During the infection and colonization process, the rice blast fungus Magnaporthe oryzae faces various challenges from hostile environment, such as nutrient limitation and carbon stress, while carbon catabolite repression (CCR) mechanism would facilitate the fungus to shrewdly and efficiently utilize carbon nutrients under fickle nutritional conditions since it ensures the preferential utilization of most preferred carbon sources through repressing the expression of enzymes required for the utilization of less preferred carbon sources. Researches on M. oryzae CCR have made some progress, however the involved regulation mechanism is still largely obscured, especially, little is known about the key carbon catabolite repressor CreA. Here we identified and characterized the biological functions of the CreA homolog MoCreA in M. oryzae. MoCreA is constitutively expressed throughout all the life stages of the fungus, and it can shuttle between nucleus and cytoplasm which is induced by glucose. Following functional analyses of MoCreA suggested that it was required for the vegetative growth, conidiation, appressorium formation and pathogenicity of M. oryzae. Moreover, comparative transcriptomic analysis revealed that disruption of MoCreA resulted in the extensive gene expression variations, including a large number of carbon metabolism enzymes, transcription factors and pathogenicity-related genes. Taken together, our results demonstrated that, as a key regulator of CCR, MoCreA plays a vital role in precise regulation of the asexual development and pathogenicity of the rice blast fungus.

CcpN: a moonlighting protein regulating catabolite repression of gluconeogenic genes in Bacillus subtilis also affects cell length and interacts with DivIVA.

CcpN is a transcriptional repressor in Bacillus subtilis that binds to the promoter region of gapB and pckA, downregulating their expression in the presence of glucose. CcpN also represses sr1, which encodes a small noncoding regulatory RNA that suppresses the arginine biosynthesis gene cluster. CcpN has homologues in other Gram-positive bacteria, including Enterococcus faecalis. We report the interaction of CcpN with DivIVA of B. subtilis as determined using bacterial two-hybrid and glutathione S-transferase pull-down assays. Insertional inactivation of CcpN leads to cell elongation and formation of straight chains of cells. These findings suggest that CcpN is a moonlighting protein involved in both gluconeogenesis and cell elongation.

The High Osmolarity Glycerol Mitogen-Activated Protein Kinase regulates glucose catabolite repression in filamentous fungi.

The utilization of different carbon sources in filamentous fungi underlies a complex regulatory network governed by signaling events of different protein kinase pathways, including the high osmolarity glycerol (HOG) and protein kinase A (PKA) pathways. This work unraveled cross-talk events between these pathways in governing the utilization of preferred (glucose) and non-preferred (xylan, xylose) carbon sources in the reference fungus Aspergillus nidulans. An initial screening of a library of 103 non-essential protein kinase (NPK) deletion strains identified several mitogen-activated protein kinases (MAPKs) to be important for carbon catabolite repression (CCR). We selected the MAPKs Ste7, MpkB, and PbsA for further characterization and show that they are pivotal for HOG pathway activation, PKA activity, CCR via regulation of CreA cellular localization and protein accumulation, as well as for hydrolytic enzyme secretion. Protein-protein interaction studies show that Ste7, MpkB, and PbsA are part of the same protein complex that regulates CreA cellular localization in the presence of xylan and that this complex dissociates upon the addition of glucose, thus allowing CCR to proceed. Glycogen synthase kinase (GSK) A was also identified as part of this protein complex and shown to potentially phosphorylate two serine residues of the HOG MAPKK PbsA. This work shows that carbon source utilization is subject to cross-talk regulation by protein kinases of different signaling pathways. Furthermore, this study provides a model where the correct integration of PKA, HOG, and GSK signaling events are required for the utilization of different carbon sources.

Enterococcus faecalis Maltodextrin Gene Regulation by Combined Action of Maltose Gene Regulator MalR and Pleiotropic Regulator CcpA.

Enterococci are Gram-positive bacteria present in the healthy human microbiota, but they are also a leading cause of nosocomial infections. Maltodextrin utilization by Enterococcus faecalis has been identified as an important factor for colonization of mammalians hosts. Here, we show that the LacI/GalR transcriptional regulator MalR, the maltose gene regulator, is also the main regulator of the operons encoding an ABC transporter (mdxEFG) and three metabolic enzymes (mmdH-gmdH-mmgT) required for the uptake and catabolism of maltotetraose and longer maltodextrins. The utilization of maltose and maltodextrins is consequently coordinated and induced by the disaccharide maltose, which binds to MalR. Carbon catabolite repression of the mdxEFG and mmdH-gmdH-mmgT operons is mediated by both P-Ser-HPr/MalR and P-Ser-HPr/CcpA. The latter complex exerts only moderate catabolite repression, which became visible when comparing maltodextrin operon expression levels of a malR- mutant (with a mutant allele for the malR gene) and a malR- ΔccpA double mutant grown in the presence of maltose, which is transported via a phosphotransferase system and, thus, favors the formation of P-Ser-HPr. Moreover, maltodextrin transport via MdxEFG slows rapidly when glucose is added, suggesting an additional regulation via inducer exclusion. This complex regulation of metabolic operons likely allows E. faecalis to fine-tune gene expression in response to changing environmental conditions.IMPORTANCEEnterococcus faecalis represents a leading cause of hospital-acquired infections worldwide. Several studies highlighted the importance of carbohydrate metabolism in the infection process of this bacterium. The genes required for maltodextrin metabolism are particularly induced during mouse infection and, therefore, should play an important role for pathogenesis. Since no data were hitherto available concerning the regulation of expression of the maltodextrin operons, we have conducted experiments to study the underlying mechanisms.

The ROK-family regulator Rok7B7 directly controls carbon catabolite repression, antibiotic biosynthesis, and morphological development in Streptomyces avermitilis.

Carbon catabolite repression (CCR) is a common phenomenon in bacteria that modulates expression of genes involved in uptake of alternative carbon sources. In the filamentous streptomycetes, which produce half of all known antibiotics, the precise mechanism of CCR is yet unknown. We report here that the ROK-family regulator Rok7B7 pleiotropically controls xylose and glucose uptake, CCR, development, as well as production of the macrolide antibiotics avermectin and oligomycin A in Streptomyces avermitilis. Rok7B7 directly repressed structural genes for avermectin biosynthesis, whereas it activated olmRI, the cluster-situated activator gene for oligomycin A biosynthesis. Rok7B7 also directly repressed the xylose uptake operon xylFGH, whose expression was induced by xylose and repressed by glucose. Both xylose and glucose served as Rok7B7 ligands. rok7B7 deletion led to enhancement and reduction of avermectin and oligomycin A production, respectively, relieved CCR of xylFGH, and increased co-uptake efficiency of xylose and glucose. A consensus Rok7B7-binding site, 5'-TTKAMKHSTTSAV-3', was identified within aveA1p, olmRIp, and xylFp, which allowed prediction of the Rok7B7 regulon and confirmation of 11 additional targets involved in development, secondary metabolism, glucose uptake, and primary metabolic processes. Our findings will facilitate methods for strain improvement, antibiotic overproduction, and co-uptake of xylose and glucose in Streptomyces species.

TpiA is a Key Metabolic Enzyme That Affects Virulence and Resistance to Aminoglycoside Antibiotics through CrcZ in Pseudomonas aeruginosa.

Carbon metabolism plays an essential role in bacterial pathogenesis and susceptibility to antibiotics. In Pseudomonas aeruginosa, Crc, Hfq, and a small RNA, CrcZ, are central regulators of carbon metabolism. By screening mutants of genes involved in carbon metabolism, we found that mutation of the tpiA gene reduces the expression of the type III secretion system (T3SS) and bacterial resistance to aminoglycoside antibiotics. TpiA is a triosephosphate isomerase that reversibly converts glyceraldehyde 3-phosphate to dihydroxyacetone phosphate, a key step connecting glucose metabolism with glycerol and phospholipid metabolisms. We found that mutation of the tpiA gene enhances the bacterial carbon metabolism, respiration, and oxidative phosphorylation, which increases the membrane potential and promotes the uptake of aminoglycoside antibiotics. Further studies revealed that the level of CrcZ is increased in the tpiA mutant due to enhanced stability. Mutation of the crcZ gene in the tpiA mutant background restored the expression of the T3SS genes and the bacterial resistance to aminoglycoside antibiotics. Overall, this study reveals an essential role of TpiA in the metabolism, virulence, and antibiotic resistance in P. aeruginosaIMPORTANCE The increase in bacterial resistance against antibiotics imposes a severe threat to public health. It is urgent to identify new drug targets and develop novel antimicrobials. Metabolic homeostasis of bacteria plays an essential role in their virulence and resistance to antibiotics. Recent studies demonstrated that antibiotic efficacies can be improved by modulating the bacterial metabolism. Pseudomonas aeruginosa is an important opportunistic human pathogen that causes various infections. The bacterium is intrinsically resistant to antibiotics. In this study, we provide clear evidence that TpiA (triosephosphate isomerase) plays an essential role in the metabolism of P. aeruginosa and influences bacterial virulence and antibiotic resistance. The significance of this work is in identifying a key enzyme in the metabolic network, which will provide clues as to the development of novel treatment strategies against infections caused by P. aeruginosa.

A Novel CreA-Mediated Regulation Mechanism of Cellulase Expression in the Thermophilic Fungus Humicola insolens.

The thermophilic fungus Humicola insolens produces cellulolytic enzymes that are of great scientific and commercial interest; however, few reports have focused on its cellulase expression regulation mechanism. In this study, we constructed a creA gene (carbon catabolite repressor gene) disruption mutant strain of H. insolens that exhibited a reduced radial growth rate and stouter hyphae compared to the wild-type (WT) strain. The creA disruption mutant also expressed elevated pNPCase (cellobiohydrolase activities), pNPGase (ß-glucosidase activities), and xylanase levels in non-inducing fermentation with glucose. Unlike other fungi, the H. insolens creA disruption mutant displayed lower FPase (filter paper activity), CMCase (carboxymethyl cellulose activity), pNPCase, and pNPGase activity than observed in the WT strain when fermentation was induced using Avicel, whereas its xylanase activity was higher than that of the parental strain. These results indicate that CreA acts as a crucial regulator of hyphal growth and is part of a unique cellulase expression regulation mechanism in H. insolens. These findings provide a new perspective to improve the understanding of carbon catabolite repression regulation mechanisms in cellulase expression, and enrich the knowledge of metabolism diversity and molecular regulation of carbon metabolism in thermophilic fungi.

CreA-independent carbon catabolite repression of cellulase genes by trimeric G-protein and protein kinase A in Aspergillus nidulans.

Cellulase production in filamentous fungi is repressed by various carbon sources. In our preliminary survey in Aspergillus nidulans, degree of de-repression differed depending on carbon sources in a mutant of creA, encoding the transcriptional repressor for carbon catabolite repression (CCR). To further understand mechanisms of CCR of cellulase production, we compared the effects of creA deletion with deletion of protein kinase A (pkaA) and G (ganB) genes, which constitute a nutrient sensing and signaling pathway. In plate culture with carboxymethyl cellulose and D-glucose, deletion of pkaA and ganB, but not creA, led to significant de-repression of cellulase production. In submerged culture with cellobiose and D-glucose or 2-deoxyglucose, both creA or pkaA single deletion led to partial de-repression of cellulase genes with the highest level by their double deletion, while ganB deletion caused de-repression comparable to that of the creA/pkaA double deletion. With ball-milled cellulose and D-glucose, partial de-repression was detected by deletion of creA but not of pkaA or ganB. The creA/pkaA or creA/ganB double deletion led to earlier expression than the creA deletion. Furthermore, the effect of each deletion with D-xylose or L-arabinose as the repressing carbon source was significantly different from that with D-glucose, D-fructose, and D-mannose. Consequently, this study revealed that PkaA and GanB participate in CreA-independent CCR and that contribution of CreA, PkaA, and GanB in CCR differs depending on the inducers, repressing carbon sources, and culture conditions (plate or submerged). Further study of CreA-independent mechanisms is needed to fully understand CCR in filamentous fungi.

Architectural principles for Hfq/Crc-mediated regulation of gene expression.

In diverse bacterial species, the global regulator Hfq contributes to post-transcriptional networks that control expression of numerous genes. Hfq of the opportunistic pathogen Pseudomonas aeruginosa inhibits translation of target transcripts by forming a regulatory complex with the catabolite repression protein Crc. This repressive complex acts as part of an intricate mechanism of preferred nutrient utilisation. We describe high-resolution cryo-EM structures of the assembly of Hfq and Crc bound to the translation initiation site of a target mRNA. The core of the assembly is formed through interactions of two cognate RNAs, two Hfq hexamers and a Crc pair. Additional Crc protomers are recruited to the core to generate higher-order assemblies with demonstrated regulatory activity in vivo. This study reveals how Hfq cooperates with a partner protein to regulate translation, and provides a structural basis for an RNA code that guides global regulators to interact cooperatively and regulate different RNA targets.

Functional analysis of the role of CcpA in Lactobacillus plantarum grown on fructooligosaccharides or glucose: a transcriptomic perspective.

BACKGROUND: The catabolite control protein A (CcpA) is a master regulator of many important cellular processes in Gram-positive bacteria. In Lactobacillus plantarum, CcpA directly or indirectly controls the transcription of a large number of genes that are involved in carbohydrate metabolism, aerobic and anaerobic growth, stress response and metabolite production, but its role in response to different carbon sources remains unclear. RESULTS: Here a combined transcriptomic and physiological approach was used to survey the global alterations that occurred during the logarithmic growth phase of wild-type and ccpA mutant strains of L. plantarum ST-III using fructooligosaccharides (FOS) or glucose as the sole carbon source. The inactivation of ccpA significantly affected the growth and production of metabolites under both carbon sources. About 15% of the total genes were significantly altered between wild-type and ccpA strains grown on glucose and the value is deceased to 12% when these two strains were compared on FOS, while only 7% were obviously changed due to the loss of CcpA when comparing strains grown on glucose and FOS. Although most of the differentially expressed genes mediated by CcpA are glucose dependent, FOS can also induce carbon catabolite repression (CCR) through the CcpA pathway. Moreover, the inactivation of ccpA led to a transformation from homolactic fermentation to mixed fermentation under aerobic conditions. CcpA can control genes directly by binding in the regulatory region of the target genes (mixed fermentation), indirectly through local regulators (fatty acid biosynthesis), or have a double effect via direct and indirect regulation (FOS metabolism). CONCLUSION: Overall, our results show that CcpA plays a central role in response to carbon source and availability of L. plantarum and provide new insights into the complex and extended regulatory network of lactic acid bacteria.

Expression of the sRNAs CrcZ and CrcY modulate the strength of carbon catabolite repression under diazotrophic or non-diazotrophic growing conditions in Azotobacter vinelandii.

Azotobacter vinelandii is a nitrogen-fixing bacterium of the Pseudomonadaceae family that prefers the use of organic acids rather than carbohydrates. Thus, in a mixture of acetate-glucose, glucose is consumed only after acetate is exhausted. In a previous work, we investigated the molecular basis of this carbon catabolite repression (CCR) process under diazotrophic conditions. In the presence of acetate, Crc-Hfq inhibited translation of the gluP mRNA, encoding the glucose transporter in A. vinelandii. Herein, we investigated the regulation in the expression of the small non-coding RNAs (sRNAs) crcZ and crcY, which are known to antagonize the repressing activity of Hfq-Crc. Our results indicated higher expression levels of the sRNAs crcZ and crcY under low CCR conditions (i.e. glucose), in relation to the strong one (acetate one). In addition, we also explored the process of CCR in the presence of ammonium. Our results revealed that CCR also occurs under non-diazotrophic conditions as we detected a hierarchy in the utilization of the supplied carbon sources, which was consistent with the higher expression level of the crcZ/Y sRNAs during glucose catabolism. Analysis of the promoters driving transcription of crcZ and crcY confirmed that they were RpoN-dependent but we also detected a processed form of CrcZ (CrcZ*) in the RpoN-deficient strain derived from a cbrB-crcZ co-transcript. CrcZ* was functional and sufficient to allow the assimilation of acetate.

[Carbon catabolite repression or how bacteria choose their favorite sugars]. / La répression catabolique ou comment les bactéries choisissent leurs sucres préférés.

Carbon catabolite repression is an important regulatory mechanism allowing bacteria, but also yeast and fungi, to preferentially use easily metabolizable carbon sources (like glucose) over relatively less favorable carbon sources (for example, organic acids and alcohols). This phenomenon is illustrated by diauxic growth during which bacteria assimilate firstly energy-efficient and rapidly metabolizable sugars then less-favored carbohydrates. A variety of molecular mechanisms are involved in carbon catabolite repression in order to control not only the expression of genes involved in the utilization of alternative carbon sources but also the expression of genes involved in several processes like virulence, competence etc. In this review, are described the main molecular mechanisms found in enterobacteria and in firmicutes and the importance of the sugar-uptake phosphotransferase system for these molecular mechanisms.

Transcriptome analysis of Δmig1Δmig2 mutant reveals their roles in methanol catabolism, peroxisome biogenesis and autophagy in methylotrophic yeast Pichia pastoris.

Two catabolite repressor genes (MIG1 and MIG2) were previously identified in Pichia pastoris, and the derepression of alcohol oxidase (AOX) expression was realized in Δmig1 or Δmig1Δmig2 mutants grown in glycerol, but not in glucose. In this study, genome-wide RNA-seq analysis of Δmig1Δmig2 and the wild-type strain grown in glycerol revealed that the expression of numerous genes was greatly altered. Nearly 7% (357 genes) of approximately 5276 genes annotated in P. pastoris were significantly upregulated, with at least a two-fold differential expression in Δmig1Δmig2; the genes were mainly related to cell metabolism. Approximately 23% (1197 genes) were significantly downregulated; these were mainly correlated with the physiological characteristics of the cell. The methanol catabolism and peroxisome biogenesis pathways were remarkably enhanced, and the genes AOX1 and AOX2 were upregulated higher than 30-fold, which was consistent with the experimental results of AOX expression. The Mig proteins had a slight effect on autophagy when cells were grown in glycerol. The expression analysis of transcription factors showed that deletion of MIG1 and MIG2 significantly upregulated the binding of an essential transcription activator, Mit1p, with the AOX1 promoter, which suggested that Mig proteins might regulate the AOX1 promoter through the regulation of Mit1p. This work provides a reference for the further exploration of the methanol induction and catabolite repression mechanisms of AOX expression in methylotrophic yeasts.

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

The 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.IMPORTANCE The production of biofuels from plant biomass has gained interest in recent years as an environmentally friendly alternative to production from petroleum-based energy sources. Filamentous fungi, which naturally thrive on decaying plant matter, are of particular interest for this process due to their ability to secrete enzymes required for the deconstruction of lignocellulosic material. A major drawback in fungal hydrolytic enzyme production is the repression of the corresponding genes in the presence of glucose, a process known as carbon catabolite repression (CCR). This report provides previously unknown mechanistic insights into CCR through elucidating part of the protein-protein interaction regulatory system that governs the CreA transcriptional regulator in the reference organism Aspergillus nidulans in the presence of glucose and the biotechnologically relevant plant polysaccharide xylan.