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D-1,2,4-butanetriol (BT) is a widely used fine chemical that can be manufactured by engineered Escherichia coli expressing heterologous pathways and using xylose as a substrate. The current study developed a glucose-xylose dual metabolic channel system in an engineered E. coli and Combinatorially optimized it using multiple strategies to promote BT production. The carbon catabolite repression effects were alleviated by deleting the gene ptsG that encodes the major glucose transporter IICBGlc and mutating the gene crp that encodes the catabolite repressor protein, thereby allowing C-fluxes of both glucose and xylose into their respective metabolic channels separately and simultaneously, which increased BT production by 33% compared with that of the original MJ133K-1 strain. Then, the branch metabolic pathways of intermediates in the BT channel were investigated, the transaminase HisC, the ketoreductases DlD, OLD, and IlvC, and the aldolase MhpE and YfaU were identified as the enzymes for the branched metabolism of 2-keto-3-deoxy-xylonate, deletion of the gene hisC increased BT titer by 21.7%. Furthermore, the relationship between BT synthesis and the intracellular NADPH level was examined, and deletion of the gene pntAB that encodes a transhydrogenase resulted in an 18.1% increase in BT production. The combination of the above approaches to optimize the metabolic network increased BT production by 47.5%, resulting in 2.67 g/L BT in 24 deep-well plates. This study provides insights into the BT biosynthesis pathway and demonstrates effective strategies to increase BT production, which will promote the industrialization of the biosynthesis of BT. • A glucose-xylose dual metabolic channel system was developed for BT biosynthesis • BT synthesis was promoted by utilizing glucose and xylose in their respective channels • The intermediate 2-keto-3-deoxy-xylonate was metabolized via multiple branch routes • Intracellular NADPH levels affect BT biosynthesis • Combinatorial optimization was an effective strategy for BT biosynthesis [ABSTRACT FROM AUTHOR]

Background: Acetate is an important chemical feedstock widely applied in the food, chemical and textile industries. It is now mainly produced from petrochemical materials through chemical processes. Conversion of lignocellulose biomass to acetate by biotechnological pathways is both environmentally beneficial and cost-effective. However, acetate production from carbohydrate in lignocellulose hydrolysate via glycolytic pathways involving pyruvate decarboxylation often suffers from the carbon loss and results in low acetate yield. Results: Escherichia coli BL21 (DE3) was confirmed to have high tolerance to acetate in this work. Thus, it was selected from seven laboratory E. coli strains for acetate production from lignocellulose hydrolysate. The byproduct-producing genes frdA, ldhA, and adhE in E. coli BL21 (DE3) were firstly knocked out to decrease the generation of succinate, lactate, and ethanol. Then, the genes pfkA and edd were also deleted and bifunctional phosphoketolase and fructose-1,6-bisphosphatase were overexpressed to construct an EP-bifido pathway in E. coli BL21 (DE3) to increase the generation of acetate from glucose. The obtained strain E. coli 5K/pFF can produce 22.89 g/L acetate from 37.5 g/L glucose with a yield of 0.61 g/g glucose. Finally, the ptsG gene in E. coli 5K/pFF was also deleted to make the engineered strain E. coli 6K/pFF to simultaneously utilize glucose and xylose in lignocellulosic hydrolysates. E. coli 6K/pFF can produce 20.09 g/L acetate from corn stover hydrolysate with a yield of 0.52 g/g sugar. Conclusion: The results presented here provide a promising alternative for acetate production with low cost substrate. Besides acetate production, other biotechnological processes might also be developed for other acetyl-CoA derivatives production with lignocellulose hydrolysate through further metabolic engineering of E. coli 6K/pFF. [ABSTRACT FROM AUTHOR]

Carbon catabolite repression (CCR) is a common transcriptional regulatory mechanism that microorganisms use to efficiently utilize carbon nutrients, which is critical for the fitness of microorganisms and for pathogenic species to cause infection. Here, we characterized two CCR genes, VdCreA and VdCreC, in Verticillium dahliae that cause cotton Verticillium wilt disease. The VdCreA and VdCreC knockout mutants displayed slow growth with decreased conidiation and microsclerotium production and reduced virulence to cotton, suggesting that VdCreA and VdCreC are involved in growth and pathogenicity in V. dahliae. We further generated 36 highly reliable and stable ΔVdCreA and ΔVdCreC libraries to comprehensively explore the dynamic expression of genes and metabolites when grown under different carbon sources and CCR conditions. Based on the weighted gene co-expression network analysis (WGCNA) and correlation networks, VdCreA is co-expressed with a multitude of downregulated genes. These gene networks span multiple functional pathways, among which seven genes, including PYCR (pyrroline-5-carboxylate reductase), are potential target genes of VdCreA. Different carbon source conditions triggered entirely distinct gene regulatory networks, yet they exhibited similar changes in metabolic pathways. Six genes, including 6-phosphogluconolactonase and 2-ODGH (2-oxoglutarate dehydrogenase E1), may serve as hub genes in this process. Both VdCreA and VdCreC could comprehensively influence the expression of plant cell wall-degrading enzyme (PCWDE) genes, suggesting that they have a role in pathogenicity in V. dahliae. The integrated expression profiles of the genes and metabolites involved in the glycolysis/gluconeogenesis and pentose phosphate pathways showed that the two major sugar metabolism-related pathways were completely changed, and GADP (glyceraldehyde-3-phosphate) may be a pivotal factor for CCR under different carbon sources. All these results provide a more comprehensive perspective for further analyzing the role of Cre in CCR. [ABSTRACT FROM AUTHOR]

The experimental results of the present study show that phenol had a certain inhibitory effect on its utilization by phenol-acclimatized activated sludge (PAAS), which was the inoculum for the microbial fuel cell (MFC) inoculation. In co-metabolic study, utilization of para-nitrophenol (PNP) at low concentrations (< 20 mg L−1) when used with phenol at a fixed concentration (250 mg L−1) favorably proceeded. The behavior was interpreted in terms of carbon catabolite repression (CCR), indicating phenol (250 mg L−1) positively affected consumption of PNP (< 20 mg L−1). The calculated values of degradation rate show the necessity of phenol presence in the system where phenol acted on the inoculum's ability to withstand the inhibitory effect of PNP. The MFC functionality in electricity generation is also definable by considering CCR applicability and the results show that the negative effect of PNP was repressed by the presence of phenol. For instance, 20 mg L−1 PNP + 250 mg L−1 phenol yielded the highest power density (66.2 mW m−2) and the lowest internal resistance (189 Ω). The PAAS performance was characterized to evaluate cells' capacity in utilizing inhibitory substrates, and several different models were used. The relevant kinetic parameters are described in terms of PAAS affinity toward the substrate (ks) and the microbe's sensitivity in responding to the toxic substrate (ki). Luong and Aiba equations were chosen to describe MFC behavior when the PAAS utilized phenol as the sole substrate. While Haldane model was more capable of addressing co-metabolic degradation of PNP. [ABSTRACT FROM AUTHOR]

Inducible expression systems are pivotal for governing gene expression in strain engineering and synthetic biotechnological applications. Therefore, a critical need persists for the development of versatile and efficient inducible expression mechanisms. In this study, the xylose-responsive promoter xylA5p and its transcriptional regulator XylR were identified in Parageobacillus thermoglucosidasius DSM 2542. By combining promoter xylA5p with its regulator XylR, fine-tuning the expression strength of XylR, and reducing the glucose catabolite repression on xylose uptake, we successfully devised a xylose-inducible and glucose-insensitive expression system, denoted as IExyl*. This system exhibited diverse promoter strengths upon induction with xylose at varying concentrations and remained unhindered in the presence of glucose. Moreover, we showed the applicability of IExyl* in P. thermoglucosidasius by redirecting metabolic flux towards riboflavin biosynthesis, culminating in a 2.8-fold increase in riboflavin production compared to that of the starting strain. This glucose-insensitive and xylose-responsive expression system provides valuable tools for designing optimized biosynthetic pathways for high-value products and facilitates future synthetic biology investigations in Parageobacillus. Key points: • A xylose-inducible and glucose-insensitive expression system IExyl* was developed. • IExyl* was applied to enhance the riboflavin production in P. thermoglucosidasius • A tool for metabolic engineering and synthetic biology research in Parageobacillus strains. [ABSTRACT FROM AUTHOR]

Fungal pathogens must exhibit strong nutritional plasticity, effectively sensing and utilizing diverse nutrients to support virulence. How the signals generated by nutritional sensing are efficiently translated to the morphogenetic machinery for optimal growth and support of virulence remains incompletely understood. Here, we show that the conserved morphogenesis-related kinase, CotA, imparts isoform-specific control over Aspergillus fumigatus invasive growth in host-mimicking environments and during infection. CotA-mediated invasive growth is responsive to exogenous carbon source quality, with only preferred carbon sources supporting hyphal morphogenesis in a mutant lacking one of two identified protein isoforms. Strikingly, we find that the CotA protein does not regulate, nor is cotA gene expression regulated by, the carbon catabolite repression system. Instead, we show that CotA partially mediates invasive growth in specific carbon sources and virulence through the conserved downstream effector and translational repressor, SsdA. Therefore, A. fumigatus CotA accomplishes its conserved morphogenetic functions to drive pathogenic growth by translating host-relevant carbon source quality signals into morphogenetic outputs for efficient tissue invasive growth. Here, Martin-Vicente et al. show that the Aspergillus fumigatus protein kinase, CotA, controls invasive growth in an isoform dependent manner in response to carbon source diversity, partially mediated by the downstream effector, SsdA. [ABSTRACT FROM AUTHOR]

Microbes have evolved intricate communication systems that enable individual cells of a population to send and receive signals in response to changes in their immediate environment. In the fission yeast Schizosaccharomyces pombe, the oxylipin nitrogen signaling factor (NSF) is part of such communication system, which functions to regulate the usage of different nitrogen sources. Yet, the pathways and mechanisms by which NSF acts are poorly understood. Here, we show that NSF physically interacts with the mitochondrial sulfide:quinone oxidoreductase Hmt2 and that it prompts a change from a fermentation- to a respiration-like gene expression program without any change in the carbon source. Our results suggest that NSF activity is not restricted to nitrogen metabolism alone and that it could function as a rheostat to prepare a population of S. pombe cells for an imminent shortage of their preferred nutrients. Synopsis: Chemical cell-to-cell communication in the fission yeast Schizosaccharomyces pombe is essential for adaption to environmental changes in nutrient availability. This study uncovers that the extracellular signaling molecule nitrogen signaling factor (NSF) functions to counter nitrogen catabolite repression, and to switch from fermentation to respiratory metabolism. Nitrogen Signaling Factor (NSF) functions in an intra-species communication system of. With increasing cell density during population growth, NSF reaches a critical concentration that triggers a switch from a fermentation- to a respiration-like gene expression program. The putative 1-acyldihydroxyacetone phosphate reductase Ayr1 counteracts NSF signaling. The mitochondrial sulfide:quinone oxidoreductase Hmt2 is a direct and functionally relevant target of NSF. NSF is a transmissible signal that activates mitochondrial respiration to maximize growth. The oxylipin NSF is a transmissible signal that activates mitochondrial respiration to maximize growth. [ABSTRACT FROM AUTHOR]

d-Xylitol is a naturally occurring sugar alcohol present in diverse plants that is used as an alternative sweetener based on a sweetness similar to sucrose and several health benefits compared to conventional sugar. However, current industrial methods for d-xylitol production are based on chemical hydrogenation of d-xylose, which is energy-intensive and environmentally harmful. However, efficient conversion of l-arabinose as an additional highly abundant pentose in lignocellulosic materials holds great potential to broaden the range of applicable feedstocks. Both pentoses d-xylose and l-arabinose are converted to d-xylitol as a common metabolic intermediate in the native fungal pentose catabolism. To engineer a strain capable of accumulating d-xylitol from arabinan-rich agricultural residues, pentose catabolism was stopped in the ascomycete filamentous fungus Aspergillus niger at the stage of d-xylitol by knocking out three genes encoding enzymes involved in d-xylitol degradation (ΔxdhA, ΔsdhA, ΔxkiA). Additionally, to facilitate its secretion into the medium, an aquaglyceroporin from Saccharomyces cerevisiae was tested. In S. cerevisiae, Fps1 is known to passively transport glycerol and is regulated to convey osmotic stress tolerance but also exhibits the ability to transport other polyols such as d-xylitol. Thus, a constitutively open version of this transporter was introduced into A. niger, controlled by multiple promoters with varying expression strengths. The strain expressing the transporter under control of the PtvdA promoter in the background of the pentose catabolism-deficient triple knock-out yielded the most favorable outcome, producing up to 45% d-xylitol from l-arabinose in culture supernatants, while displaying minimal side effects during osmotic stress. Due to its additional ability to extract d-xylose and l-arabinose from lignocellulosic material via the production of highly active pectinases and hemicellulases, A. niger emerges as an ideal candidate cell factory for d-xylitol production from lignocellulosic biomasses rich in both pentoses. In summary, we are showing for the first time an efficient biosynthesis of d-xylitol from l-arabinose utilizing a filamentous ascomycete fungus. This broadens the potential resources to include also arabinan-rich agricultural waste streams like sugar beet pulp and could thus help to make alternative sweetener production more environmentally friendly and cost-effective. [ABSTRACT FROM AUTHOR]

Trichoderma species have been reported as masters in producing cellulolytic enzymes for the biodegradation of lignocellulolytic biomass and biocontrol agents against plant pathogens and pests. In our previous study, a novel Trichoderma strain LZ117, which shows potent capability in cellulase production, was isolated. Herein, we conducted multilocus phylogenetic analyses based on DNA barcodes and performed time-scaled phylogenomic analyses using the whole genome sequences of the strain, annotated by integrating transcriptome data. Our results suggest that this strain represents a new species closely related to T. atrobrunneum (Harzianum clade). Genes encoding carbohydrate-active enzymes (CAZymes), transporters, and secondary metabolites were annotated and predicted secretome in Trichoderma sp. LZ117 was also presented. Furthermore, genetic manipulation of this strain was successfully achieved using PEG-mediated protoplast transformation. A putative transporter gene encoding maltose permease (Mal1) was overexpressed, which proved that this transporter does not affect cellulase production. Moreover, overexpressing the native Cre1 homolog in LZ117 demonstrated a more pronounced impact of glucose-caused carbon catabolite repression (CCR), suggesting the importance of Cre1-mediated CCR in cellulase production of Trichoderma sp. LZ117. The results of this study will benefit further exploration of the strain LZ117 and related species for their applications in bioproduction. [ABSTRACT FROM AUTHOR]

Pseudomonas putida have emerged as promising biocatalysts for the conversion of sugars and aromatic compounds obtained from lignocellulosic biomass. Understanding the role of carbon catabolite repression (CCR) in these strains is critical to optimize biomass conversion to fuels and chemicals. The CCR functioning in P. putida M2, a strain capable of consuming both hexose and pentose sugars as well as aromatic compounds, was investigated by cultivation experiments, proteomics, and CRISPRi-based gene repression. Strain M2 co-utilized sugars and aromatic compounds simultaneously; however, during cultivation with glucose and aromatic compounds (p-coumarate and ferulate) mixture, intermediates (4-hydroxybenzoate and vanillate) accumulated, and substrate consumption was incomplete. In contrast, xylose-aromatic consumption resulted in transient intermediate accumulation and complete aromatic consumption, while xylose was incompletely consumed. Proteomics analysis revealed that glucose exerted stronger repression than xylose on the aromatic catabolic proteins. Key glucose (Eda) and xylose (XylX) catabolic proteins were also identified at lower abundance during cultivation with aromatic compounds implying simultaneous catabolite repression by sugars and aromatic compounds. Reduction of crc expression via CRISPRi led to faster growth and glucose and p-coumarate uptake in the CRISPRi strains compared to the control, while no difference was observed on xylose+p-coumarate. The increased abundances of Eda and amino acid biosynthesis proteins in the CRISPRi strain further supported these observations. Lastly, small RNAs (sRNAs) sequencing results showed that CrcY and CrcZ homologues levels in M2, previously identified in P. putida strains, were lower under strong CCR (glucose+p-coumarate) condition compared to when repression was absent (p-coumarate or glucose only).IMPORTANCEA newly isolated Pseudomonas putida strain, P. putida M2, can utilize both hexose and pentose sugars as well as aromatic compounds making it a promising host for the valorization of lignocellulosic biomass. Pseudomonads have developed a regulatory strategy, carbon catabolite repression, to control the assimilation of carbon sources in the environment. Carbon catabolite repression may impede the simultaneous and complete metabolism of sugars and aromatic compounds present in lignocellulosic biomass and hinder the development of an efficient industrial biocatalyst. This study provides insight into the cellular physiology and proteome during mixed-substrate utilization in P. putida M2. The phenotypic and proteomics results demonstrated simultaneous catabolite repression in the sugar-aromatic mixtures, while the CRISPRi and sRNA sequencing demonstrated the potential role of the crc gene and small RNAs in carbon catabolite repression.

Background: Sugarcane molasses, rich in sucrose, glucose, and fructose, offers a promising carbon source for industrial fermentation due to its abundance and low cost. However, challenges arise from the simultaneous utilization of multiple sugars and carbon catabolite repression (CCR). Despite its nutritional content, sucrose metabolism in Escherichia coli, except for W strain, remains poorly understood, hindering its use in microbial fermentation. In this study, E. coli W was engineered to enhance sugar consumption rates and overcome CCR. This was achieved through the integration of a synthetically designed csc operon and the optimization of glucose and fructose co-utilization pathways. These advancements facilitate efficient utilization of sugarcane molasses for the production of 3-hydroxypropionic acid (3-HP), contributing to sustainable biochemical production processes. Results: In this study, we addressed challenges associated with sugar metabolism in E. coli W, focusing on enhancing sucrose consumption and improving glucose-fructose co-utilization. Through targeted engineering of the sucrose utilization system, we achieved accelerated sucrose consumption rates by modulating the expression of the csc operon components, cscB, cscK, cscA, and cscR. Our findings revealed that monocistronic expression of the csc genes with the deletion of cscR, led to optimal sucrose utilization without significant growth burden. Furthermore, we successfully alleviated fructose catabolite repression by modulating the binding dynamics of FruR with the fructose PTS regulon, enabling near-equivalent co-utilization of glucose and fructose. To validate the industrial applicability of our engineered strain, we pursued 3-HP production from sugarcane molasses. By integrating heterologous genes and optimizing metabolic pathways, we achieved improvements in 3-HP titers compared to previous studies. Additionally, glyceraldehyde-3-phosphate dehydrogenase (gapA) repression aids in carbon flux redistribution, enhancing molasses conversion to 3-HP. Conclusions: Despite limitations in sucrose metabolism, the redesigned E. coli W strain, adept at utilizing sugarcane molasses, is a valuable asset for industrial fermentation. Its synthetic csc operon enhances sucrose consumption, while mitigating CCR improves glucose-fructose co-utilization. These enhancements, coupled with repression of gapA, aim to efficiently convert sugarcane molasses into 3-HP, addressing limitations in sucrose and fructose metabolism for industrial applications. [ABSTRACT FROM AUTHOR]

Filamentous fungi with their diverse inventory of carbohydrate‐active enzymes promise a holistic usage of lignocellulosic residues. A major challenge for application is the inherent repression of enzyme production by carbon catabolite repression (CCR). In the presence of preferred carbon sources, the transcription factor CreA/CRE‐1 binds to specific but conserved motifs in promoters of genes involved in sugar metabolism, but the status of CCR is notoriously difficult to quantify. To allow for a real‐time evaluation of CreA/CRE‐1‐mediated CCR at the transcriptional level, we developed a luciferase‐based construct, representing a dynamic, highly responsive reporter system that is inhibited by monosaccharides in a quantitative fashion. Using this tool, CreA/CRE‐1‐dependent CCR triggered by several monosaccharides could be measured in Neurospora crassa, Aspergillus niger and Aspergillus nidulans over the course of hours, demonstrating distinct and dynamic regulatory processes. Furthermore, we used the reporter to visualize the direct impacts of multiple CreA truncations on CCR induction. Our reporter thus offers a widely applicable quantitative approach to evaluate CreA/CRE‐1‐mediated CCR across diverse fungal species and will help to elucidate the multifaceted effects of CCR on fungal physiology for both basic research and industrial strain engineering endeavours. [ABSTRACT FROM AUTHOR]

The high cost of cellulolytic enzyme complexes (CECs) has been a significant impediment to the commercial production of bioproducts from lignocellulose biomass. This study aimed to develop a cost‐effective CEC derived from Penicillium ucsense (former Penicillium echinulatum), utilizing diverse forms of pretreated sugarcane bagasse as the primary carbon/inductor source. Among the different pretreatments used, the hydrothermal pretreatment followed by NaOH delignification (BHD) produced higher FPase and xylanase activities (4.5 FPU mL–1 and 120 IU mL–1) in bioreactor experiments at 20 g BHD L–1 initial concentration. A batch‐mode assay conducted across a range of initial carbon source (5 to 60 g L–1) confirmed the highest FPase activity (4.0 to 5.0 FPU mL–1 at 120 h), in the range of 20–40 g BHD L–1. During these assays the agitation rate, controlled by dissolved O2, tended to stabilize at lower levels, indicating substrate limitation. Conversely, higher initial carbon source concentrations led to an excess of glucose, likely triggering carbon catabolite repression and inhibiting cellulase production. This insight prompted the development of a controlled pulsed fed‐batch strategy, resulting in FPase activity of 11 FPU mL–1 at 220 h using 90 g L–1 BHD controlled fed into the bioreactor. An enzymatic hydrolysis procedure using the generated CEC was also optimized using a central composite rotational design (CCRD). The optimized enzyme hydrolysis conditions achieved a reducing sugar concentration of 80.9 g L–1 in 48 h using 170 g L–1 of BHD as the substrate at a ratio of 15 FPU of enzyme substrate per g of BHD. A preliminary economic assessment demonstrated that, for a first‐ and second‐generation (1G + 2G) ethanol biorefinery, the cost contribution of enzymes would be about US$0.2/L of biofuel. In conclusion, an efficient and highly productive procedure was developed successfully for the production of a CEC. It was particularly effective for the enzymatic hydrolysis of pretreated sugarcane bagasse. [ABSTRACT FROM AUTHOR]

One unique attribute of Cryptococcus neoformans is its ability to procure essential monomers from its surroundings to survive in diverse environments. Preferentially, sugars are the energy sources for this opportunistic pathogenic fungus under the carbon catabolite repression (CCR); however, sugar restriction induces alternative use of low molecular weight alcohol, organic acids, and amino acids. The expression of transmembrane amino acid permeases (Aaps) allows C. neoformans to utilize different amino acids and their conjugates, notwithstanding under the nitrogen catabolite repression (NCR). Being referred to as global permeases, there is a notion that all cryptococcal Aaps are important to survival and virulence. This functional divergence makes alternative drug targeting against Cryptococcus a challenge. We examine the functions and regulations of C. neoformans Aap variants with the aim of rationalizing their relevance to cryptococcal cell survival and virulence. Based on nutrient bioavailability, we linked the Cac1 pathway to Ras1 activation for thermotolerance that provides a temperature cushion for Aap activity under physiological conditions. Lastly, mutants of Aaps are examined for significant phenotypic deficiencies/advantages, which buttress the specific importance of limited numbers of Aaps involved in cryptococcal infections. [ABSTRACT FROM AUTHOR]

Functional microbial communities producing β-glucosidases differentially regulate the expression of glucose/non-glucose-tolerant β-glucosidases in response to carbon catabolite repression (CCR), which is a common phenomenon. To better understand their biological roles in a composting environment, four representative β-glucosidase genes were cloned from the metatranscriptome of the compost for expression and characterization. BGLA contains conserved sites Trp168 and Leu173, associated with glucose tolerance. In the presence of 100 mM glucose, BGLA's hydrolysis activity increases by 80%. BGLB also contains Trp168 and Leu173; however, its hydrolysis activity is significantly inhibited by glucose. On the contrary, Trp168 of BGLC was replaced by Phe, yet its hydrolysis activity increased by 20% in the presence of 25 mM glucose. However, BGLD did not exhibit any enzymatic activity. Molecular docking and dynamic simulations revealed the structural basis of glucose tolerance. Metatranscriptomic analysis of the four genes showed that functional microbial communities upregulated or downregulated gene expression in response to different CCR environments to maintain overall carbon metabolic balance. These findings demonstrate that the enzymatic activity characteristics of individual glucose/non-glucose-tolerant β-glucosidases are consistent with their transcriptional regulation under CCR in the complex composting environment. [Display omitted] • Four new GH1 family β-glucosidases cloned from compost. • BGLA activity increased by 80% with added glucose. • Computational biology elucidates β-glucosidase glucose tolerance mechanism. • Glucose tolerance of β-glucosidases correlates with their expression level. [ABSTRACT FROM AUTHOR]

Skin serves as both barrier and interface between body and environment. Skin microbes are intermediaries evolved to respond, transduce, or act in response to changing environmental or physiological conditions. We quantified genomewide changes in gene expression levels for one abundant skin commensal, Staphylococcus epidermidis, in response to an internal physiological signal, glucose levels, and an external environmental signal, temperature. We found 85 of 2,354 genes change up to ~34-fold in response to medically relevant changes in glucose concentration (0-17 mM; adj p ≤0.05). We observed carbon catabolite repression in response to a range of glucose spikes, as well as upregulation of genes involved in glucose utilization in response to persistent glucose. We observed 366 differentially expressed genes in response to a physiologically relevant change in temperature (37-45°C; adj p ≤ 0.05) and an S. epidermidis heat-shock response that mostly resembles the heat-shock response of related staphylococcal species. DNA motif analysis revealed CtsR and CIRCE operator sequences arranged in tandem upstream of dnaK and groESL operons. We identified and curated 38 glucose-responsive genes as candidate ON or OFF switches for use in controlling synthetic genetic systems. Such systems might be used to instrument the in-situ skin microbiome or help control microbes bioengineered to serve as embedded diagnostics, monitoring, or treatment platforms. [ABSTRACT FROM AUTHOR]

Pyruvate dehydrogenase (PDH) catalyses the irreversible decarboxylation of pyruvate to acetyl‐CoA, which feeds the tricarboxylic acid cycle. We investigated how the loss of PDH affects metabolism in Pseudomonas putida. PDH inactivation resulted in a strain unable to utilize compounds whose assimilation converges at pyruvate, including sugars and several amino acids, whereas compounds that generate acetyl‐CoA supported growth. PDH inactivation also resulted in the loss of carbon catabolite repression (CCR), which inhibits the assimilation of non‐preferred compounds in the presence of other preferred compounds. Pseudomonas putida can degrade many aromatic compounds, most of which produce acetyl‐CoA, making it useful for biotransformation and bioremediation. However, the genes involved in these metabolic pathways are often inhibited by CCR when glucose or amino acids are also present. Our results demonstrate that the PDH‐null strain can efficiently degrade aromatic compounds even in the presence of other preferred substrates, which the wild‐type strain does inefficiently, or not at all. As the loss of PDH limits the assimilation of many sugars and amino acids and relieves the CCR, the PDH‐null strain could be useful in biotransformation or bioremediation processes that require growth with mixtures of preferred substrates and aromatic compounds. [ABSTRACT FROM AUTHOR]

Colletotrichum lindemuthianum is a phytopathogenic fungus that causes anthracnose in common beans (Phaseolus vulgaris) and presents a great diversity of pathotypes with different levels of virulence against bean varieties worldwide. The purpose of this study was to establish whether pathotypic diversity is associated with differences in the mycelial growth and secretion of plant-cell-wall-degrading enzymes (PCWDEs). We evaluated growth, hemicellulase and cellulase activity, and PCWDE secretion in four pathotypes of C. lindemuthianum in cultures with glucose, bean hypocotyls and green beans of P. vulgaris, and water hyacinth (Eichhornia crassipes). The results showed differences in the mycelial growth, hemicellulolytic activity, and PCWDE secretion among the pathotypes. Glucose was not the preferred carbon source for the best mycelial growth in all pathotypes, each of which showed a unique PCWDE secretion profile, indicating different levels of carbon catabolite regulation (CCR). The pathotypes showed a high differential hemicellulolytic capacity to degrade host and water hyacinth tissues, suggesting CCR by pentoses and that there are differences in the absorption and metabolism of different monosaccharides and/or disaccharides. We propose that different levels of CCR could optimize growth in different host tissues and could allow for consortium behavior in interactions with bean crops. [ABSTRACT FROM AUTHOR]

Carbon catabolite repression (CCR) is a highly conserved mechanism that regulates carbon source utilization in Streptomyces. CCR has a negative impact on secondary metabolite fermentation, both in industrial and research settings. In this study, CCR was observed in the daunorubicin (DNR)-producing strain Streptomyces coeruleorubidus DM, which was cultivated in high concentration of carbohydrates. Unexpectedly, DM exhibited a high ability for anthraquinone glucuronidation biotransformation under CCR conditions with a maximum bioconversion rate of 95% achieved at pH 6, 30°C for 24 h. The co-utilization of glucose and sucrose resulted in the highest biotransformation rate compared to other carbon source combinations. Three novel anthraquinone glucuronides were obtained, with purpurin-O-glucuronide showing significantly improved water solubility, antioxidant activity, and antibacterial bioactivity. Comparative transcript analysis revealed that glucose and sucrose utilization were significantly upregulated as DM cultivated under CCR condition, which strongly enhance the biosynthetic pathway of the precursors Uridine diphosphate glucuronic acid (UDPGA). Meanwhile, the carbon metabolic flux has significantly enhanced the fatty acid biosynthesis, the exhaust of acetyl coenzyme A may lead to the complete repression of the biosynthesis of DNR, Additionally, the efflux transporter genes were simultaneously downregulated, which may contribute to the anthraquinones intracellular glucuronidation. Overall, our findings demonstrate that utilizing CCR can be a valuable strategy for enhancing the biotransformation efficiency of anthraquinone O-glucuronides by DM. This approach has the potential to improve the bioavailability and therapeutic potential of these compounds, opening up new possibilities for their pharmaceutical applications. [ABSTRACT FROM AUTHOR]

The Streptomyces genus comprises Gram-positive bacteria known to produce over two-thirds of the antibiotics used in medical practice. The biosynthesis of these secondary metabolites is highly regulated and influenced by a range of nutrients present in the growth medium. In Streptomyces coelicolor, glucose inhibits the production of actinorhodin (ACT) and undecylprodigiosin (RED) by a process known as carbon catabolite repression (CCR). However, the mechanism mediated by this carbon source still needs to be understood. It has been observed that glucose alters the transcriptomic profile of this actinobacteria, modifying different transcriptional regulators, including some of the one- and two-component systems (TCSs). Under glucose repression, the expression of one of these TCSs SCO6162/SCO6163 was negatively affected. We aimed to study the role of this TCS on secondary metabolite formation to define its influence in this general regulatory process and likely establish its relationship with other transcriptional regulators affecting antibiotic biosynthesis in the Streptomyces genus. In this work, in silico predictions suggested that this TCS can regulate the production of the secondary metabolites ACT and RED by transcriptional regulation and protein–protein interactions of the transcriptional factors (TFs) with other TCSs. These predictions were supported by experimental procedures such as deletion and complementation of the TFs and qPCR experiments. Our results suggest that in the presence of glucose, the TCS SCO6162/SCO6163, named GarR/GarS, is an important negative regulator of the ACT and RED production in S. coelicolor. Key points: • GarR/GarS is a TCS with domains for signal transduction and response regulation • GarR/GarS is an essential negative regulator of the ACT and RED production • GarR/GarS putatively interacts with and regulates activators of ACT and RED [ABSTRACT FROM AUTHOR]

Background: Filamentous fungi have long been recognized for their exceptional enzyme production capabilities. Among these, Trichoderma reesei has emerged as a key producer of various industrially relevant enzymes and is particularly known for the production of cellulases. Despite the availability of advanced gene editing techniques for T. reesei, the cultivation and characterization of resulting strain libraries remain challenging, necessitating well-defined and controlled conditions with higher throughput. Small-scale cultivation devices are popular for screening bacterial strain libraries. However, their current use for filamentous fungi is limited due to their complex morphology. Results: This study addresses this research gap through the development of a batch cultivation protocol using a microbioreactor for cellulase-producing T. reesei strains (wild type, RutC30 and RutC30 TR3158) with offline cellulase activity analysis. Additionally, the feasibility of a microscale fed-batch cultivation workflow is explored, crucial for mimicking industrial cellulase production conditions. A batch cultivation protocol was developed and validated using the BioLector microbioreactor, a Round Well Plate, adapted medium and a shaking frequency of 1000 rpm. A strong correlation between scattered light intensity and cell dry weight underscores the reliability of this method in reflecting fungal biomass formation, even in the context of complex fungal morphology. Building on the batch results, a fed-batch strategy was established for T. reesei RutC30. Starting with a glucose concentration of 2.5 g l - 1 in the batch phase, we introduced a dual-purpose lactose feed to induce cellulase production and prevent carbon catabolite repression. Investigating lactose feeding rates from 0.3 to 0.75 g (l h) - 1 , the lowest rate of 0.3 g (l h) - 1 revealed a threefold increase in cellobiohydrolase and a fivefold increase in β -glucosidase activity compared to batch processes using the same type and amount of carbon sources. Conclusion: We successfully established a robust microbioreactor batch cultivation protocol for T. reesei wild type, RutC30 and RutC30 TR3158, overcoming challenges associated with complex fungal morphologies. The study highlights the effectiveness of microbioreactor workflows in optimizing cellulase production with T. reesei, providing a valuable tool for simultaneous assessment of critical bioprocess parameters and facilitating efficient strain screening. The findings underscore the potential of microscale fed-batch strategies for enhancing enzyme production capabilities, revealing insights for future industrial applications in biotechnology. [ABSTRACT FROM AUTHOR]

Sialidases (neuraminidases) catalyze the removal of terminal sialic acid residues from glycoproteins. Novel enzymes from non-clinical isolates are of increasing interest regarding their application in the food and pharmaceutical industry. The present study aimed to evaluate the participation of carbon catabolite repression (CCR) in the regulation of cold-active sialidase biosynthesis by the psychrotolerant fungal strain Penicillium griseofulvum P29, isolated from Antarctica. The presence of glucose inhibited sialidase activity in growing and non-growing fungal mycelia in a dose- and time-dependent manner. The same response was demonstrated with maltose and sucrose. The replacement of glucose with glucose-6-phosphate also exerted CCR. The addition of cAMP resulted in the partial de-repression of sialidase synthesis. The CCR in the psychrotolerant strain P. griseofulvum P29 did not depend on temperature. Sialidase might be subject to glucose repression by both at 10 and 25 °C. The fluorescent assay using 4MU-Neu5Ac for enzyme activity determination under increasing glucose concentrations evidenced that CCR may have a regulatory role in sialidase production. The real-time RT-PCR experiments revealed that the sialidase gene was subject to glucose repression. To our knowledge, this is the first report that has studied the effect of CCR on cold-active sialidase, produced by an Antarctic strain. [ABSTRACT FROM AUTHOR]

In wine, lactic acid bacteria (LAB) are responsible for the bioconversion of malic acid to lactic acid, malolactic fermentation that mainly aims at reducing wine acidity. Two LAB strains isolated from the red wine microbiota (Oenococcus oeni 13-7 and Lactobacillus plantarum R1-1), were tested for their ability to exhibit the carbon catabolite repression (CCR) mechanism, that allows the rapid use of certain carbohydrates, over other carbon sources. Bacterial cells were inoculated in 0.1 M glycine buffer (pH 3.5), incubated at 30°C, with different carbohydrates (45 mM) and malic acid (45 mM). For both strains, the presence of glucose significantly inhibited malic acid metabolization (-60%), a similar effect being observed for galactose, mannose and maltose. The highest rate of malic acid conversion was shown in fructose/malate medium. Obtained results showed that malolactic strains can control the utilization of carbon sources via CCR, further studies being necessary to elucidate the mechanisms underlying this process. [ABSTRACT FROM AUTHOR]

ABSTRACT The human pathogen Pseudomonas aeruginosa, a leading cause of hospital-acquired infections, inhabits and forms sessile antibiotic-resistant communities called biofilms in a wide range of biotic and abiotic environments. In this study, we examined how two global sensory signaling pathways—the RhlR quorum-sensing system and the CbrA/CbrB nutritional adaptation system—intersect to control biofilm development. Previous work has shown that individually these two systems repress biofilm formation. Here, we used biofilm analyses, RNA-seq, and reporter assays to explore the combined effect of information flow through RhlR and CbrA on biofilm development. We find that the ΔrhlRΔcbrA double mutant exhibits a biofilm morphology and an associated transcriptional response distinct from wildtype and the parent ΔrhlR and ΔcbrA mutants indicating codominance of each signaling pathway. The ΔrhlRΔcbrA mutant gains suppressor mutations that allow biofilm expansion; these mutations map to the crc gene resulting in loss of function of the carbon catabolite repression protein Crc. Furthermore, the combined absence of RhlR and CbrA leads to a drastic reduction in the abundance of the Crc antagonist small RNA CrcZ. Thus, CrcZ acts as the molecular convergence point for quorum- and nutrient-sensing cues. We find that in the absence of antagonism by CrcZ, Crc promotes the expression of biofilm matrix components—Pel exopolysaccharide, and CupB and CupC fimbriae. Therefore, this study uncovers a regulatory link between nutritional adaption and quorum sensing with potential implications for anti-biofilm targeting strategies.IMPORTANCEBacteria often form multicellular communities encased in an extracytoplasmic matrix called biofilms. Biofilm development is controlled by various environmental stimuli that are decoded and converted into appropriate cellular responses. To understand how information from two distinct stimuli is integrated, we used biofilm formation in the human pathogen Pseudomonas aeruginosa as a model and studied the intersection of two global sensory signaling pathways—quorum sensing and nutritional adaptation. Global transcriptomics on biofilm cells and reporter assays suggest parallel regulation of biofilms by each pathway that converges on the abundance of a small RNA antagonist of the carbon catabolite repression protein, Crc. We find a new role of Crc as it modulates the expression of biofilm matrix components in response to the environment. These results expand our understanding of the genetic regulatory strategies that allow P. aeruginosa to successfully develop biofilm communities.

The multifunctional carbon catabolite repression negative on TATA-box-less complex (CCR4-NOT) is a multi-subunit complex present in all eukaryotes, including fungi. This complex plays an essential role in gene expression; however, a functional study of the CCR4-NOT complex in the rice blast fungus Magnaporthe oryzae has not been conducted. Seven genes encoding the putative CCR4-NOT complex were identified in the M. oryzae genome. Among these, a homologous gene, MoNOT3, was overexpressed during appressorium development in a previous study. Deletion of MoNOT3 in M. oryzae resulted in a significant reduction in hyphal growth, conidiation, abnormal septation in conidia, conidial germination, and appressorium formation compared to the wild-type. Transcriptional analyses suggest that the MoNOT3 gene affects conidiation and conidial morphology by regulating COS1 and COM1 in M. oryzae. Furthermore, Δmonot3 exhibited a lack of pathogenicity, both with and without wounding, which is attributable to deficiencies in the development of invasive growth in planta. This result was also observed in onion epidermal cells, which are non-host plants. In addition, the MoNOT3 gene was involved in cell wall stress responses and heat shock. Taken together, these observations suggest that the MoNOT3 gene is required for fungal infection-related cell development and stress responses in M. oryzae. [ABSTRACT FROM AUTHOR]

The rice false smut disease, caused by Ustilaginoidea virens , has emerged as a significant global threat to rice production. The mechanism of carbon catabolite repression plays a crucial role in the efficient utilization of carbon nutrients and enzyme regulation in the presence of complex nutritional conditions. Although significant progress has been made in understanding carbon catabolite repression in fungi such as Aspergillus nidulans and Magnaporthe oryzae , its role in U. virens remains unclear. To address this knowledge gap, we identified UvCreA , a pivotal component of carbon catabolite repression, in U. virens. Our investigation revealed that UvCreA localized to the nucleus. Deletion of UvCreA resulted in decreased growth and pathogenicity in U. virens. Through RNA-seq analysis, it was found that the knockout of UvCreA led to the up-regulation of 514 genes and down-regulation of 640 genes. Moreover, UvCreA was found to be involved in the transcriptional regulation of pathogenic genes and genes associated with carbon metabolism in U. virens. In summary, our findings indicated that UvCreA is important in fungal development, virulence, and the utilization of carbon sources through transcriptional regulation, thus making it a critical element of carbon catabolite repression. [ABSTRACT FROM AUTHOR]

Summary: Transposable elements (TEs) are mobile genetic elements that can impair the host genome stability and integrity. It has been well documented that activated transposons in plants are suppressed by small interfering (si) RNAs. However, transposon repression by the cytoplasmic RNA surveillance system is unknown.Here, we show that mRNA deadenylation is critical for controlling transposons in Arabidopsis. Trimming of poly(A) tail is a rate‐limiting step that precedes the RNA decay and is primarily mediated by the CARBON CATABOLITE REPRESSION 4 (CCR4)‐NEGATIVE ON TATA‐LESS (NOT) complex.We found that the loss of CCR4a leads to strong derepression and mobilization of TEs in Arabidopsis. Intriguingly, CCR4a regulates a largely distinct set of TEs from those controlled by RNA‐dependent RNA Polymerase 6 (RDR6), a key enzyme that produces cytoplasmic siRNAs. This indicates that the cytoplasmic RNA quality control mechanism targets the TEs that are poorly recognized by the previously well‐characterized RDR6‐mediated pathway, and thereby augments the host genome stability.Our study suggests a hitherto unknown mechanism for transposon repression mediated by RNA deadenylation and unveils a complex nature of the host's strategy to maintain the genome integrity. [ABSTRACT FROM AUTHOR]

Corn cob is a major waste mass-produced in corn agriculture. Corn cob hydrolysate containing xylose, arabinose, and glucose is the hydrolysis product of corn cob. Herein, a recombinant Escherichia coli strain BT-10 was constructed to transform corn cob hydrolysate into 1,2,4-butanetriol, a platform substance with diversified applications. To eliminate catabolite repression and enhance NADPH supply for alcohol dehydrogenase YqhD catalyzed 1,2,4-butanetriol generation, ptsG encoding glucose transporter EIICBGlc and pgi encoding phosphoglucose isomerase were deleted. With four heterologous enzymes including xylose dehydrogenase, xylonolactonase, xylonate dehydratase, α-ketoacid decarboxylase and endogenous YqhD, E. coli BT-10 can produce 36.63 g/L 1,2,4-butanetriol with a productivity of 1.14 g/[L·h] using xylose as substrate. When corn cob hydrolysate was used as the substrate, 43.4 g/L 1,2,4-butanetriol was generated with a productivity of 1.09 g/[L·h] and a yield of 0.9 mol/mol. With its desirable characteristics, E. coli BT-10 is a promising strain for commercial 1,2,4-butanetriol production. [ABSTRACT FROM AUTHOR]

As advances are made toward the industrial feasibility of mass-producing biofuels and commodity chemicals with sugar-fermenting microbes, high feedstock costs continue to inhibit commercial application. Hydrolyzed lignocellulosic biomass represents an ideal feedstock for these purposes as it is cheap and prevalent. However, many microbes, including Escherichia coli, struggle to efficiently utilize this mixture of hexose and pentose sugars due to the regulation of the carbon catabolite repression (CCR) system. CCR causes a sequential utilization of sugars, rather than simultaneous utilization, resulting in reduced carbon yield and complex process implications in fed-batch fermentation. A mutant of the gene encoding the cyclic AMP receptor protein, crp*, has been shown to disable CCR and improve the co-utilization of mixed sugar substrates. Here, we present the strain construction and characterization of a site-specific crp* chromosomal mutant in E. coli BL21 star (DE3). The crp* mutant strain demonstrates simultaneous consumption of glucose and xylose, suggesting a deregulated CCR system. The proteomics further showed that glucose was routed to the C5 carbon utilization pathways to support both de novo nucleotide synthesis and energy production in the crp* mutant strain. Metabolite analyses further show that overflow metabolism contributes to the slower growth in the crp* mutant. This highly characterized strain can be particularly beneficial for chemical production by simultaneously utilizing both C5 and C6 substrates from lignocellulosic biomass. [ABSTRACT FROM AUTHOR]

Efficient utilization of galactose by microorganisms can lead to the production of valuable bio-products and improved metabolic processes. While Bacillus subtilis has inherent pathways for galactose metabolism, there is potential for enhancement via evolutionary strategies. This study aimed to boost galactose utilization in B. subtilis using adaptive laboratory evolution (ALE) and to elucidate the genetic and metabolic changes underlying the observed enhancements. The strains of B. subtilis underwent multiple rounds of adaptive laboratory evolution (approximately 5000 generations) in an environment that favored the use of galactose. This process resulted in an enhanced specific growth rate of 0.319 ± 0.005 h−1, a significant increase from the 0.03 ± 0.008 h−1 observed in the wild-type strains. Upon selecting the evolved strain BSGA14, a comprehensive whole-genome sequencing revealed the presence of 63 single nucleotide polymorphisms (SNPs). Two of them, located in the coding sequences of the genes araR and glcR, were found to be the advantageous mutations after reverse engineering. The strain with these two accumulated mutations, BSGALE4, exhibited similar specific growth rate on galactose to the evolved strain BSGA14 (0.296 ± 0.01 h−1). Furthermore, evolved strain showed higher productivity of protease and β-galactosidase in mock soybean biomass medium. ALE proved to be a potent tool for enhancing galactose metabolism in B. subtilis. The findings offer valuable insights into the potential of evolutionary strategies in microbial engineering and pave the way for industrial applications harnessing enhanced galactose conversion. Key Points: Following adaptive laboratory evolution in a minimal medium with galactose serving as the selective pressure, the B. subtilis BSGA14 strain was successfully developed, exhibiting a specific growth rate of 0.319 ± 0.005 h−1. Among SNPs after resequencing, mutation in the araR gene enhances the uptake of galactose in Bacillus subtilis, leading to increased galactose utilization. The nonsense mutation in the glcR gene, which is involved in carbon catabolite repression (CCR) and sugar phosphorylation, influences galactose metabolism in B. subtilis. [ABSTRACT FROM AUTHOR]

The discovery and characterization of bacterial carbohydrate-active enzymes is a fundamental component of biotechnology innovation, particularly for renewable fuels and chemicals; however, these studies have increasingly transitioned to exploring the complex regulation required for recalcitrant polysaccharide utilization. This pivot is largely due to the current need to engineer and optimize enzymes for maximal degradation in industrial or biomedical applications. Given the structural simplicity of a single cellulose polymer, and the relatively few enzyme classes required for complete bioconversion, the regulation of cellulases in bacteria has been thoroughly discussed in the literature. However, the diversity of hemicelluloses found in plant biomass and the multitude of carbohydrate-active enzymes required for their deconstruction has resulted in a less comprehensive understanding of bacterial hemicellulase-encoding gene regulation. Here we review the mechanisms of this process and common themes found in the transcriptomic response during plant biomass utilization. By comparing regulatory systems from both Gram-negative and Gram-positive bacteria, as well as drawing parallels to cellulase regulation, our goals are to highlight the shared and distinct features of bacterial hemicellulase-encoding gene regulation and provide a set of guiding questions to improve our understanding of bacterial lignocellulose utilization. Key points: • Canonical regulatory mechanisms for bacterial hemicellulase-encoding gene expression include hybrid two-component systems (HTCS), extracytoplasmic function (ECF)-σ/anti-σ systems, and carbon catabolite repression (CCR). • Current transcriptomic approaches are increasingly being used to identify hemicellulase-encoding gene regulatory patterns coupled with computational predictions for transcriptional regulators. • Future work should emphasize genetic approaches to improve systems biology tools available for model bacterial systems and emerging microbes with biotechnology potential. Specifically, optimization of Gram-positive systems will require integration of degradative and fermentative capabilities, while optimization of Gram-negative systems will require bolstering the potency of lignocellulolytic capabilities. [ABSTRACT FROM AUTHOR]

Metabolically versatile bacteria exhibit a global regulatory response known as carbon catabolite repression (CCR), which prioritizes some carbon sources over others when all are present in sufficient amounts. This optimizes growth by distributing metabolite fluxes, but can restrict yields in biotechnological applications. The molecular mechanisms and preferred substrates for CCR vary between bacterial groups. Escherichia coli prioritizes glucose whereas Pseudomonas sp. prefer certain organic acids or amino acids. A significant issue in understanding (and potentially bypassing) CCR is the lack of information about the signals that trigger this regulatory response. In E. coli, several key compounds act as flux sensors, governing the flow of metabolites through catabolic pathways and preventing imbalances. These flux sensors can also modulate the CCR response. It has been suggested that the order of substrate preference is determined by carbon uptake flux rather than substrate identity. For Pseudomonas, much less information is available, as the signals that induce CCR are poorly understood. This article briefly discusses the available evidence on the signals that trigger CCR and the questions that remain to be answered in Pseudomonas. [ABSTRACT FROM AUTHOR]

ABSTRACT Cross-feeding of metabolites between subpopulations can affect cell phenotypes and population-level behaviors. In chronic Pseudomonas aeruginosa lung infections, subpopulations with loss-of-function (LOF) mutations in the lasR gene are common. LasR, a transcription factor often described for its role in virulence factor expression, also impacts metabolism, which, in turn, affects interactions between LasR+ and LasR− genotypes. Prior transcriptomic analyses suggested that citrate, a metabolite secreted by many cell types, induces virulence factor production when both genotypes are together. An unbiased analysis of the intracellular metabolome revealed broad differences including higher levels of citrate in lasR LOF mutants. Citrate consumption by LasR− strains required the CbrAB two-component system, which relieves carbon catabolite repression and is elevated in lasR LOF mutants. Within mixed communities, the citrate-responsive two-component system TctED and its gene targets OpdH (porin) and TctABC (citrate transporter) that are predicted to be under catabolite repression control were induced and required for enhanced RhlR/I-dependent signaling, pyocyanin production, and fitness of LasR− strains. Citrate uptake by LasR− strains markedly increased pyocyanin production in co-culture with Staphylococcus aureus, which also secretes citrate and frequently co-infects with P. aeruginosa. This citrate-induced restoration of virulence factor production by LasR− strains in communities with diverse species or genotypes may offer an explanation for the contrast observed between the markedly deficient virulence factor production of LasR− strains in monocultures and their association with the most severe forms of cystic fibrosis lung infections. These studies highlight the impact of secreted metabolites in mixed microbial communities.IMPORTANCECross-feeding of metabolites can change community composition, structure, and function. Here, we unravel a cross-feeding mechanism between frequently co-observed isolate genotypes in chronic Pseudomonas aeruginosa lung infections. We illustrate an example of how clonally derived diversity in a microbial communication system enables intra- and inter-species cross-feeding. Citrate, a metabolite released by many cells including P. aeruginosa and Staphylococcus aureus, was differentially consumed between genotypes. Since these two pathogens frequently co-occur in the most severe cystic fibrosis lung infections, the cross-feeding-induced virulence factor expression and fitness described here between diverse genotypes exemplify how co-occurrence can facilitate the development of worse disease outcomes.

The fungus Trichoderma reesei is an essential producer of enzymes that degrade lignocellulosic biomass to produce value-added bioproducts. The cellulolytic system of T. reesei is controlled by several transcription factors (TFs) that efficiently regulate the production of these enzymes. Recently, a new TF named Azf1 was identified as a positive regulator of cellulase expression. Here, we investigated novel regulatory functions of Azf1 by its overexpression. In the mutant strain OEazf1, overexpression of azf1 was achieved under both repression and induction conditions. Although azf1 was more abundant in transcript and protein, overexpression of this TF did not activate transcription of the cellulase gene in the presence of the repressor glucose, suggesting that Azf1 may be subject to posttranslational regulation. In cellulose, the expression of swo, encoding the accessory protein swollenin, and the β-glucosidases cel1a, cel1b, cel3b, and cel3g increases in the early stages of cultivation. The increased production of these β-glucosidases increases the hydrolysis rate of cellobiose and sophorose, which activates carbon catabolite repression (CCR) and causes repression of cellulase genes and the regulator Xyr1 in the later stages of cultivation. Moreover, overexpression of azf1 led to increased cellulase activity in T. reesei during long-term cultivation in cellulose and sugarcane bagasse. Our results provide new insights into the mechanisms regulating Azf1 and novel genes that are important targets of this TF. This work contributes to a better understanding of the complex mechanisms regulating cellulase expression in T. reesei. It will contribute to the development of strains with higher production of these essential enzymes. [ABSTRACT FROM AUTHOR]

The exopolysaccharide galactosaminogalactan (GAG) contributes to biofilm formation and virulence in the pathogenic fungus Aspergillus fumigatus. Increasing evidence indicates that GAG production is inversely linked with asexual development. However, the mechanisms underlying this regulatory relationship are unclear. In this study, we found that the dysfunction of CreA, a conserved transcription factor involved in carbon catabolite repression in many fungal species, causes abnormal asexual development (conidiation) under liquid‐submerged culture conditions specifically in the presence of glucose. The loss of creA decreased GAG production independent of carbon sources. Furthermore, CreA contributed to asexual development and GAG production via distinct pathways. CreA promoted A. fumigatus GAG production by positively regulating GAG biosynthetic genes (uge3 and agd3). CreA suppressed asexual development in glucose liquid‐submerged culture conditions via central conidiation genes (brlA, abaA, and wetA) and their upstream activators (flbC and flbD). Restoration of brlA expression to the wild‐type level by flbC or flbD deletion abolished the abnormal submerged conidiation in the creA null mutant but did not restore GAG production. The C‐terminal region of CreA was crucial for the suppression of asexual development, and the repressive domain contributed to GAG production. Overall, CreA is involved in GAG production and asexual development in an inverse manner. [ABSTRACT FROM AUTHOR]

As a global regulatory mechanism, carbon catabolite repression allows bacteria and eukaryal microbes to preferentially utilize certain substrates from a mixture of carbon sources. The mechanism varies among different species. In Pseudomonas spp., it is mainly mediated by the Crc-Hfq complex which binds to the 5′ region of the target mRNAs, thereby inhibiting their translation. This molecular mechanism enables P. putida to rapidly adjust and fine-tune gene expression in changing environments. Hfq is an RNA-binding protein that is ubiquitous and highly conserved in bacterial species. Considering the characteristics of Hfq, and the widespread use and rapid response of Crc-Hfq in P. putida , this complex has the potential to become a general toolbox for post-transcriptional multiplex regulation. In this study, we demonstrate for the first time that transplanting the pseudomonal catabolite repression protein, Crc, into E. coli causes multiplex gene repression. Under the control of Crc, the production of a diester and its precursors was significantly reduced. The effects of Crc introduction on cell growth in both minimal and rich media were evaluated. Two potential factors - off-target effects and Hfq-sequestration - could explain negative effects on cell growth. Simultaneous reduction of off-targeting and increased sequestration of Hfq by the introduction of the small RNA CrcZ, indicated that Hfq sequestration plays a more prominent role in the negative side-effects. This suggests that the negative growth effect can be mitigated by well-controlled expression of Hfq. This study reveals the feasibility of controlling gene expression using heterologous regulation systems. [Display omitted] • Expression of the Crc system resulted in multiplex gene repression in E. coli. • Hfq from E. coli is in vivo compatible with Crc from P. putida. • Hfq sequestration by Crc negatively affects cell growth. • Crc-Hfq was used for post-transcriptional regulation in a non-native host. [ABSTRACT FROM AUTHOR]

Trichoderma reesei is the most well-known cellulase producer in the biorefinery industry. Its cellulase biosynthesis is repressed by glucose via carbon catabolite repression (CCR), making CCR-releasing strains with cellulase hyperproduction desirable. Here, we employed a microfluidic droplet platform to culture and screen T. reesei mutants capable of CCR release and cellulase overproduction from extensive mutagenesis libraries. With 3 mutagenesis rounds, about 6.20 × 10 3 droplets were sorted from a population of 1.51 × 10 6 droplets in a period of 4.4 h; 76 recovery mutants were screened on flask fermentation, and 2 glucose uptake retarded mutants, MG-9-3 and MG-9-3-30, were eventually isolated. We also generated a hypercellulase producer, M-5, with CCR release via a single mutagenesis round. The hyphal morphology and molecular mechanisms in the mutants were analyzed. This versatile approach combined with a comprehensive understanding of CCR release mechanisms will provide innovative and effective strategies for low-cost cellulase production. A repeated high-throughput mutagenesis workflow for strain improvements of Trichoderma reesei using the droplet-based microfluidic platform [ABSTRACT FROM AUTHOR]

Members of the Paracoccidioides complex are the causative agents of Paracoccidioidomycosis (PCM), a human systemic mycosis endemic in Latin America. Upon initial contact with the host, the pathogen needs to uptake micronutrients. Nitrogen is an essential source for biosynthetic pathways. Adaptation to nutritional stress is a key feature of fungi in host tissues. Fungi utilize nitrogen sources through Nitrogen Catabolite Repression (NCR). NCR ensures the scavenging, uptake and catabolism of alternative nitrogen sources, when preferential ones, such as glutamine or ammonium, are unavailable. The NanoUPLC-MSE proteomic approach was used to investigate the NCR response of Paracoccidioides lutzii after growth on proline or glutamine as a nitrogen source. A total of 338 differentially expressed proteins were identified. P. lutzii demonstrated that gluconeogenesis, β-oxidation, glyoxylate cycle, adhesin-like proteins, stress response and cell wall remodeling were triggered in NCR-proline conditions. In addition, within macrophages, yeast cells trained under NCR-proline conditions showed an increased ability to survive. In general, this study allows a comprehensive understanding of the NCR response employed by the fungus to overcome nutritional starvation, which in the human host is represented by nutritional immunity. In turn, the pathogen requires rapid adaptation to the changing microenvironment induced by macrophages to achieve successful infection. [ABSTRACT FROM AUTHOR]

Within biofilms, gradients of electron acceptors such as oxygen stimulate the formation of physiological subpopulations. This heterogeneity can enable cross-feeding and promote drug resilience, features of the multicellular lifestyle that make biofilm-based infections difficult to treat. The pathogenic bacterium Pseudomonas aeruginosa produces pigments called phenazines that can support metabolic activity in hypoxic/anoxic biofilm subzones, but these compounds also include methylated derivatives that are toxic to their producer under some conditions. In this study, we uncover roles for the global regulators RpoS and Hfq/Crc in controlling the beneficial and detrimental effects of methylated phenazines in biofilms. Our results indicate that RpoS controls phenazine methylation by modulating activity of the carbon catabolite repression pathway, in which the Hfq/Crc complex inhibits translation of the phenazine methyltransferase PhzM. We find that RpoS indirectly inhibits expression of CrcZ, a small RNA that binds to and sequesters Hfq/Crc, specifically in the oxic subzone of P. aeruginosa biofilms. Deletion of rpoS or crc therefore leads to overproduction of methylated phenazines, which we show leads to increased metabolic activity—an apparent beneficial effect—in hypoxic/anoxic subpopulations within biofilms. However, we also find that under specific conditions, biofilms lacking RpoS and/or Crc show increased sensitivity to phenazines indicating that the increased metabolic activity in these mutants comes at a cost. Together, these results suggest that complex regulation of PhzM allows P. aeruginosa to simultaneously exploit the benefits and limit the toxic effects of methylated phenazines. [ABSTRACT FROM AUTHOR]

Magnaporthe oryzae is a pathogenic fungus that seriously harms rice production. Phosphatases and carbon metabolism play crucial roles in the growth and development of eukaryotes. However, it remains unclear how serine/threonine phosphatases regulate the catabolism of triglycerides, a major form of stored lipids. In this study, we identified a serine/threonine protein phosphatase regulatory subunit, Smek1, which is required for the growth, conidiation, and virulence of M. oryzae. Deletion of SMEK1 led to defects in the utilization of lipids, arabinose, glycerol, and ethanol. In glucose medium, the expression of genes involved in lipolysis, long‐chain fatty acid degradation, β‐oxidation, and the glyoxylate cycle increased in the Δsmek1 mutant, which is consistent with ΔcreA in which a carbon catabolite repressor CREA was deleted. In lipid medium, the expression of genes involved in long‐chain fatty acid degradation, β‐oxidation, the glyoxylate cycle, and utilization of arabinose, ethanol, or glycerol decreased in the Δsmek1 mutant, which is consistent with Δcrf1 in which a transcription activator CRF1 required for carbon metabolism was deleted. Lipase activity, however, increased in the Δsmek1 mutant in both glucose and lipid media. Moreover, Smek1 directly interacted with CreA and Crf1, and dephosphorylated CreA and Crf1 in vivo. The phosphatase Smek1 is therefore a dual‐function regulator of the lipid and carbohydrate metabolism, and controls fungal development and virulence by coordinating the functions of CreA and Crf1 in carbon catabolite repression (CCR) and derepression (CCDR). [ABSTRACT FROM AUTHOR]

Poly(butylene adipate-co-terephthalate) (PBAT), a promising biodegradable aliphatic-aromatic copolyester material, can be applied as an alternative material to reduce the adverse effects of conventional plastics. However, the degradation of PBAT plastics in soil is time-consuming, and effective PBAT-degrading microorganisms have rarely been reported. In this study, the biodegradation properties of PBAT by an elite fungal strain and related mechanisms were elucidated. Four PBAT-degrading fungal strains were isolated from farmland soils, and Purpureocillium lilacinum strain BA1S showed a prominent degradation rate. It decomposed approximately 15 wt.% of the PBAT films 30 days after inoculation. Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and Liquid chromatography mass spectrometry (LC‒MS) were conducted to analyze the physicochemical properties and composition of the byproducts after biodegradation. In the presence of PBAT, the lipolytic enzyme activities of BA1S were remarkably induced, and its cutinase gene was also significantly upregulated. Of note, the utilization of PBAT in BA1S cells was closely correlated with intracellular cytochrome P450 (CYP) monooxygenase. Furthermore, CreA-mediated carbon catabolite repression was confirmed to be involved in regulating PBAT-degrading hydrolases and affected the degradation efficiency. This study provides new insight into the degradation of PBAT by elite fungal strains and increases knowledge on the mechanism, which can be applied to control the biodegradability of PBAT films in the future. Key points: • Purpureocillium lilacinum strain BA1S was isolated from farmland soils and degraded PBAT plastic films at a prominent rate. • The lipolytic enzyme activities of strain BA1S were induced during coculture with PBAT, and the cutinase gene was significantly upregulated during PBAT degradation. • CreA-mediated carbon catabolite repression of BA1S plays an essential role in regulating the expression of PBAT-degrading hydrolases. [ABSTRACT FROM AUTHOR]

Carbohydrate active enzymes (CAZymes) are vital for the lignocellulose-based biorefinery. The development of hypersecreting fungal protein production hosts is therefore a major aim for both academia and industry. However, despite advances in our understanding of their regulation, the number of promising candidate genes for targeted strain engineering remains limited. Here, we resequenced the genome of the classical hypersecreting Neurospora crassa mutant exo-1 and identified the causative point of mutation to reside in the F-box protein-encoding gene, NCU09899. The corresponding deletion strain displayed amylase and invertase activities exceeding those of the carbon catabolite derepressed strain Δcre-1, while glucose repression was still mostly functional in Δexo-1 Surprisingly, RNA sequencing revealed that while plant cell wall degradation genes are broadly misexpressed in Δexo-1, only a small fraction of CAZyme genes and sugar transporters are up-regulated, indicating that EXO-1 affects specific regulatory factors. Aiming to elucidate the underlying mechanism of enzyme hypersecretion, we found the high secretion of amylases and invertase in Δexo-1 to be completely dependent on the transcriptional regulator COL-26. Furthermore, misregulation of COL-26, CRE-1, and cellular carbon and nitrogen metabolism was confirmed by proteomics. Finally, we successfully transferred the hypersecretion trait of the exo-1 disruption by reverse engineering into the industrially deployed fungus Myceliophthora thermophila using CRISPR-Cas9. Our identification of an important F-box protein demonstrates the strength of classical mutants combined with next-generation sequencing to uncover unanticipated candidates for engineering. These data contribute to a more complete understanding of CAZyme regulation and will facilitate targeted engineering of hypersecretion in further organisms of interest.

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.

ABSTRACT Bacteria adapt to nutrient availability by regulating the synthesis of enzymes. Transcriptome- and multi-sugar growth studies suggest that Lactococcus cremoris represses genes involved in the catabolization of lower growth rate-supporting (lower quality) sugars in a hierarchical order. Furthermore, L. cremoris appears to always express genes involved in the catabolization of higher growth rate-supporting sugars (higher quality) relative to the sugar it is growing on. Here, we unraveled the sugar catabolization hierarchy by determining the sugar catabolizing capacity and the proteome of cells exponentially growing on glucose (µmax = 0.72 h−1), lactose (µmax = 0.6 h−1), galactose (µmax = 0.44 h−1), or maltose (µmax = 0.43 h−1). We found that L. cremoris can grow on 14 of the 96 sugars in a Biolog plate, with µmax ranging from 0.32 to 0.72 h−1. Proteome and catabolization rate measurements show that L. cremoris consistently prepares for the catabolization of higher-quality sugars, except trehalose. While cells were not prepared for the catabolization of most lower-quality sugars, some proteins related to fructose and lactose consumption were always present. Moreover, reducing the growth rate of glucose through salt stress had only a minor influence on the sugars that L. cremoris could catabolize. These findings demonstrate that the catabolization hierarchy is not strictly linked to absolute growth rate or sugar quality. Cells instantly catabolizing a higher-quality sugar require enhanced expression of ribosomal and nucleotide metabolism functions for growth rate maximization, whereas transitioning to lower-quality sugars requires enhanced synthesis of proteins related to arginine catabolism and mixed acid fermentation, besides sugar-specific catabolic proteins. IMPORTANCE The availability of nutrients to microorganisms varies considerably between different environments, and changes can occur rapidly. As a general rule, a fast growth rate—typically growth on glucose—is associated with the repression of other carbohydrate utilization genes, but it is not clear to what extent catabolite repression is exerted by other sugars. We investigated the hierarchy of sugar utilization after substrate transitions in Lactococcus cremoris. For this, we determined the proteome and carbohydrate utilization capacity after growth on different sugars. The results show that the preparedness of cells for the utilization of “slower” sugars is not strictly determined by the growth rate. The data point to individual proteins relevant for various sugar transitions and suggest that the evolutionary history of the organism might be responsible for deviations from a strictly growth rate-related sugar catabolization hierarchy.

Background: Plastics are an indispensable part of our daily life. However, mismanagement at their end-of-life results in severe environmental consequences. The microbial conversion of these polymers into new value-added products offers a promising alternative. In this study, we engineered the soil-bacterium Comamonas testosteroni KF-1, a natural degrader of terephthalic acid, for the conversion of the latter to the high-value product 2-pyrone-4,6-dicarboxylic acid. Results: In order to convert terephthalic acid to 2-pyrone-4,6-dicarboxylic acid, we deleted the native PDC hydrolase and observed only a limited amount of product formation. To test whether this was the result of an inhibition of terephthalic acid uptake by the carbon source for growth (i.e. glycolic acid), the consumption of both carbon sources was monitored in the wild-type strain. Both carbon sources were consumed at the same time, indicating that catabolite repression was not the case. Next, we investigated if the activity of pathway enzymes remained the same in the wild-type and mutant strain. Here again, no statistical differences could be observed. Finally, we hypothesized that the presence of a pmdK variant in the degradation operon could be responsible for the observed phenotype and created a double deletion mutant strain. This newly created strain accumulated PDC to a larger extent and again consumed both carbon sources. The double deletion strain was then used in a bioreactor experiment, leading to the accumulation of 6.5 g/L of product in 24 h with an overall productivity of 0.27 g/L/h. Conclusions: This study shows the production of the chemical building block 2-pyrone-4,6-dicarboxylic acid from terephthalic acid through an engineered C. testosteroni KF-1 strain. It was observed that both a deletion of the native PDC hydrolase as well as a pmdK variant is needed to achieve high conversion yields. A product titer of 6.5 g/L in 24 h with an overall productivity of 0.27 g/L/h was achieved. [ABSTRACT FROM AUTHOR]

Pirin family proteins perform a variety of biological functions and widely exist in all living organisms. A few studies have shown that Pirin family proteins may be involved in the biosynthesis of antibiotics in actinomycetes. However, the function of Pirin-like proteins in S. spinosa is still unclear. In this study, the inactivation of the sspirin gene led to serious growth defects and the accumulation of H2O2. Surprisingly, the overexpression and knockout of sspirin slightly accelerated the consumption and utilization of glucose, weakened the TCA cycle, delayed sporulation, and enhanced sporulation in the later stage. In addition, the overexpression of sspirin can enhance the β-oxidation pathway and increase the yield of spinosad by 0.88 times, while the inactivation of sspirin hardly produced spinosad. After adding MnCl2, the spinosad yield of the sspirin overexpression strain was further increased to 2.5 times that of the wild-type strain. This study preliminarily revealed the effects of Pirin-like proteins on the growth development and metabolism of S. spinosa and further expanded knowledge of Pirin-like proteins in actinomycetes. Key points: • Overexpression of the sspirin gene possibly triggers carbon catabolite repression (CCR) • Overexpression of the sspirin gene can promote the synthesis of spinosad • Knockout of the sspirin gene leads to serious growth and spinosad production defects [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is a ubiquitous nosocomial opportunistic pathogen that harbors many virulence determinants. Part of P. aeruginosa success colonizing a variety of habitats resides in its metabolic robustness and plasticity, which are the basis of its capability of adaptation to different nutrient sources and ecological conditions, including the infected host. Given this situation, it is conceivable that P. aeruginosa virulence might be, at least in part, under metabolic control, in such a way that virulence determinants are produced just when needed. Indeed, it has been shown that the catabolite repression control protein Crc, which together with the RNA chaperon Hfq regulates the P. aeruginosa utilization of carbon sources at the post-transcriptional level, also regulates, directly or indirectly, virulence-related processes in P. aeruginosa. Among them, Crc regulates P. aeruginosa cytotoxicity, likely by modulating the activity of the Type III Secretion System (T3SS), which directly injects toxins into eukaryotic host cells. The present work shows that the lack of Crc produces a Type III Secretion-defective phenotype in P. aeruginosa. The observed impairment is a consequence of a reduced expression of the genes encoding the T3SS, together with an impaired secretion of the proteins involved. Our results support that the impaired T3SS activity of the crc defective mutant is, at least partly, a consequence of a defective protein export, probably due to a reduced proton motive force. This work provides new information about the complex regulation of the expression and the activity of the T3SS in P. aeruginosa. Our results highlight the need of a robust bacterial metabolism, which is defective in the ∆crc mutant, to elicit complex and energetically costly virulence strategies, as that provided by the T3SS. [ABSTRACT FROM AUTHOR]

The current study investigated the feasibility of developing and adopting a few state-of-the-art fermentation techniques to maximize the efficiency of the lignocellulosic waste bioconversion. There have been various efforts towards utilizing the fermentable sugars released from the specific parts of lignocellulose, i.e., cellulose and hemicellulose. However, complete utilization of carbon sources derived from lignocellulosic biomass remains challenging owing to the generated glucose in the presence of β-glucosidase, which is known as glucose-induced carbon catabolite repression (CCR). To overcome this obstacle, a novel simultaneous saccharification and fermentation (SSF) of lactic acid was designed by using Celluclast 1.5L as a hydrolytic enzyme to optimize the generation and utilization of pentose and hexose. Under the optimal enzyme loading and pH condition, 53.1 g/L optically pure L-lactic acid with a maximum volumetric productivity of 3.65 g/L/h was achieved during the SSF from the brewer's spent grain without any nutrient supplementation. This study demonstrated the potential of lactic acid production from the designed lignocellulosic substrate. [ABSTRACT FROM AUTHOR]

Filamentous fungi, such as Neurospora crassa, are very efficient in deconstructing plant biomass by the secretion of an arsenal of plant cell wall-degrading enzymes, by remodeling metabolism to accommodate production of secreted enzymes, and by enabling transport and intracellular utilization of plant biomass components. Although a number of enzymes and transcriptional regulators involved in plant biomass utilization have been identified, how filamentous fungi sense and integrate nutritional information encoded in the plant cell wall into a regulatory hierarchy for optimal utilization of complex carbon sources is not understood. Here, we performed transcriptional profiling of N. crassa on 40 different carbon sources, including plant biomass, to provide data on how fungi sense simple to complex carbohydrates. From these data, we identified regulatory factors in N. crassa and characterized one (PDR-2) associated with pectin utilization and one with pectin/hemicellulose utilization (ARA-1). Using in vitro DNA affinity purification sequencing (DAP-seq), we identified direct targets of transcription factors involved in regulating genes encoding plant cell wall-degrading enzymes. In particular, our data clarified the role of the transcription factor VIB-1 in the regulation of genes encoding plant cell wall-degrading enzymes and nutrient scavenging and revealed a major role of the carbon catabolite repressor CRE-1 in regulating the expression of major facilitator transporter genes. These data contribute to a more complete understanding of cross talk between transcription factors and their target genes, which are involved in regulating nutrient sensing and plant biomass utilization on a global level.