Combinatorial control of Pseudomonas aeruginosa biofilm development by quorum-sensing and nutrient-sensing regulators.

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.

CRP improves the survival and competitive fitness of Salmonella Typhimurium under starvation by controlling the cellular maintenance rate.

Catabolite repression is a mechanism of selectively utilizing preferred nutrient sources by redirecting the metabolic pathways. Therefore, it prevents non-essential energy expenditure by repressing the genes and proteins involved in the metabolism of other less favored nutrient sources. Catabolite repressor protein (CRP) is a chief mediator of catabolite repression in microorganisms. In this context, we investigated the role of CRP in starvation tolerance, at both cell physiology and molecular level, by comparing the growth, survival, competitive fitness, maintenance rate, and gene and protein expression of wild type (WT) and ∆crp of Salmonella Typhimurium, under nutrient-rich and minimal medium condition. The ∆crp shows slow growth upon the arrival of nutrient-limiting conditions, poor survival under prolong-starvation, and inability to compete with its counterpart WT strain in nutrient-rich [Luria broth (LB)] and glucose-supplemented M9 minimal medium. Surprisingly, we observed that the survival and competitive fitness of ∆crp are influenced by the composition of the growth medium. Consequently, compared to the glucose-supplemented M9 medium, ∆crp shows faster death and a higher maintenance rate in the LB medium. The comparative gene and protein expression studies of WT and ∆crp in LB medium show that ∆crp has partial or complete loss of repression from CRP-controlled genes, resulting in a high abundance of hundreds of proteins in ∆crp compared to WT. Subsequently, the addition of metabolizable sugar or fresh nutrients to the competing culture showed extended survival of ∆crp. Therefore, our results suggest that CRP-mediated gene repression improves starvation tolerance and competitive fitness of Salmonella Typhimurium by adapting its cellular maintenance rate to environmental conditions.IMPORTANCESalmonella Typhimurium is a master at adapting to chronic starvation conditions. However, the molecular mechanisms to adapt to such conditions are still unknown. In this context, we have evaluated the role of catabolite repressor protein (CRP), a dual transcriptional regulator, in providing survival and competitive fitness under starvation conditions. Also, it showed an association between CRP and nutrient composition. We observed that Δcrp growing on alternate carbon sources has lower survival and competitive fitness than Δcrp growing on glucose as a carbon source. We observed that this is due to the loss of repression from the glucose and CRP-controlled genes, resulting in elevated cellular metabolism (a high maintenance rate) of the Δcrp during growth in a medium having a carbon source other than glucose (e.g., Luria broth medium).

Structure-activity relationship study of nitrogen signaling factors.

We have synthesized 10 analogs of oxylipins, which are nitrogen signaling factors (NSFs) that mediate cell-to-cell communication in the fission yeast Schizosaccharomyces pombe, and evaluated their structure-activity relationships with the aim of developing molecular probes for NSFs. We found that the OH or OAc group at C10 could be replaced with a compact amide (17) or carbamate (19). Introducing an alkyne as a detection tag at C10 led to decreased, though still sufficient, activity. Introducing an alkyne at the C18 position showed a similar trend, suggesting tolerance is relatively low even for compact functional groups such as alkynes. Although introduction of a diazirine moiety as a photoreactive group at the C5 position decreased the activity, we found that introducing diazirine at the C13 position was acceptable, and compound 38 exhibited potent NSF activity. These findings will be helpful in the development of molecular probes for NSFs.

Utilization of carbon catabolite repression for efficiently biotransformation of anthraquinone O-glucuronides by Streptomyces coeruleorubidus DM.

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.

Virtually identical does not mean exactly identical: Discrepancy in energy metabolism between glucose and fructose fermentation influences the reproductive potential of yeast cells.

The physiological efficiency of cells largely depends on the possibility of metabolic adaptations to changing conditions, especially on the availability of nutrients. Central carbon metabolism has an essential role in cellular function. In most cells is based on glucose, which is the primary energy source, provides the carbon skeleton for the biosynthesis of important cell macromolecules, and acts as a signaling molecule. The metabolic flux between pathways of carbon metabolism such as glycolysis, pentose phosphate pathway, and mitochondrial oxidative phosphorylation is dynamically adjusted by specific cellular economics responding to extracellular conditions and intracellular demands. Using Saccharomyces cerevisiae yeast cells and potentially similar fermentable carbon sources i.e. glucose and fructose we analyzed the parameters concerning the metabolic status of the cells and connected with them alteration in cell reproductive potential. Those parameters were related to the specific metabolic network: the hexose uptake - glycolysis and activity of the cAMP/PKA pathway - pentose phosphate pathway and biosynthetic capacities - the oxidative respiration and energy generation. The results showed that yeast cells growing in a fructose medium slightly increased metabolism redirection toward respiratory activity, which decreased pentose phosphate pathway activity and cellular biosynthetic capabilities. These differences between the fermentative metabolism of glucose and fructose, lead to long-term effects, manifested by changes in the maximum reproductive potential of cells.

Co-utilization of carbon sources in microorganisms for the bioproduction of chemicals.

Carbon source is crucial for the cell growth and metabolism in microorganisms, and its utilization significantly affects the synthesis efficiency of target products in microbial cell factories. Compared with a single carbon source, co-utilizing carbon sources provide an alternative approach to optimize the utilization of different carbon sources for efficient biosynthesis of many chemicals with higher titer/yield/productivity. However, the efficiency of bioproduction is significantly limited by the sequential utilization of a preferred carbon source and secondary carbon sources, attributed to carbon catabolite repression (CCR). This review aimed to introduce the mechanisms of CCR and further focus on the summary of the strategies for co-utilization of carbon sources, including alleviation of CCR, engineering of the transport and metabolism of secondary carbon sources, compulsive co-utilization in single culture, co-utilization of carbon sources via co-culture, and evolutionary approaches. The findings of representative studies with a significant improvement in the bioproduction of chemicals via the co-utilization of carbon sources were discussed in this review. It suggested that by combining rational metabolic engineering and irrational evolutionary approaches, co-utilizing carbon sources can significantly contribute to the bioproduction of chemicals.

Stress-Induced Changes in Nucleocytoplasmic Localization of Crucial Factors in Gene Expression Regulation.

This study investigates the toxic effect of harmful materials, unfiltered by the placenta, on neonatal umbilical cord (UC) vessels, focusing on stress-induced adaptations in transcriptional and translational processes. It aims to analyze changes in pathways related to mRNA condensate formation, transcriptional regulation, and DNA damage response under maternal smoking-induced stress. UC vessels from neonates born to smoking (Sm) and nonsmoking mothers (Ctr) were examined. Immunofluorescence staining and confocal microscopy assessed the localization of key markers, including Transcription Complex Subunit 1 (CNOT1) and the largest subunit of RNA polymerase II enzyme (RPB1). Additionally, markers of DNA damage response, such as Poly(ADP-ribose) polymerase-1, were evaluated. In Sm samples, dissolution of CNOT1 granules in UC vessels was observed, potentially aiding stalled translation and enhancing transcription via RPB1 assembly and translocation. Control vessels showed predominant cytoplasmic RPB1 localization. Despite adaptive responses, Sm endothelial cells exhibited significant damage, indicated by markers like Poly(ADP-ribose) polymerase-1. Ex vivo metal treatment on control vessels mirrored Sm sample alterations, emphasizing marker roles in cell survival under toxic exposure. Maternal smoking induces specific molecular adaptations in UC vessels, affecting mRNA condensate formation, transcriptional regulation, and DNA damage response pathways. Understanding these intricate molecular mechanisms could inform interventions to improve neonatal health outcomes and mitigate adverse effects of toxic exposure during pregnancy.

Understanding operon architecture using LEGO bricks.

Many undergraduates struggle to interpret abstract concepts in molecular biology. Modeling can facilitate learning by making these abstract concepts tangible. Here, we present an exercise based on the lac operon designed for undergraduate students using LEGO bricks. The lac operon is a classic example of transcriptional regulation taught in a variety of undergraduate biology courses and is fundamental to understanding the regulation of gene expression. This easy-to-implement active learning exercise demonstrates how the various components of the lac operon are oriented under a variety of nutritional conditions to control gene expression. In addition, higher-order concepts, such as the effect of mutation on lac operon expression, can be readily modeled. Overall, students not only found this exercise to be enjoyable but also helpful as a tool to engage with this course material.

Glucose Catabolite Repression Participates in the Regulation of Sialidase Biosynthesis by Antarctic Strain Penicillium griseofulvum P29.

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.

Quality and flavor development of solid-state fermented surimi with Actinomucor elegans: A perspective on the impacts of carbon and nitrogen sources.

The influence of four carbon and nitrogen substrates on the quality and flavor of a novel surimi-based product fermented with Actinomucor elegans (A. elegans) was investigated, with a focus on carbon and nitrogen catabolite repression. The results showed that the substrate significantly affected mycelial growth, enzyme activities, and the metabolites of A. elegans. Although glucose significantly promoted A. elegans growth by 116.69%, it decreased enzyme secretion by 69.79% for α-amylase and 59.80% for protease, most likely by triggering the carbon catabolite repression pathway. Starch, soy protein, and wheat gluten substantially affected the textural properties of the fermented surimi. Furthermore, wheat gluten significantly promoted the protease activity (102.70%) and increased protein degradation during surimi fermentation. The fishy odor of surimi was alleviated through fermentation, and a correlation between the volatile compounds and A. elegans metabolism was observed. These results explore fermentation substrates in filamentous fungi metabolism from a catabolite repression perspective.

Regulation of Aerobic Succinate Transporter dctA of E. coli by cAMP-CRP, DcuS-DcuR, and EIIAGlc: Succinate as a Carbon Substrate and Signaling Molecule.

INTRODUCTION: C4-dicarboxylates (C4-DC) have emerged as significant growth substrates and signaling molecules for various Enterobacteriaceae during their colonization of mammalian hosts. Particularly noteworthy is the essential role of fumarate respiration during colonization of pathogenic bacteria. To investigate the regulation of aerobic C4-DC metabolism, the study explored the transcriptional control of the main aerobic C4-DC transporter, dctA, under different carbohydrate conditions. In addition, mutants related to carbon catabolite repression (CCR) and C4-DC regulation (DcuS-DcuR) were examined to better understand the regulatory integration of aerobic C4-DC metabolism into CCR. For initial insight into posttranslational regulation, the interaction between the aerobic C4-DC transporter DctA and EIIAGlc from the glucose-specific phosphotransferase system was investigated. METHODS: The expression of dctA was characterized in the presence of various carbohydrates and regulatory mutants affecting CCR. This was accomplished by fusing the dctA promoter (PdctA) to the lacZ reporter gene. Additionally, the interaction between DctA and EIIAGlc of the glucose-specific phosphotransferase system was examined in vivo using a bacterial two-hybrid system. RESULTS: The dctA promoter region contains a class I cAMP-CRP-binding site at position -81.5 and a DcuR-binding site at position -105.5. DcuR, the response regulator of the C4-DC-activated DcuS-DcuR two-component system, and cAMP-CRP stimulate dctA expression. The expression of dctA is subject to the influence of various carbohydrates via cAMP-CRP, which differently modulate cAMP levels. Here we show that EIIAGlc of the glucose-specific phosphotransferase system strongly interacts with DctA, potentially resulting in the exclusion of C4-DCs when preferred carbon substrates, such as sugars, are present. In contrast to the classical inducer exclusion known for lactose permease LacY, inhibition of C4-DC uptake into the cytoplasm affects only its role as a substrate, but not as an inducer since DcuS detects C4-DCs in the periplasmic space ("substrate exclusion"). The work shows an interplay between cAMP-CRP and the DcuS-DcuR regulatory system for the regulation of dctA at both transcriptional and posttranslational levels. CONCLUSION: The study highlights a hierarchical interplay between global (cAMP-CRP) and specific (DcuS-DcuR) regulation of dctA at the transcriptional and posttranslational levels. The integration of global and specific transcriptional regulation of dctA, along with the influence of EIIAGlc on DctA, fine-tunes C4-DC catabolism in response to the availability of other preferred carbon sources. It attributes DctA a central role in the control of aerobic C4-DC catabolism and suggests a new role to EIIAGlc on transporters (control of substrate uptake by substrate exclusion).

Independent metabolism of oligosaccharides is the keystone of synchronous utilization of cellulose and hemicellulose in Myceliophthora.

The effective utilization of cellulose and hemicellulose, the main components of plant biomass, is a key technical obstacle that needs to be overcome for the economic viability of lignocellulosic biorefineries. Here, we firstly demonstrated that the thermophilic cellulolytic fungus Myceliophthora thermophila can simultaneously utilize cellulose and hemicellulose, as evidenced by the independent uptake and intracellular metabolism of cellodextrin and xylodextrin. When plant biomass serviced as carbon source, we detected the cellodextrin and xylodextrin both in cells and in the culture medium, as well as high enzyme activities related to extracellular oligosaccharide formation and intracellular oligosaccharide hydrolysis. Sugar consumption assay revealed that in contrast to inhibitory effect of glucose on xylose and cellodextrin/xylodextrin consumption in mixed-carbon media, cellodextrin and xylodextrin were synchronously utilized in this fungus. Transcriptomic analysis also indicated simultaneous induction of the genes involved in cellodextrin and xylodextrin metabolic pathway, suggesting carbon catabolite repression (CCR) is triggered by extracellular glucose and can be eliminated by the intracellular hydrolysis and metabolism of oligosaccharides. The xylodextrin transporter MtCDT-2 was observed to preferentially transport xylobiose and tolerate high cellobiose concentrations, which helps to bypass the inhibition of xylobiose uptake. Furthermore, the expression of cellulase and hemicellulase genes was independently induced by their corresponding inducers, which enabled this strain to synchronously utilize cellulose and hemicellulose. Taken together, the data presented herein will further elucidate the degradation of plant biomass by fungi, with implications for the development of consolidated bioprocessing-based lignocellulosic biorefinery.

XylR Overexpression in Escherichia coli Alleviated Transcriptional Repression by Arabinose and Enhanced Xylitol Bioproduction from Xylose Mother Liquor.

Xylitol is a polyol widely used in food, pharmaceuticals, and light industries. It is currently produced through the chemical catalytic hydrogenation of xylose and generates xylose mother liquor as a substantial byproduct in the procedure of xylose extraction. If xylose mother liquor could also be efficiently bioconverted to xylitol, the greenness and atom economy of xylitol production would be largely improved. However, xylose mother liquor contains a mixture of glucose, xylose, and arabinose, raising the issue of carbon catabolic repression in its utilization by microbial conversion. Targeting this challenge, the transcriptional activator XylR was overexpressed in a previously constructed xylitol-producing E. coli strain CPH. The resulting strain CPHR produced 16.61 g/L of xylitol in shake-flask cultures from the mixture of corn cob hydrolysate and xylose mother liquor (1:1, v/v) with a xylose conversion rate of 90.1%, which were 2.23 and 2.15 times higher than the starting strain, respectively. Furthermore, XylR overexpression upregulated the expression levels of xylE, xylF, xylG, and xylH genes by 2.08-2.72 times in arabinose-containing medium, suggesting the alleviation of transcriptional repression of xylose transport genes by arabinose. This work lays the foundation for xylitol bioproduction from xylose mother liquor.

Citrate cross-feeding by Pseudomonas aeruginosa supports lasR mutant fitness.

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.

Current models in bacterial hemicellulase-encoding gene regulation.

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.

Carbon metabolism characteristics of quorum quenching bacteria Rhodococcus sp. BH4 determine the bioaugmentation efficiency under different carbon source conditions.

Carbon sources are critical factors influencing bacterial bioaugmentation, however, the underlying mechanisms, particularly the metabolic characteristics of bioaugmented bacteria remain poorly understood. The bioaugmented bacterium Rhodococcus sp. BH4 secretes the quorum quenching (QQ) enzyme QsdA to disrupt the quorum sensing (QS) in the activated sludge (AS) process, reducing AS yield in-situ. This study investigated the carbon metabolic characteristics of BH4 and explored the effects on bioaugmentation with different influent carbon sources. Because of the absence of glucose-specific phosphoenol phosphotransferase system (PTS), BH4 prefers sodium acetate to glucose. However, the lactones produced during extracellular glucose metabolism enhance BH4 qsdA expression. Moreover, BH4 possess carbon catabolite repression (CCR), acetate inhibits glucose utilization. BH4 microbeads were added to reactors with different carbon sources (R1: sodium acetate; R2: glucose; R3: a mixture of sodium acetate and glucose) for in-situ AS yield reduction. During operation, AS reduction efficiency decreased in the following order: R1 > R3 > R2. R2 and R3 microbeads exhibited similar QQ activity to R1, with less BH4 biomass at 5 d. 13C labeling and Michaelis-Menten equation showed that, due to differences in the competitiveness of carbon sources, R1 BH4 obtained the most carbon, whereas R2 BH4 obtained the least carbon. Moreover, acetate inhibited glucose utilization of R3 BH4. Transcriptome analysis showed that R1 BH4 qsdA expression was the lowest, R2 BH4 was the most serious form of programmed cell death, and the R3 BH4 PTS pathway was inhibited. At 10 d, R1 BH4 biomass and microbead QQ activity were higher than that in R3, and the R2 BH4 lost viability and QQ activity. This study provides new insights into bioaugmentation from the perspectives of carbon source competitiveness, carbon metabolism pathways, and CCR.

Simultaneous glucose and xylose utilization by an Escherichia coli catabolite repression mutant.

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.IMPORTANCEAs the need for renewable biofuel and biochemical production processes continues to grow, there is an associated need for microbial technology capable of utilizing cheap, widely available, and renewable carbon substrates. This work details the construction and characterization of the first B-lineage Escherichia coli strain with mutated cyclic AMP receptor protein, Crp*, which deregulates the carbon catabolite repression (CCR) system and enables the co-utilization of multiple sugar sources in the growth medium. In this study, we focus our analysis on glucose and xylose utilization as these two sugars are the primary components in lignocellulosic biomass hydrolysate, a promising renewable carbon feedstock for industrial bioprocesses. This strain is valuable to the field as it enables the use of mixed sugar sources in traditional fed-batch based approaches, whereas the wild-type carbon catabolite repression system leads to biphasic growth and possible buildup of non-preferential sugars, reducing process efficiency at scale.

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

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

Synergistic co-utilization of biomass-derived sugars enhances aromatic amino acid production by engineered Escherichia coli.

Efficient co-utilization of mixed sugar feedstocks remains a biomanufacturing challenge, thus motivating ongoing efforts to engineer microbes for improved conversion of glucose-xylose mixtures. This study focuses on enhancing phenylalanine production by engineering Escherichia coli to efficiently co-utilize glucose and xylose. Flux balance analysis identified E4P flux as a bottleneck which could be alleviated by increasing the xylose-to-glucose flux ratio. A mutant copy of the xylose-specific activator (XylR) was then introduced into the phenylalanine-overproducing E. coli NST74, which relieved carbon catabolite repression and enabled efficient glucose-xylose co-utilization. Carbon contribution analysis through 13 C-fingerprinting showed a higher preference for xylose in the engineered strain (NST74X), suggesting superior catabolism of xylose relative to glucose. As a result, NST74X produced 1.76 g/L phenylalanine from a model glucose-xylose mixture; a threefold increase over NST74. Then, using biomass-derived sugars, NST74X produced 1.2 g/L phenylalanine, representing a 1.9-fold increase over NST74. Notably, and consistent with the carbon contribution analysis, the xylR* mutation resulted in a fourfold greater maximum rate of xylose consumption without significantly impeding the maximum rate of total sugar consumption (0.87 vs. 0.70 g/L-h). This study presents a novel strategy for enhancing phenylalanine production through the co-utilization of glucose and xylose in aerobic E. coli cultures, and highlights the potential synergistic benefits associated with using substrate mixtures over single substrates when targeting specific products.

Engineering a Synthetic Escherichia coli Coculture for Compartmentalized de novo Biosynthesis of Isobutyl Butyrate from Mixed Sugars.

Short-chain esters are versatile chemicals that can be used as flavors, fragrances, solvents, and fuels. The de novo ester biosynthesis consists of diverging and converging pathway submodules, which is challenging to engineer to achieve optimal metabolic fluxes and selective product synthesis. Compartmentalizing the pathway submodules into specialist cells that facilitate pathway modularization and labor division is a promising solution. Here, we engineered a synthetic Escherichia coli coculture with the compartmentalized sugar utilization and ester biosynthesis pathways to produce isobutyl butyrate from a mixture of glucose and xylose. To compartmentalize the sugar-utilizing pathway submodules, we engineered a xylose-utilizing E. coli specialist that selectively consumes xylose over glucose and bypasses carbon catabolite repression (CCR) while leveraging the native CCR machinery to activate a glucose-utilizing E. coli specialist. We found that the compartmentalization of sugar catabolism enabled simultaneous co-utilization of glucose and xylose by a coculture of the two E. coli specialists, improving the stability of the coculture population. Next, we modularized the isobutyl butyrate pathway into the isobutanol, butyl-CoA, and ester condensation submodules, where we distributed the isobutanol submodule to the glucose-utilizing specialist and the other submodules to the xylose-utilizing specialist. Upon compartmentalization of the isobutyl butyrate pathway submodules into these sugar-utilizing specialist cells, a robust synthetic coculture was engineered to selectively produce isobutyl butyrate, reduce the biosynthesis of unwanted ester byproducts, and improve the production titer as compared to the monoculture.