This study uncovers a previously unappreciated mechanism of Tps1-mediated heat adaptation in C. deneoformans, by revealing that Tps1 functions as a critical metabolic gatekeeper that safeguards glycolytic flux to sustain growth at elevated temperatures.
Abstract
Growth at physiologically relevant temperatures is essential for fungal pathogenesis and is controlled by several cellular factors. The evolution of fungal thermotolerance is concerning as warming environments may promote the emergence of new pathogens. Trehalose, a disaccharide absent in mammals, plays a central role in thermotolerance by stabilizing proteins and membranes during heat stress. Trehalose is synthesized from glucose-6-phosphate (G6P) and uridine-diphosphate-glucose (UDPG) in two steps catalyzed by trehalose-6-phosphate synthase (Tps1) and trehalose-6-phosphate phosphatase (Tps2). Here, we investigated genetic suppression of Tps1 function in Cryptococcus deneoformans, a species in the Cryptococcus pathogenic species complex. Tps1 is essential for growth at 37°C in C. deneoformans and spontaneous suppressor mutations restored the growth of tps1Δ mutants at 37°C. Whole-genome sequencing followed by variant calling analysis primarily identified loss-of-function mutations in the gene encoding hexokinase 1 (Hxk1). Targeted gene deletion mutants further showed that loss of either HXK1 or HXK2 can bypass tps1Δ in a carbon source-dependent manner. The tps1Δ mutant exhibited elevated hexokinase activity, accumulation of G6P and glycogen, and ATP depletion after heat shock. Deletion of HXK1 or HXK2 restored hexokinase activity and partially restored G6P and ATP levels in the tps1Δ mutant, while glycogen remained elevated, indicating that excess glycolytic flux underlies the tps1Δ high-temperature growth defect. Overall, our study uncovers a previously unappreciated mechanism of Tps1-mediated heat adaptation in C. deneoformans, by revealing that Tps1 functions as a critical metabolic gatekeeper that safeguards glycolytic flux to sustain growth at elevated temperatures. Article summary Trehalose is crucial for fungal thermal adaptation and mutants lacking trehalose are inviable at 37°C. This study examined how genetic suppressors restore viability at 37°C in mutants lacking TPS1, which encodes trehalose-6-phosphate synthase. Through whole-genome sequencing of spontaneous suppressor isolates and variant calling analysis, mutations were identified in HXK1. Gene deletion mutants and biochemical assays show these mutations alter glycolytic flux. We show that tps1Δ mutants exhibit unbridled glycolysis, and their growth at 37°C is restored by hxk1Δ mutations that reduce glycolytic flux. This study highlights the interdependence between Tps1 and Hxk1, which may have broader relevance across organisms.
The proteomic basis of thermal adaptation in P. plecoglossicida is revealed and molecular targets for engineering thermotolerant strains to optimize industrial 2-KGA fermentation are provided.
Daming Wang, Yu-Ming Jing, Qing-hong Wang et al.· 3 Biotech· 0 citations
This study uncovers that moderate reactive oxygen species (ROS) signaling mediates aeration-dependent thermotolerance in Kluyveromyces marxianus, challenging the long-held paradigm that ROS function solely as toxic metabolic byproducts. Through integrated transcriptomic profiling and RT-qPCR validation, 19 key transcription factors regulating this adaptive response were identified, and functional assays demonstrated that targeted knockout of GSF2 and RIM101 coupled with MED15 overexpression significantly enhances hypoxic thermotolerance. The ERG6-overexpressing strain YZB559 yields 72.45 g/L xylitol at 45 °C under medium-high oxygen (MHO) condition, marking a 20% improvement over the parental strain, while the combinatorially engineered YZB639 (ΔGSF2::MED15) achieves 74.13 g/L xylitol with complete xylose consumption at 46 °C and 30.64 g/L ethanol under micro-oxygen conditions, representing a 23% increase in ethanol production. Notably, at 47 °C, the highest temperature reported for xylitol fermentation, YZB639 accumulates 51.68 g/L xylitol under constant MHO condition, and an optimized two-stage oxygen supply strategy further elevates the titer to 67.78 g/L, with robust performance also observed when using industrial xylose mother liquor as feedstock to produce 57.34 g/L xylitol. This work resolves the fundamental oxygen contradiction between thermotolerance enhancement and product biosynthesis in high-temperature fermentation, providing a transformative strategy for cost-effective and sustainable industrial biomanufacturing.
Zhongmei Hu, Yanjie Li, Na Dong et al.· Bioresource Technology· 0 citations
ABSTRACT Thermotolerance is fundamental to fungal ecology and survival. Although heat stress triggers extensive metabolic reprogramming, the function of these changes for thermotolerance has remained poorly understood. Here, we identify glutamine synthetase (GS), a central nitrogen metabolism enzyme, as a critical determinant of thermotolerance in Ganoderma lucidum. Silencing of gs significantly enhanced fungal tolerance under heat stress and reduced the relative inhibition rate of mycelial growth to 9.71%, compared with 20.7% in the wild-type (WT) strain. Heat stress also increased reactive oxygen species and H₂O₂ levels by 1.49- and 1.38-fold in the WT strain, whereas the increments were markedly lower in gs-silenced strains. Subsequently, under heat stress, α-ketoglutarate contents in WT increased by 1.49-fold. Inhibition of GS further increased the accumulation, which was achieved by upregulating glutamate dehydrogenase to promote the conversion of glutamate to α-ketoglutarate. This metabolic response was correlated with the generation of adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide (NADH). Moreover, under heat stress, the level of GS protein in WT strains decreased by 29.0%, compared with that under normal conditions, due to accelerated degradation via the 26S proteasome. Our findings reveal that a ubiquitin-dependent signal instantaneously rebalances central carbon-nitrogen metabolism, offering a direct and rapid link between central metabolism and thermotolerance in fungi. IMPORTANCE Understanding how organisms adapt to heat stress is of increasing urgency in the context of global warming. While the roles of heat-shock proteins and antioxidant systems are well established, how microbes actively reprogram central metabolism to survive thermal challenge remains a fundamental, unanswered question. This study reveals that the central nitrogen metabolism enzyme glutamine synthetase (GS) is degraded by the ubiquitin-proteasome system and that this degradation acts as a metabolic switch to enhance thermotolerance in Ganoderma lucidum. We discovered that heat stress induces ubiquitin-proteasome system-dependent GS degradation, leading to redirected central nitrogen flux that elevates α-ketoglutarate content. This metabolic shift boosts ATP and NADH production. In summary, our findings represent a significant advance beyond classical protein chaperone systems and reactive oxygen species-scavenging systems, highlighting a direct and rapid link between metabolic flux and thermotolerance. Understanding how organisms adapt to heat stress is of increasing urgency in the context of global warming. While the roles of heat-shock proteins and antioxidant systems are well established, how microbes actively reprogram central metabolism to survive thermal challenge remains a fundamental, unanswered question. This study reveals that the central nitrogen metabolism enzyme glutamine synthetase (GS) is degraded by the ubiquitin-proteasome system and that this degradation acts as a metabolic switch to enhance thermotolerance in Ganoderma lucidum. We discovered that heat stress induces ubiquitin-proteasome system-dependent GS degradation, leading to redirected central nitrogen flux that elevates α-ketoglutarate content. This metabolic shift boosts ATP and NADH production. In summary, our findings represent a significant advance beyond classical protein chaperone systems and reactive oxygen species-scavenging systems, highlighting a direct and rapid link between metabolic flux and thermotolerance.
Jinjin Qiao, Huajun Li, Yuzhen Yang et al.· Applied and Environmental Mi...· 0 citations
In the model bacterium Escherichia coli, the Embden-Meyerhof-Parnas (EMP) glycolysis pathway is the primary route for carbohydrate metabolism. However, alternative metabolic routes can be activated depending on genetic configurations and available carbon sources. In this study, E. coli strains lacking key enzymes of the lower EMP pathway-phosphoglycerate kinase or enolase-were subjected to long-term adaptive evolution in continuous culture under a medium swap regime, for growth on D-xylose as the sole carbon and energy source. Through metabolic 13C-labeling experiments on evolved isolates, we found that carbon flux was predominantly rerouted via the Entner-Doudoroff pathway, known to remain silent when wild type cells are fed with D-xylose. To investigate the molecular basis of this growth adaptation, we identified the mutations fixed in the genomes of evolved prototrophic isolates and conducted comprehensive transcriptomic and proteomic analyses. Our study revealed that mutations in key enzymes and transcriptional regulators at various metabolic branching points were essential for effective growth on D-xylose, enabling optimized partitioning of carbon through central metabolism despite the loss of the canonical EMP glycolytic route. The multi-layered regulatory and metabolic adaptations identified in the evolved strains demonstrate the complex nature of evolutionary trajectories and underscore the potential of adaptive evolution to optimize metabolic network function and enzyme utilization in ways that extend rational engineering approaches.
C. Iacometti, Valérie A. Delmas, Mélodie Cadillon et al.· Metabolic Engineering· 0 citations
BACKGROUND
Prokaryotic isocitrate dehydrogenase (PIDH) is a conserved and highly versatile enzyme regulating carbon flux between the TCA cycle and the glyoxylate shunt. While most mesophilic organisms encode a single PIDH, the industrial chassis Pseudomonas putida KT2440 harbours two isoforms: a monomeric (IDH) and a dimeric (ICD). The physiological significance of this dual-system in mesophiles remains poorly understood, yet it may provide a framework for understanding metabolic flexibility and flux regulation in biotechnological applications.
RESULTS
Here, we biochemically characterized both PIDHs and, through modelling and site-directed mutagenesis, identified key residues (Ser133, Asn136, and Arg140) critical for monomeric catalytic activity and substrate binding. Genetic analysis revealed that the monomeric IDH is essential for growth, whereas the dimeric ICD appears to facilitate high-flux oxidative metabolism. System-level analysis integrating transcriptomics and flux modelling further demonstrated that these isoforms might fuel distinct TCA functional modes. Our results suggest that IDH sustains a basal, carbon-independent mode critical for biosynthetic precursor supply. In contrast, ICD drives a high-flux oxidative mode under glycolytic conditions and is post-translationally inactivated under gluconeogenic conditions, favouring the glyoxylate cycle.
CONCLUSIONS
Taken together, these findings support a model in which specialized isoenzymes with conditional redundancy, rather than simple redundancy, in P. putida. This dual-enzyme system enables the cell to balance energy production with biosynthetic demands, which might facilitate metabolic adaptation under fluctuating environmental conditions. Our results provide a potential modular framework for engineering central metabolism to optimize precursor provisioning in microbial cell factories.
M. tuberculosis, an intracellular pathogen, survives within the membrane-bound vacuole, the phagosome with acidic pH and limited access to nutrients. To survive and replicate within human host, M. tuberculosis must adapt and fulfil its nutritional requirements. To investigate how the intracellular bacillus scavenges nutrients from its host, we studied mycobacterial acquisition of host-derived amino acids, the preferred nitrogen sources. We discovered that PhoP, a key determinant of mycobacterial adaptation to phagosomal acidification, controls expression of AnsP1 and AnsP2 to facilitate acquisition of host aspartate and asparagine, respectively. Thus, macrophage-infected WT-H37Rv showed a significantly higher level of intra-bacterial Asn compared to the phoPR-KO mutant and a complemented mutant could restore Asn level to that of WT-H37Rv. Under acidic conditions, elevated DNA binding of PhoP within the promoters lead to direct activation of ansP1 and ansP2. Consistently, phoPR-KO is unable to utilize Asn under acidic condition, and over-expression of ansP1 or ansP2 in the mutant restored intracellular growth defect of the mutant. These findings uncover the regulatory network allowing utilization of organic nitrogen sources by the pathogen during infection.
Bhanwar Bamniya, Kajal, Khushboo Mehta et al.· Journal of Biological Chemis...· 0 citations