Transcription factor YALI1_A16891g contributes to enhanced glycolytic flux and mevalonate production at elevated temperature in engineered Yarrowia lipolytica.
It is demonstrated that temperature can effectively reshape metabolic flux distribution and highlight transcription factor-mediated regulation as a practical target for improving precursor supply in engineered Y. lipolytica.
Abstract
Yarrowia lipolytica (Y. lipolytica) is an attractive host for terpenoid biosynthesis due to its high acetyl-CoA availability and robust genetic tractability. However, how fermentation temperature influences intracellular metabolic states remains insufficiently characterized. Here, the engineered mevalonate-producing strain YL17 was employed as a platform to characterize temperature-dependent metabolic responses in Y. lipolytica. Fermentation experiments across a range of temperatures revealed a clear growth-production trade-off, whereby increasing temperature enhanced mevalonate synthesis at the expense of cellular growth. 13C metabolic flux analysis revealed that higher temperature promoted a significant redistribution of carbon flux toward glycolysis, increasing acetyl-CoA generation and precursor supply. Notably, transcriptomic analysis indicated a global downregulation of genes involved in glycolysis, indicating a decoupling between transcriptional levels and flux regulation. To explore the regulatory basis underlying this phenotype, differentially expressed transcription factors were systematically analyzed, leading to the identification of YALI1_A16891g as a key regulatory candidate associated with glycolytic flux redistribution. Further analysis suggested that YALI1_A16891g may regulate downstream glycolysis-related genes under elevated temperature conditions, thereby influencing glycolytic carbon allocation and mevalonate production specifically at high temperature in Y. lipolytica. These results demonstrate that temperature can effectively reshape metabolic flux distribution and highlight transcription factor-mediated regulation as a practical target for improving precursor supply in engineered Y. lipolytica.
Z. mellis adapts to high-glucose stress by coordinating changes in gene expression associated with respiratory activity, lipid metabolism, and cell division processes, suggesting structural reinforcement under osmotic stress.
T. Tamura· Letters in Applied Microbiol...· 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
Lipomyces is a genus of oleaginous yeasts with potential for contributing to reliable biomanufacturing supply chains. However, progress in advanced strain designs and engineering efforts are still constrained by a lack of understanding of the underlying molecular drivers of Lipomyces phenotypes. To address this gap, we collected a suite of multi-omic data to dissect how carbon source availability reshapes the metabolic network, lipid allocation, and regulatory architecture of Lipomyces starkeyi. We observed that glucose promotes biosynthetic and proliferative processes supported by abundant energy and carbon intermediates, xylose enhances redox-balancing mechanisms centered on the pentose phosphate pathway, and glycerol activates respiratory metabolism, β-oxidation, and the glyoxylate cycle. Lipid species distributions remained consistent in both nitrogen replete and depleted conditions across the carbon sources, indicating robust production mechanisms. Regulatory protein identification and network analysis revealed glycerol-driven respiratory growth favors regulatory programs integrating stress tolerance, redox balance, and lipid-associated metabolism, whereas xylose growth activates compensatory transcriptional responses aimed at maintaining mitochondrial function. Nitrogen limitation modulates the strength of these responses but does not fundamentally alter their direction, reinforcing carbon source as the dominant driver of regulatory architecture. Taken together, this data enhances the understanding of Lipomyces molecular rearrangements and provides a foundation for further development of predictive phenotypic tools in this genus.
L. Monteiro, Xiaolu Li, Kyle R. Pomraning et al.· Scientific Reports· 0 citations
Systematic mining of heat stress factors is pivotal for constructing robust microbial cell factories. However, the genetic basis underlying thermotolerance remains poorly understood in C1 gas-utilizing chassis. Here, we identified LexA as a key regulator governing thermotolerance and glycogen accumulation in the methanotroph Methylotuvimicrobium buryatense 5GB1C. Through ultraviolet mutagenesis coupled with adaptive laboratory evolution, we developed a thermotolerant evolved strain 5C27. Whole-genome resequencing and transcriptomic analysis revealed global transcriptional reprogramming underlying the heat tolerance phenotype. Notably, LexA was sharply downregulated at 40 °C and identified as a core regulatory factor for thermoadaptation. Transcriptomic analysis indicated that the acquired thermotolerance arises from coordinated regulation of oxidative stress response, energy metabolism, and cellular homeostasis. Subsequent validation experiments demonstrated that lexA deficiency simultaneously conferred enhanced thermotolerance and redirected intracellular carbon flux, resulting in increased glycogen production that exceeded wild-type levels even at the optimal growth temperature of 30 °C. Collectively, our work demonstrated for the first time that LexA plays a critical regulatory role in thermotolerance of methanotrophs. This discovery also provides a foundation for advancing the development of thermotolerant cell factories and promotes the deployment of C1-gas industrial biomanufacturing.
Xylitol is a highly functional sweetener with extensive applications. Sustainable biosynthesis from glucose is desirable yet metabolically challenging. Here, we engineered Yarrowia lipolytica as a cell factory by constructing a core biosynthetic route via combinatorial screening and multicopy integration of d-arabitol dehydrogenases (ArDH) and an NADPH-dependent xylitol dehydrogenase (XDH) in the robust chassis NBRC1631. To further drive the metabolic flux and alleviate bottlenecks, we employed a synergistic push-and-pull strategy: overexpressing glucose transporters (YH3 and YH4), while upregulating pentose phosphate pathway enzymes (ZWF1 and GND1) to enhance NADPH regeneration, matching the redox demand of the synthetic cascade. Following two-stage pH-controlled fed-batch fermentation in a 3 L bioreactor, the final engineered strain achieved a record-high xylitol titer of 39.0 g/L with a yield of 0.09 g/g glucose. This study establishes a productive platform for microbial de novo xylitol biosynthesis from glucose, offering a green and economically viable route for industrial production.
Bingbing Liu, Xi Yao, Jianping Lin et al.· Journal of Agricultural and...· 0 citations
Deciphering the metabolic basis of high-yield antibiotic production in Streptomyces is crucial for strain optimization. Atmospheric and room-temperature plasma (ARTP) mutagenesis of Streptomyces xinghaiensis sf106 generated a mutant with a 30% increase in tylosin-equivalent concentration (μg/mL). 4D-FastDIA quantitative proteomics identified 279 differentially abundant proteins enriched in the Type I polyketide synthase (PKS) pathway, with increased abundance of key macrolide-biosynthesis-related proteins. Lysine-acetylome profiling identified 1152 differentially abundant acetylation sites and revealed altered acetylation of enzymes involved in fatty acid metabolism and the tricarboxylic acid (TCA) cycle, suggesting adjustments in central metabolism associated with acyl-CoA precursor availability and energy generation. Integration of proteomic and acetylomic data suggests coordinated changes in protein abundance and lysine acetylation associated with the increased tylosin-equivalent concentration. These results highlight candidate nodes for rational metabolic engineering of S. xinghaiensis.
C. Jia, Cilang Ma, Yuting Jiang et al.· Microbiology Research· 0 citations