This 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.
Abstract
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.
Formate is a promising, sustainable feedstock for microbial conversion into value-added products via the Wood–Ljungdahl pathway. Based on the identification of three distinct formate dehydrogenases in
Clostridium
sp. AWRP, this study investigated the potential of this acetogen for formate-based growth. Although the wild-type strain exhibited an extended lag phase due to formate toxicity, adaptive laboratory evolution yielded the adapted strain F30T, which demonstrated a significantly enhanced growth rate and tolerance to formate concentrations up to 300 mM. Whole-genome and transcriptomic analyses of the F30T strain revealed a sophisticated, multi-layered strategy for mitigating organic acid stress and optimizing energy conservation. Adaptive laboratory evolution resulted in six key non-synonymous mutations, including those in
ackA
and
adhE1
, and triggered the differential expression of 948 genes out of 4199 genes under the formate-supplemented condition. Specifically, F30T achieved intracellular pH buffering and supplemental ATP generation by upregulating the arginine deiminase pathway, histidine biosynthesis, and ethanolamine utilization. To maintain growth near the thermodynamic limit, the strain implemented a stringent energy-saving program by downregulating the F₀F₁-type ATP synthase and the methyl-branch of the Wood–Ljungdahl pathway, while simultaneously enhancing stress resilience through molecular chaperone upregulation. In a pH-stat fed-batch system using formic acid, the adapted F30T strain showed a higher biomass yield while achieving a similar acetate yield from formate as the wild-type strain. This study demonstrated that adaptive laboratory evolution is a highly effective strategy for enhancing the formate utilization and stress tolerance of
Clostridium
sp. AWRP. Through integrated genomic and transcriptomic analyses, the molecular basis of the F30T strain’s improved performance was elucidated, identifying key mutations and coordinated metabolic and regulatory mechanisms that maintain cellular homeostasis and maximize energy efficiency. These findings provide valuable molecular insights to aid the targeted design and optimization of formate-utilizing acetogens for use in microbial cell factories aimed at sustainable C1-based bioproduction.
Soo Jae Kwon, Seongeun Yang, Sung-Mok Lee et al.· Biotechnology for Biofuels a...· 0 citations
Acetate is an attractive renewable two-carbon substrate for microbial biotechnology, but its toxicity limits growth and carbon-use efficiency at process-relevant concentrations. Here, we used adaptive laboratory evolution to improve acetate tolerance in a genome-reduced strain of Pseudomonas putida and combined whole-genome sequencing, reverse engineering, transcriptomics, proteomics, and 13C-acetate fluxomics to resolve the underlying adaptation mechanisms. Evolution under increasing acetate concentrations selected recurrent mutations in gacA and fabB, which encode a global response regulator and a fatty acid biosynthesis enzyme, respectively. Reverse engineering of these mutations recovered most of the evolved phenotype, including shorter lag phase and substantially higher biomass yield from acetate. Multi-omic analyses showed repression of type VI secretion systems, carbohydrate storage functions, fatty acid metabolism, and oxidative stress-associated proteins, indicating resource reallocation away from costly stress and non-essential programs. Fluxomics further revealed reduced EDEMP cycling and increased glyoxylate shunt flux, consistent with improved acetate-carbon retention in biomass. These results establish acetate tolerance in P. putida as a resource-efficiency phenotype and identify gacA and fabB as actionable targets for acetate-based bioproduction.
N. Gurdo, A. Srinivasan, Tommaso Tagliani et al.· bioRxiv· 0 citations
O-Succinyl-L-homoserine (OSH) plays a pivotal role in L-methionine biosynthesis. Microbial cell factories for high-yield OSH production have been progressively optimized, achieving substantial improvements in fermentation titers. In this study, a multi-step progressive optimization strategy was adopted to construct a high-yield OSH-producing strain. First, the feedback inhibition of the key enzyme HST was relieved, and the key genes involved in the byproduct metabolic pathways were knocked out. This modification enabled engineered strain to produce 9.77 ± 0.27g/L OSH in shake-flask fermentation. Second, ribosome binding site (RBS) engineering, promoter engineering, and dynamic metabolic regulation were integrated to strengthen and balance the intracellular supply of the two core precursors, L-homoserine and succinyl-CoA. These strategies greatly increased the OSH titer to 18.54 ± 0.03g/L. Finally, global optimization of cofactor and energy optimization was carried out to further enhance strain performance, and the engineered strain OSHM40 achieved the OSH titer of 20.15 ± 0.21g/L via shake-flask cultivation, and 104.09 ± 2.06g/L in a 5-L bioreactor under fed-batch fermentation, with a sugar-acid conversion rate of 64.99% and a volumetric productivity of 1.43g/L/h. Notably, the OSH titer and sugar-acid conversion rate of this strain represent the highest levels reported to date among all plasmid-free OSH-producing strains. The plasmid-free system constructed in this study effectively avoids the plasmid-induced metabolic burden and genetic instability. This work demonstrates the prominent advantages and great application potential of plasmid-free modular engineering for the efficient biosynthesis of OSH and other high-value amino acids.
Si-Min Huang, Xu-Yue He, Ruo-Nan Wang et al.· Journal of Biotechnology· 0 citations
This review addresses current knowledge on the regulatory networks controlling glycerol metabolism in yeasts, compassing transport mechanisms, metabolic pathways, transcriptional control and enzyme regulation, and pointed out promising avenues for future research and biotechnological innovation.
Juliana Silva Carneiro Fonseca, W. B. da Silveira· World Journal of Microbiolog...· 0 citations
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.
V. Yadav, Kahlia A. Carl, J. Heitman et al.· bioRxiv· 0 citations
Efficient co-utilization of hexose and pentose sugars from lignocellulose is essential for microbial bioconversion, yet engineered catabolic pathways can be unstable or suboptimal in complex resource environments. Here, we use a Pseudomonas putida strain engineered to catabolize xylose and arabinose to examine how resource abundance, temporal availability, and subculturing shape evolutionary outcomes. Using an automated adaptive laboratory evolution (ALE) platform, we evolve the strain under simple single-substrate and complex multi-substrate selection pressures. These environments drive divergence between catabolic specialists and generalists. Weak or absent selection for xylose frequently leads to loss of xylose catabolism, whereas carbon-limited mixed-sugar environments promote stable retention and coordinated optimization of multiple catabolic pathways, enhancing growth and substrate utilization. Genomic, proteomic, and biochemical analyses show that pathway-specific fitness costs determine evolutionary stability. A generalist clone also shows improved indigoidine production from mixed sugars relative to the parental strain. Together, these findings show how resource dynamics shape fitness landscapes that govern catabolic specialization, generalization, evolutionary trade-offs, and engineering of bioconversion. Efficient co-utilization of sugars from lignocellulose is essential for microbial bioconversion. Here the authors perform laboratory evolution of P. putida to reveal how selection shapes retention or loss of catabolic pathways, offering design rules for biomanufacturing phenotypes.
Sunghwa Woo, H. Lim, B. Norton-Baker et al.· Nature Communications· 0 citations
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