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.
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
Carbon catabolite repression (CCR) is a widespread regulatory strategy across diverse microorganisms that prioritises the utilisation of preferred carbon sources. In industrial bioprocesses, however, CCR-mediated substrate hierarchy can delay the utilisation of secondary carbon sources in mixed feedstock, thereby extending fermentation time and limiting carbon conversion efficiency toward target products. Existing reviews have largely addressed CCR in a microorganism-specific manner, potentially obscuring conserved principles, limiting cross-species comparisons, and constraining generalisable engineering; computational modelling and engineering applications also remain underrepresented. This review therefore first summarises CCR mechanisms across diverse microorganisms through a common systems-level framework, in which transport-linked sensing converts carbon flux into intracellular signals, transcriptional regulators reprogramme genome-wide expression, RNA-based mechanisms fine-tune the timing and magnitude of responses, and protein stability shapes their persistence. Multi-omics approaches, particularly interactomics and single-cell omics, are then highlighted for revealing CCR as a dynamic and multilayered regulatory system by resolving interaction networks, temporal changes, and cell-to-cell heterogeneity beyond bulk measurements. Quantitative computational modelling is also discussed, with emphasis on kinetic and constraint-based frameworks that connect regulatory mechanisms to metabolic flux and predict system behaviour under changing carbon substrates. Engineering strategies, including adaptive laboratory evolution, transcription factor engineering, transporter and promoter engineering, and pathway rewiring, are further compared for the relief of CCR and improvement of substrate co-utilisation and production performance. Finally, these advances are presented within a Design-Build-Test-Learn framework, through which mechanistic discovery and omics-scale analysis are used to inform model development, and modelling and engineering are iteratively coupled to improve microbial performance. We propose that such an integrative paradigm is emerging as a unifying direction for CCR research, and provides a foundation for rational strain design and more efficient, sustainable biomanufacturing.
Meng-Tian Lu, Hao-Ran Ding, Hong-Yu Zhang et al.· Synthetic and Systems Biotec...· 0 citations
The acid-tolerant yeast
Issatchenkia orientalis
is a promising platform for the sustainable production of organic acids. However, the inefficient conversion of lignocellulosic biomass-derived sugars, primarily due to carbon catabolite repression (CCR), reduces overall production efficiency and limits its industrial application. In this study, we established a targeted genetic framework for efficient glucose–xylose co-utilization by coordinating hexokinase (HXK) modulation and transport-level engineering. Sequential fed-batch fermentations revealed that a xylose-initiated feeding strategy achieved a 2.04-fold higher lactic acid yield than simultaneous fermentation. To bypass carbon catabolite repression, endogenous hexokinases were characterized, and single deletions (
hxk
1Δ,
hxk
2Δ, or
hxk
3Δ) were conducted to attenuate glucose dominance. While this approach improved lactic acid yields, it simultaneously imposed severe kinetic bottlenecks. To address these limitations, heterologous sugar transporters, plant-derived
AtSWEET7
and yeast-derived
LST1
, were integrated. Characterization in the intact background revealed that the Major Facilitator Superfamily (MFS)-type
LST1
from
Lipomyces starkeyi
outperformed
AtSWEET7
. Double-copy integration of
LST1
yielded the engineered SD108XL-LST2 strain, which achieved a lactic acid titer of 53.4 g/L within 56 h from a mixed-sugar substrate containing approximately 45 g/L glucose and 44 g/L xylose. Notably, the final yield (0.63 g/g) and volumetric productivity (0.96 g/L·h) represented 57.5% and 47.7% increases over the parental SD108XL strain, respectively. This transport-driven strategy effectively overrides native metabolic hierarchies while preserving essential glycolytic signaling, offering a robust framework for high-efficiency lignocellulosic biorefineries for organic acid production.
Ye-Jin Lin, Ayoung Kim, Daeun Lee et al.· Frontiers in Microbiology· 0 citations
Anaerobic gut fungi (AGF) are key degraders of plant biomass in ruminants, yet there is limited knowledge of how AGF respond to mixtures of plant-derived sugars. Here, we assessed monosaccharide and disaccharide utilisation by Neocallimastix frontalis CoB3, Caecomyces communis SHB, and Piromyces edwardsiae SHC, which are abundant in the rumen microbiome. While all AGF isolates shared a core set of sugars that supported growth, they had different hierarchies of uptake. Co-substrate experiments using glucose and lignocellulose-derived sugars revealed species-specific responses, with N. frontalis displaying a novel concentration-dependent co-utilisation of glucose and mannose, whereas growth of P. edwardsiae was inhibited under the same conditions, and C. communis exhibited growth inhibition in glucose and xylose co-substrate cultures. Together, these findings demonstrate functional diversity in monosaccharide and disaccharide metabolism amongst the AGF investigated here. Understanding such sugar utilisation phenotypes provides a foundation for evaluating AGF isolate suitability for lignocellulosic biomass valorisation.
J. L. Matthews, Hannah Haupt, Stephen C. Fry et al.· bioRxiv· 0 citations
Molasses is an abundant and low-cost fermentation feedstock, but its direct utilization is often constrained by its complex composition and inhibitory components. In this study, a molasses-utilizing yeast strain, M0, was isolated from tea rhizosphere soil and identified as Pichia ethanolica. M0 grew directly in untreated molasses diluted only with water, and adaptive laboratory evolution (ALE) was performed under stepwise increasing molasses concentrations. After 70 transfers, the evolved population reached an OD560 of 26.41, representing a 73.1% increase relative to the parental strain, and a superior isolate was designated M70. M70 showed lower residual sucrose after 48 h in 100 g/L molasses medium (16.02 vs. 21.11 g/L) and improved growth under high-molasses, -temperature, and -NaCl conditions, with additional growth advantages under selected ethanol and oxidative-stress conditions. Genome resequencing identified five small candidate variants and one interchromosomal duplicative insertion, including alterations involving RIB1, SOL3, and a CET1-like/REX2-like region. Transcriptomic and metabolomic analyses identified 179 differentially expressed genes and 169 differentially accumulated metabolites. Integrated analysis highlighted coordinated changes in D-amino acid, arginine and proline, beta-alanine, and glyoxylate and dicarboxylate metabolism. These findings indicate multilevel genomic, transcriptional, and metabolic remodeling associated with improved molasses adaptation in P. ethanolica.
Peng Yang, Yu-Tian Fan, Bing-Qi Hu et al.· Microorganisms· 0 citations
Branched-chain amino acids (BCAAs), comprising L-valine, L-leucine, and L-isoleucine, are essential amino acids with extensive applications in food, feed, pharmaceuticals, and cosmetics. Escherichia coli and Corynebacterium glutamicum, the two predominant industrial workhorses have been extensively engineered for high-level BCAAs biosynthesis. This review presents a comprehensive analysis of recent advances in microbial BCAAs production through a Mechanism-Module-Process framework. From the mechanism dimension, the intricate BCAA biosynthetic architectures are delineated, encompassing allosteric feedback inhibition, transcriptional attenuation, and transport system. From the module dimension, modular metabolic engineering strategies are dissected, including precursor supply enhancement, NADPH cofactor rebalancing, biosensor-driven dynamic regulation, and adaptive laboratory evolution for strain robustness. From the process dimension, how oxygen availability reprograms cellular metabolism across aerobic, microaerobic, and anaerobic fermentation is systematically examined, and how two-stage fermentation coupled with cofactor rebalancing resolves the growth-production trade-off to achieve near-theoretical yields is discussed. Furthermore, the biosynthetic strategies and current production status of BCAAs derivatives are summarized, highlighting the importance of multidimensional framework engineering. Collectively, this Mechanism-Module-Process framework provides a holistic roadmap for constructing superior BCAAs cell factories and extending their metabolic potential toward derivative biosynthesis.
Guihong Zhao, Si-Yu Tian, Jiong-Ran Li et al.· Biotechnology Advances· 0 citations
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