This study presents a systematic comparison of sugar catabolic pathways that enabled development of strains suited for the tradeoffs between rate and yield and indicates that these performance metrics can reduce the minimum selling price of muconate-derived adipic acid and greenhouse gas emissions.
Abstract
Engineering heterologous utilization of substrates requires selection of catabolic pathways that balance strain performance and product biosynthesis. Here, we compare the oxidative and isomerase arabinose utilization pathways in Pseudomonas putida strains engineered for cis,cis-muconic acid production from glucose and xylose. Based on the point of entry into central carbon metabolism, we hypothesized that the oxidative arabinose pathway would enable higher productivity while the arabinose isomerase pathway would enable higher muconate yield. In both strains, additional modifications were engineered to improve muconic acid production including sugar transporter tuning, catechol 1,2-dioxygenase overexpression, a feedback-resistant DAHP synthase, and a flux-stabilizing gltA variant. Consistent with our hypothesis, the oxidative arabinose pathway supported faster growth and higher productivity (0.58 g/L/h), whereas the arabinose isomerase pathway improved carbon efficiency, achieving muconate yields of up to 50 C-mol% in fed-batch bioreactors. Process modeling indicates that these performance metrics can reduce the minimum selling price of muconate-derived adipic acid to $2.74/kg and greenhouse gas emissions to 1.31 kg CO2e/kg, approaching cost parity and reducing emissions by 86% relative to fossil carbon-derived adipic acid. Overall, this study presents a systematic comparison of sugar catabolic pathways that enabled development of strains suited for the tradeoffs between rate and yield.
l-arabinose, a valuable C5 pentose sugar in xylose mother liquor (a low-cost cellulose hydrolysis byproduct), remains underutilized due to costly separation requirements, resulting in significant waste of fermentable carbon resources. In this study, Escherichia coli W3110 was systematically engineered to efficiently convert l-arabinose into xylitol, a low-calorie sweetener with significant commercial value. Firstly, the l-arabinose metabolic network was reconstructed and glucose catabolic repression was alleviated through coordinated pathway modifications, enabling simultaneous utilization of both l-arabinose and glucose. Subsequently, a xylitol synthesis module consisting of l-arabinose isomerase (AraA), l-xylulose reductase (LXR), and d-psicose-3-epimerase (DPE) was systematically optimized via gene arrangement, promoter and RBS engineering. The optimized pathway was integrated into the E. coli W3110 genome at the IS5 locus using MUCICAT technology, generating a plasmid-free production strain and reducing plasmid-segregation concerns. Fed-batch fermentation in a 3 L bioreactor yielded 64.07 g/L xylitol at a productivity of 1.46 g/L/h with 90.77% l-arabinose conversion in 44 h. This achievement overcomes two critical metabolic bottlenecks: (1) glucose catabolite repression, which normally prevents pentose utilization in the presence of glucose, and (2) the successful stoichiometric balancing of three enzymatic steps (AraA, LXR, DPE). The engineered strain achieves simultaneous glucose-arabinose co-metabolism, and glucose co-utilization supports xylitol formation in a manner consistent with an endogenous reducing-power contribution, thereby eliminating the requirement for exogenous glycerol supplementation in the optimized process. This work establishes a defined-substrate engineering platform for l-arabinose-to-xylitol conversion and provides a strategic basis for future evaluation using complex industrial carbohydrate streams.
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
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
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
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
Vanillin is an important flavor compound widely used in the food, fragrance, and pharmaceutical industries. Current biotransformation processes from ferulic acid or eugenol are limited by high substrate cost and low carbon efficiency, motivating de novo biosynthesis from glucose. This study employed Escherichia coli as the chassis organism to establish a modular vanillin biosynthesis system based on the phenylpropanoid metabolic pathway. Heterologous expression of sam8, sam5, and comt established a biosynthetic module for the sequential conversion of l-tyrosine to p-coumaric acid, then to caffeic acid, and finally to ferulic acid. This module enabled the production of 15.86 mg/L ferulic acid from glucose. Two ferulic acid-to-vanillin modules were compared: a CoA-dependent deacetylation pathway (fcs/ech) and an oxidative decarboxylation pathway (fdc/cso2). With ferulic acid feeding, the deacetylation route produced 445.78 mg/L vanillin, far exceeding the 3.49 mg/L obtained via oxidative decarboxylation. When integrated with the upstream module, the deacetylation pathway enabled de novo vanillin production from glucose at 4.46 mg/L, whereas the oxidative decarboxylation route yielded only 0.46 mg/L, indicating better performance of the former under the tested conditions. Metabolite profiling indicated accumulation of caffeic acid and limited ferulic acid levels, identifying O-methylation and S-adenosyl-L-methionine (SAM) supply as major bottlenecks. Implementation of SAM regeneration modules revealed that mtn overexpression enhanced the vanillin titer by about 3-fold, to 12.36 mg/L, while luxS overexpression had a negligible effect. In summary, this study establishes a functional de novo phenylpropanoid pathway for vanillin in E. coli, underscores the critical role of terminal‑pathway selection, and demonstrates that SAM regeneration effectively improves vanillin production from glucose.
Yue Wang, Tian-Jie Han, Yan-Xiang Bao et al.· Biotechnology and applied bi...· 0 citations
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