Overcoming protocatechuate and catechol accumulation in muconic acid production via adaptive laboratory evolution and metabolic engineering in Pseudomonas putida
Results indicate that catechol, not PCA, is the principal bottleneck in muconate production via the PCA decarboxylation route originally demonstrated by Draths et al., refining the understanding of pathway limitations and offering new strategies for improving rate, yield, and strain resilience in muconate bioproduction.
Abstract
Muconic acid is an industrially valuable molecule that can be biologically produced from diverse biogenic and waste-derived feedstocks, including sugars and lignin- and plastic-derived aromatic compounds. However, accumulation of protocatechuate (PCA) has been observed in multiple microbes engineered for muconate production when the PCA decarboxylase, AroY, is used. This raises the question of whether PCA decarboxylation represents a rate-limiting step and how this bottleneck might be alleviated, especially given the toxicity and reactivity of PCA and catechol intermediates. To address this, we performed adaptive laboratory evolution (ALE) on a strain of Pseudomonas putida originally engineered for muconate production from aromatic compounds, but with catBC restored, to select for improved conversion of PCA and, in separate lineages, 4-hydroxybenzoate. Contrary to our expectations, the predominant beneficial mutations localized to the catA1 cassette encoding catechol 1,2-dioxygenase, rather than aroY or its associated cofactor biosynthesis genes. Transcriptomic analysis revealed elevated catA1 expression in evolved isolates from ALE, and introduction of these mutations improved productivity in strains designed for muconate production from both aromatic and sugar substrates. Quantitative proteomics and biochemical assays demonstrated that the mutations also led to increased CatA1 protein abundance and modest enhancements in catalytic efficiency, respectively, with strain phenotypes largely driven by high CatA1 levels and potentially synergistic kinetic improvements. Additional reverse-engineering studies identified variants with modest effects on muconate accumulation, including those with potential to enhance biosynthesis of the prenylated FMN cofactor of AroY. Collectively, these results indicate that catechol, not PCA, is the principal bottleneck in muconate production via the PCA decarboxylation route originally demonstrated by Draths et al., refining our understanding of pathway limitations and offering new strategies for improving rate, yield, and strain resilience in muconate bioproduction. Highlights Accumulation of metabolic intermediates was alleviated by adaptive laboratory evolution Sequencing, proteomics, and enzyme kinetics revealed mechanisms for adaptation Increased CatA1 expression reduced bottlenecks and improved muconate production
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
Lignin has potential as a sustainable feedstock to replace fossil fuels in chemical manufacturing. The coupling of chemical fractionation and biocatalysis has emerged as a promising technology to realize this potential. In this process, chemocatalytic fractionation of biomass or lignin yields heterogeneous mixtures of lignin-derived aromatic compounds (LDACs), which are subsequently funneled to target chemicals by microbial cell factories. The recent expansion of genetic toolkits for non-model bacteria offers burgeoning possibilities for engineering bespoke biocatalysts using natural LDAC degraders such as Rhodococcus aromaticivorans RHA1. Herein, we describe the development of an RHA1 biocatalyst to convert a softwood kraft lignin stream containing vanillin, vanillate and acetovanillone to muconic acid by leveraging Serine integrase-Assisted Genome Engineering (SAGE). We increased vanillin metabolism by co-expressing ligV, encoding a vanillin dehydrogenase, and RHA1's endogenous vanACB, encoding a vanillate O-demethylase, partially overcoming the vanillate bottleneck observed when expressing ligV alone. To funnel aromatics to muconic acid, we tested two aromatic acid decarboxylases, finding that AroY with EcdBD efficiently decarboxylated protocatechuate to catechol. We then integrated the Hpe pathway of Rhodococcus rhodochrous GD02 to enable acetovanillone conversion. Finally, deletion of catB enabled muconic acid accumulation. Our biocatalyst, strain RHAAL14, transformed the LDACs derived from the oxidation of softwood kraft lignin to muconic acid with a 97% molar yield and a titer of 1.4 g/L. The iterative, integrated metabolic engineering strategies described in this work advance the development of rhodococcal strains for microbial cell factories.
Anne T. Lalande, Logan D. Robeck, Dillon T. Hofsommer et al.· Metabolic Engineering· 1 citation
Caffeic acid (CA) is a valuable phenylpropanoid with applications in food, pharmaceutical, and chemical industries. Microbial production of CA is often limited by the terminal hydroxylation catalyzed by 4-hydroxyphenylacetate 3-monooxygenase (HpaBC). Here, we constructed a de novo CA biosynthetic pathway in Escherichia coli and enhanced production through systematic metabolic and spatial engineering. Optimization of l-tyrosine supply and HpaBC expression increased CA production to 61.0 mg/L. However, further enhancement of glucose uptake and precursor supply was insufficient, indicating that hydroxylation remained a major limitation. To address this, HpaBC was relocated to the periplasm via the Tat pathway, increasing CA production 4.9-fold to 299.1 mg/L with reduced byproduct formation. Further lpp+14 mediated periplasmic remodeling and fermentation optimization increased shake-flask production to 463.7 mg/L. Finally, fed-batch fermentation achieved 5.1 g/L CA in a 3 L bioreactor. This study highlights periplasmic engineering as an effective strategy for improving oxidation-dependent phenylpropanoid biosynthesis.
Shangyi Wang, Yuqi Zhuo, Jamila A Tuly et al.· Journal of Agricultural and...· 0 citations
Synthetic consortia is an emerging field in biotechnology, offering advantages such as division of labor between the individual members and reduced risk of contamination. Consortia combining phototrophic and heterotrophic bacteria are attractive, as phototrophs can utilize CO₂ and light energy to sustain growth. Several studies have explored such systems where sucrose is most commonly used as the provided carbon source. An alternative is acetate, a smaller two-carbon molecule produced as a by-product by many microorganisms, including the model cyanobacterium Synechocystis PCC 6803 (thereafter Synechocystis). Although acetate production during phototrophic growth is typically low, previous studies have demonstrated that metabolic engineering—specifically the introduction of phosphoketolase (PK) and overexpression of phosphotransacetylase (Pta)—enabled the development of a high-producing strain (WT_PKPa_RBS_BsPta_Δacs) capable of secreting significant levels of acetate into the medium (Roussou and Lindblad 2026). In this study, Escherichia coli and Pseudomonas taiwanensis were engineered to produce 1-butanol, an industrially relevant bulk chemical, and cultivated using acetate as the sole carbon source. These strains were then individually co-cultivated with the previously engineered Synechocystis strain, WT_PKPa_RBS_BsPta_Δacs, forming two distinct consortia that were maintained for 42 days. Growth dynamics were successfully monitored, and acetate concentrations in the consortia were lower than in a corresponding axenic Synechocystis culture. At the same time, 1-butanol production was detected in the two co-cultures, demonstrating the feasibility of coupling photosynthetically derived acetate to heterotrophic production of a value-added bulk chemical. •Photosynthetic/heterotrophic synthetic consortia for 1-butanol production. • Engineered Synechocystis PCC 6803 cells produce acetate from CO2. • Modified Escherichia coli and Pseudomonas taiwanensis cells grow on acetate and produce 1-butanol.
Stamatina Roussou, Peter Lindblad· Applied Microbiology and Bio...· 0 citations
4-hydroxybenzoate (HBA) is an aromatic component that forms the basis of great number of specialty plastics and fine chemicals. In pursuit of a greener and sustainable production, this article described Pseudomonas sp. NGC7, which has been metabolically engineered for the production of HBA from a mixture of aromatic compounds from sugarcane bagasse fractionated by alkaline treatment. While biomass-derived saccharides will continue to be in use, alternative carbon feedstocks for bioproduction are also desirable. For this reason, our work focused on producing HBA from a complex mixture of aromatics derived from lignocellulosic biomass. Following a mild alkaline treatment of sugarcane bagasse, the extracts principally comprised an aromatic mixture of p-coumarate, ferulate, 4-hydroxybenzaldehyde, HBA, syringaldehyde, syringate, vanillin, and vanillate. An HBA hydroxylase gene (pobA) knockout of NGC7 could produce HBA while utilizing the alkaline extract as a carbon source. The regulator gene (ferR) associated with the degradation pathway of hydroxycinnamate derivatives in NGC7 was identified, and its disruption enhanced the production rate of HBA from the extract. The engineered ⊿pobA⊿ferR strain proliferated and produced >6 g/L HBA with a yield of exceeding 90 mol% efficiently from the alkaline extract in the absence of saccharides. This study proved that "glucose-free HBA production" is attainable through metabolic flux manipulation in NGC7.
Yue-Ling Wang, Zen Ookawa, Yudai Higuchi et al.· Bioresource Technology· 0 citations
Acetate is a promising carbon source for microbial biotechnology as it can be produced sustainably from lignocellulosic biomass or C1 gases. Since acetate is directly activated to acetyl-CoA, it is especially suitable for producing acetyl-CoA-derived products, showcased here with the production of 3-(3-hydroxyalkanoyloxy) alkanoic acids (HAAs). P. putida KT2440 can natively metabolize acetate, but the weak acid has also inhibitory effects on microbial growth. We present an in-depth study on the physiology of P. putida KT2440 using acetate as carbon and energy source and evaluate acetate as feedstock for the biosynthesis of HAAs. Initially, a rational engineering approach to overexpress acetyl-CoA synthetase for acetate activation resulted in an improved growth rate of 16% and reduced lag phase by six hours. To further increase the performance of P. putida KT2440 on acetate, adaptive laboratory evolution was performed. This resulted in an improvement in the growth rate from 0.4 h-1 to 0.6 h-1 and enabled growth on up to 12.5 g L-1 acetate with a shortened lag phase compared to the wild type. Whole-genome sequencing revealed mutations in proteins involved in gene expression regulation and signal transduction. This evolutionary engineering approach informed the deletions of gacS and crc, which resulted in a reduction in the lag phase from seven hours to one hour and an improvement of the growth rate by 25 %, matching the growth properties of the evolved clones. Using the evolved strains for the production of HAAs resulted in faster biomass and product formation with product titers reaching up to 94 % of that of the wild type. In conclusion, we identified mechanisms in the acetate metabolism of P. putida KT2440 and improved the growth performance of the strain by rational and evolutionary engineering, demonstrating the potential of the promising, but challenging 3rd generation feedstock acetate.
M. Filbig, Luisa Wachtendonk, Luca Hampe et al.· bioRxiv· 0 citations
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