Metabolic rewiring overcomes physiological constraints in Sphingobium lignivorans SYK-6 for valorization of industrial lignin streams.

Engineered Pseudomonas putida reconfigures metabolic fluxes to support energy demands during muconate bioproduction from lignin-related aromatics

Muconic acid is a versatile platform chemical that can be biologically produced from lignocellulosic substrates, including from lignin-related aromatic compounds. Pseudomonas putida has been previously engineered to convert lignin-related aromatic compounds to muconate at quantitative molar yields. This high atom efficiency requires a supplemental carbon and energy source to support bacterial growth, and central carbon metabolic efficiency and its interaction with aromatic catabolism are underexplored. Here, we applied proteomics, metabolomics, and 13C-fluxomics to quantitatively compare central carbon and energy metabolism in wild-type P. putida KT2440 and a muconate-producing strain, P. putida CJ781. During cultivation on glucose and 4-hydroxybenzoate, CJ781 showed increased glucose uptake, reconfigured central fluxes, and increased extracellular leakage of aliphatic acids relative to wild type. These altered fluxes supported a 3-fold higher ATP pool, in excess of demand. Pyruvate and acetate secretion in CJ781 was mitigated by debottlenecking TCA-cycle entry via citrate synthase overexpression. Furthermore, tuned expression of the catechol dioxygenase and protocatechuate decarboxylase enabled the production of 36.3 g L-1 muconate at 1.1 g L-1 h-1. Overall, this work reveals how P. putida redirects carbon and energy fluxes to support aromatic bioconversion for improved bioproduction from renewable feedstocks.

Overcoming protocatechuate and catechol accumulation in muconic acid production via adaptive laboratory evolution and metabolic engineering in Pseudomonas putida

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

Metabolic engineering of Rhodococcus aromaticivorans RHA1 for muconic acid production from lignin.

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.

Metabolic engineering strategies of Corynebacterium glutamicum for sulfur-containing amino acid production: current progress and future directions

Substrate-informed metabolic engineering enables a genome-encoded seven-sugar chassis of Corynebacterium glutamicum for spent sulfite liquor valorization.

Efficient microbial conversion of heterogeneous carbohydrate mixtures remains a central bottleneck in industrial biotechnology, yet scalable chassis architectures capable of coordinated multi-sugar utilization are limited. Recently, substrate-informed metabolic alignment enabled balanced glucose-xylose co-utilization in Corynebacterium glutamicum. Here, we examine whether this alignment principle is scalable and transferable to complex industrial feedstocks. We progressively expanded the substrate spectrum of C. glutamicum through genome-encoded integration of mannose, xylose, arabinose, galactose, and rhamnose utilization modules, establishing a stable seven-sugar chassis. Rather than relying on adaptive evolution or high-copy expression, pathway capacities were quantitatively aligned with native uptake and central metabolism. Progressive expansion increased total volumetric carbon uptake and attenuated substrate hierarchy without compromising transcriptional stability. Complementary 13C tracer experiments showed that carbon from all seven sugars was incorporated into biomass in proportion to substrate utilization while revealing characteristic precursor-level incorporation patterns consistent with their distinct metabolic entry routes. Using authentic spent sulfite liquor (SSL) as a chemically heterogeneous validation substrate, the engineered strain achieved near-complete depletion of all fermentable sugars and substantially accelerated conversion kinetics compared to the wild type. Transfer of the aligned substrate module into a glucose-optimized glutarate production strain enabled sustained fed-batch conversion of SSL to 30 g L-1 glutarate without pathway erosion or performance decline. Together, these results demonstrate that substrate-informed metabolic engineering can provide a scalable and industrially transferable strategy for chassis development and position C. glutamicum as a robust multi-substrate platform for heterogeneous renewable feedstocks.

Simultaneous optimization of lignocellulosic sugar catabolism via systematic laboratory evolution under complex selection pressure

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.