Metabolic Engineering

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.

Extracellular vesicles (EVs) are mediators of intercellular communication, yet their impact on Chinese Hamster Ovary (CHO) cell physiology and bioprocess performance remains poorly understood. Here, we investigated whether small EVs (sEVs) and large EVs (LgEVs) that accumulate during fed-batch and perfusion cultures modulate CHO cell growth, metabolism, apoptosis, and monoclonal antibody (mAb) production. EVs isolated from early- and late-stage cultures were added to fresh CHO cultures grown with or without glutamine supplementation. Only LgEVs had a significant impact. Late-stage LgEVs markedly altered CHO-cell behavior, reducing cell proliferation, increasing apoptosis under glutamine-limited conditions, and substantially enhancing mAb productivity in a dose-dependent manner. Glutamine supplementation largely alleviated the growth-inhibitory and pro-apoptotic effects of LgEVs while preserving their positive impact on productivity, suggesting that glutamine decouples EV-mediated stress from productivity enhancement. Metabolic analyses revealed increased glucose consumption, a glutamine-dependent shift between glycine and alanine overflow metabolism, and remodeling of amino-acid utilization. Metabolic flux analysis further demonstrated enhanced glycolytic overflow and increased reliance on amino acid-supported anaplerosis. Conversely, selective removal of LgEVs from perfusion medium significantly improved cell expansion without reducing antibody production, supporting an inhibitory role for late-stage LgEVs. These LgEVs were enriched in let-7 family miRNAs and miR-21, consistent with RNAseq analyses demonstrating stress-associated enrichment of these miRNAs in CHO EVs and with functional studies showing that let-7a and miR-21reduce CHO-cell growth. Together, these observations suggest that selective miRNA loading contributes to the growth, metabolic, and productivity phenotypes elicited by late-stage LgEVs. Our findings identify LgEVs as endogenous regulators of CHO-cell physiology and potential targets for optimizing high-density fed-batch and perfusion biomanufacturing processes. HighlightsO_LIEndogenous late-stage Large Extracellular Vesicles (LgEVs) reduce CHO cell growth but boost specific mAb productivity. C_LIO_LIGlutamine supplementation rescues LgEV-mediated growth inhibition and apoptosis. C_LIO_LIMetabolic Flux Analysis (MFA) based on the dynamic behavior of amino acid and other metabolite and substrate concentrations reveals the pyruvate node as a metabolic bottleneck and the associated lactate overflow metabolism as resulting from LgEV exposure. C_LIO_LIStress-associated let-7 and miR-21 microRNAs are highly enriched on a per-EV basis in late-stage LgEVs. C_LIO_LISelective removal of LgEVs improves perfusion cell growth without impacting antibody titer. C_LI

While in vivo synthesis of biologic therapeutics has been broadly successful, it is limited by biological constraints of the cells and by the complexity, time, and cost of implementing the pipeline from discovery through manufacturing. Cell-free expression systems (CFES), which use cellular transcription and translation machinery to express proteins in vitro, offer a promising alternative approach that could improve robustness and modularity in that pipeline. However, current benchmark CFES productivity is well below the theoretical capacity of the input nucleotides and amino acids. Efforts to address this issue are hindered by limited understanding of the extent of enzymatic activity in CFES beyond gene expression, as previous work has shown that metabolic enzymes in cell-free lysates cause substantial background metabolic activity that influences protein expression. Here, we hypothesized that the inflection point of protein expression is a critical timescale for CFES metabolism. We performed metabolomics characterization of CFES reactions, finding significant metabolic changes at the inflection point. Driven by these findings, we sought to identify supplements that could be added to the cell-free reaction to avoid metabolic limitations. We found that amino acid supplementation increased expression productivity and lifetime only when added after the inflection point, and actually hurt expression when added before the inflection point. We found similar supplementation timing impacts for some other metabolites as well. These findings show that endogenous metabolism and supplementation timing are deeply interconnected and are critical considerations in CFES optimization, and that metabolomics-informed fed-batch supplementation is a potentially valuable strategy to improve reaction productivity.

Methanol is a reduced, soluble one-carbon (C1) feedstock for sustainable bioproduction, but converting this potential into robust microbial growth remains difficult. Several synthetic C1 assimilation routes depend on autocatalytic cycles, whose operation requires coordinated control of redox balance, toxic intermediates, substrate regeneration, and host regulation. Here, we implemented the serine-threonine cycle (STC) in the soil bacterium Pseudomonas putida and used growth-coupled selection with adaptive laboratory evolution (ALE) to transition from mixotrophic C1 incorporation to strict methylotrophy. The evolved strain grew with methanol as the sole carbon and energy source under atmospheric CO2 with a doubling time of ca. 40 h. Whole-genome sequencing, reverse genetics, biosensors, isotope labelling, and comparative RNA sequencing showed that evolution repeatedly targeted native pyrroloquinoline quinone (PQQ)-dependent methanol oxidation, membrane-bound transhydrogenase activity, glycine regeneration, STC enzyme balance, and global regulatory nodes. Additional ALE under glycine-methanol co-feeding increased growth rates and exposed further targets for improving cycle flux. These results establish P. putida as a chassis for strict synthetic methylotrophy and define actionable engineering routes toward C1 biomanufacturing. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=63 SRC="FIGDIR/small/739708v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1dce5a6org.highwire.dtl.DTLVardef@16910eaorg.highwire.dtl.DTLVardef@d59018org.highwire.dtl.DTLVardef@e7287f_HPS_FORMAT_FIGEXP M_FIG C_FIG

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. HighlightsO_LIAccumulation of metabolic intermediates was alleviated by adaptive laboratory evolution C_LIO_LISequencing, proteomics, and enzyme kinetics revealed mechanisms for adaptation C_LIO_LIIncreased CatA1 expression reduced bottlenecks and improved muconate production C_LI

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.

Fed-batch production in Chinese hamster ovary cells is usually optimised empirically, yet the intracellular mechanisms that determine whether cells sustain productivity or enter terminal decline remain poorly resolved. Here, we combine longitudinal proteomics and intracellular metabolomics across differential ambr250 fed-batch processes to define the metabolic programmes associated with CHO cell viability and antibody production. Across media/feed combinations and an intensified seeding regime, culture progression followed a conserved trajectory from proliferation to metabolic transition and terminal stress. The high-stress state was characterised by a switch toward mitochondrial and peroxisomal fatty acid {beta}-oxidation, lipid remodelling, oxidative burden and activation of ferroptosis-associated pathways. Enriched feed conditions delayed this transition through enhanced redox and glutathione-linked defence, but did not proportionally increase antibody titre, revealing a trade-off in which cellular resources are diverted from recombinant protein production toward survival. These data identify fatty acid metabolism and ferroptosis as key constraints on late-stage CHO fed-batch performance and provide a mechanistic framework for rational feed design and host-cell engineering.

Nicotine accumulates in the gut and drives non-alcoholic steatohepatitis (NASH) via the gut-liver axis, yet no effective clinical intervention is currently available. To address this challenge, the probiotic Escherichia coli Nissle 1917 (EcN) was engineered for in situ nicotine clearance in the gut. Mutational screening of nicotine oxidoreductase 2 (PpNicA2) identified a highly active variant, PpNicA2A107R. Its incorporation into EcN together with an electron transfer protein (CycN) and a newly identified transporter (T3/T7) yielded 80% nicotine-degrading activity. Chromosomal integration of this module generated a stable strain, EcN-N12, which in NASH mouse models depleted intestinal nicotine, rescued hepatic lipid metabolism, alleviated tissue damage, and intercepted the nicotine-mediated gut-liver axis pathological progression. This work thus offers an effective and clinically translatable approach for nicotine-associated diseases.

Improving carbon recovery during sugar fermentation remains a major challenge because a substantial fraction of substrate carbon is lost as CO2 during central metabolism. To overcome this limitation, Clostridium sp. JS66 (JS66), an acetogen producing hexanoic acid from glucose, was subjected to adaptive laboratory evolution under CO2/H2 conditions to enhance H2-assisted CO2 reassimilation during glucose fermentation. The evolved strain, ALECO2, exhibited CO2 consumption without a lag phase under autotrophic conditions and reached a 9.5-fold higher CO2 uptake rate than JS66. Under fed-batch conditions, glucose-only fermentation yielded a carbon molar yield (Cmetabolite/Csugar, CM/CS) of 0.60, whereas H2 supplementation increased CM/CS to 0.91 and redirected carbon flux toward C6 products (hexanoic acid and hexanol), which accounted for 49% of total C_output. With additional CO2 supplementation, ALECO2 further assimilated externally supplied CO2, increasing the CM/CS to 1.10 and demonstrating carbon-negative fermentation. Assimilation of externally supplied CO2 further redirected carbon flux toward chain elongation, producing 7.14 g/L hexanoic acid and increasing the C6 carbon fraction to 57% of total C_output. Constraint-based flux analysis supported increased acetyl-CoA formation through the Wood-Ljungdahl pathway and enhanced flux toward reverse {beta}-oxidation under H2- and CO2/H2-supplemented conditions. Genome analysis identified mutations including genes encoding a putative HytB homolog and a LysR-type transcriptional regulator. These results establish ALECO2 as a promising evolved anaerobic non-photosynthetic (ANP) mixotrophy platform that links CO2 reassimilation and external CO2 assimilation with chain elongation, enabling carbon-neutral and carbon-negative production of value-added C6 products from glucose.

Halophilic chassis has emerged as a promising biomanufacturing platform for industrial polyhydroxyalkanoate (PHA) production. However, challenges still remain in improving the production capacity, scalability and robustness, thereby lowering cost to meet market demands. Here, a high-performing halophilic strain Halomonas LY03 was isolated with over 38% glucose- to-PHA conversion rate and broad non-grain substrate utilization capability. Multidimensional tools, including algorithm-guided high-expression neutral integration site (HENIS) screening toolkit designated SiteSeek, stop codon (TAA)-dependent enhancement of gene expression and recombinase-mediated large-fragment (> 9 kb) genomic integration, were then developed to enable precise, efficient and interference-free genomic integrative expression. Using these tools, various chromosomally engineered strains were rapidly constructed to achieve high-level production of poly-3-hydroxybutyrate (PHB, 151 g L-{superscript 1}) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P34HB, 139 g L-{superscript 1}) under high cell-density fermentation (up to 186 g L-1 cell dry weight) in a 5-L bioreactor. Scalability was demonstrated at 2-m3 and 20-m3 industry-scale fermentations, yielding up to 134 g L-{superscript 1} PHB and 127 g L-{superscript 1} P34HB (6.1 mol% 4HB). Building on the proven robustness, a two-stage continuous fermentation (TCF) process was developed using a twin-bioreactor system at 5-L and 20-m3 scales, where stable and sustained PHA production lasted over 260 h and 160 h, respectively. Techno-economic analysis revealed a substantial cost-reduction space of 48% compared with conventional fed-batch process. This study demonstrates a successful paradigm for engineering a newly isolated strain toward robust, high-titer and cost-competitive PHA production across lab-to-industry scales.

Building high-performance microbial cell factories requires dynamic coordination of resource allocation among cellular growth, target-product biosynthesis, and endogenous host metabolism. However, existing polyploid engineering strategies rely primarily on static manipulation of chromosome copy number. Although increasing gene dosage can enhance biosynthetic capacity, static designs cannot readily accommodate the changing metabolic demands encountered during fermentation. Here, we developed a metabolite-responsive dynamic polyploid engineering strategy that couples chromosome ploidy to the cellular metabolic state. We first constructed a high-performance L-threonine biosensor and used it to sense intracellular L-threonine levels and regulate ftsZ expression, a key cell-division gene, thereby establishing a dynamic polyploid system that requires neither exogenous inducers nor antibiotics. This system enabled engineered cells to progressively transition from polyploid to haploid during fermentation, accompanied by stage-specific remodeling of cellular physiology and metabolism. Physiological characterization revealed a marked increase in cell size and alterations in cell-envelope properties during the polyploid phase, followed by a gradual decrease in chromosome copy number as fermentation progressed. Transcriptomic and metabolomic analyses further demonstrated that dynamic ploidy transitions induced global metabolic network rewiring, remodeling the tricarboxylic acid cycle and amino acid metabolism while redirecting carbon flux toward the biosynthesis of aspartate-family amino acids. Ultimately, dynamic polyploid engineering substantially enhanced L-threonine production, enabling the engineered strain to achieve an L-threonine titer of 183.1 g/L and a yield of 0.67 g/g glucose in 5-L fed-batch fermentation without antibiotics or exogenous inducers. These findings show that dynamic regulation of chromosome ploidy can couple gene-dosage control with remodeling of cellular physiology and metabolic networks, providing a new engineering strategy to overcome the limitations of static polyploid designs and build high-performance microbial cell factories.

Multi-stage continuous bioprocessing can increase volumetric productivity, operational consistency, and process throughput, but its design is complicated by coupling among dilution rate, reactor volume, feed allocation, and cellular physiology. Here, we present ContiDesigner, available at https://chemnettools.anc.univie.ac.at/ContiDesigner/, a mechanistic steady-state framework and interactive web tool for the system-level design of continuous fermentation cascades. Comparing one- and two-stage configurations at equal total reactor volume and outlet flow, ContiDesigner reveals how internal flow and reactor volume allocation shape space-time yield and identifies productivity-maximizing operating conditions. Compared with one-stage processes, two-stage cascades favor lower over-all dilution rates, thereby preserving residence time in the production stage. The first-stage dilution rate approaches the corresponding one-stage productivity optimum, but the cascade optimum occurs earlier, reflecting a system-level compromise between biomass generation and production-stage residence time. However, two-stage operation outperforms optimized one-stage operation only when non-growth-associated production in the second stage is sufficiently strong, whereas increasing growth coupling favors one-stage operation. Two case studies demonstrate both the potential and limits of process intensification. An optimized two-stage design is predicted to achieve a more than 1.5 fold increase in space-time yield for poly-R-3-hydroxybutyrate (PHB) production compared with a published experimental five-stage cascade, whereas the lactic acid case study identifies conditions under which staging offers no advantage. ContiDesigner translates these design principles into an accessible workflow to explore feasible operating regions and prioritize cascade designs for experimental evaluation. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/743657v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@ef58faorg.highwire.dtl.DTLVardef@1ba88a4org.highwire.dtl.DTLVardef@160edd3org.highwire.dtl.DTLVardef@9dda34_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LIContiDesigner enables system-level design of continuous fermentation cascades C_LIO_LIHigh stage-one dilution supports biomass generation C_LIO_LILow stage-two dilution preserves productive residence time C_LIO_LIYet two-stage cascades favor lower overall dilution than one-stage systems C_LIO_LITwo-stage advantage requires strong non-growth-associated production in stage two C_LI

Biomanufacturing offers sustainable alternatives to chemical synthesis under lower temperatures and pressures than traditional catalytic methods. However, the slow pace and iterative engineering bottlenecks of cell strain development restrict the feasible biological design space. Cell-free systems circumvent these constraints, providing a flexible and high-throughput screening approach to accelerate pathway prototyping and enzyme optimization but are not typically used for manufacturing scale-up. To understand the scalability of cell-free biosynthesis, we establish an end-to-end fully cell-free architecture to discover, develop, and scale the biosynthesis of 1,2,4-butanetriol (BT), a high-value industrial platform chemical. First, we systematically screened ~150 enzymes across the 4-step pathway from xylose to BT to identify highly active homologs for each reaction. Next, we applied statistical Design of Experiments to optimize reaction formulations for cost and titer. Finally, the maximum-titer and minimum-cost formulations were scaled up across five orders of magnitude, from 10-{micro}L to 1-L reactions. This resulted in peak volumetric productivities of ~1 g/L/h and yields over 13 g of BT in a single 1-L reaction, with raw substrate costs totaling just $3.00 per liter. This work expands the diversity of enzymes tested for BT synthesis and establishes a blueprint for advancing industrial biochemical manufacturing fully in vitro.

The trend towards concentrated animal feeding operations (CAFOs) has served to concentrate not only livestock animals but the waste they produce to comparatively smaller areas. The point-source nature of this waste is an opportunity for the recovery and valorization of the nitrogen therein. Such a process would be viable on small to intermediate scales and require minimal inputs at the CAFO. In this study, we demonstrate the potential of the biopolymer cyanophycin to serve as a medium for manure-nitrogen recovery. In the first step, genetically modified strains of Escherichia coli produce intracellular cyanophycin from mock manure hydrolysates. Next, cyanophycin is recovered from microbial biomass via acid solubilization and base precipitation using electrochemically generated acids and bases. Finally, to improve both the yield and recoverable fraction of cyanophycin produced, we leverage the tunability of our genetically engineered system to probe the impacts of cyanophycin synthetase solubility, N-domain activity, and cyanophycin molecular weight on cyanophycin recoverability. Collectively, this work serves as a proof of concept for nitrogen recovery from agricultural waste, aligning with global sustainability initiatives.

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.

Many valuable plant metabolites are synthesized by type III polyketide synthases (PKS) that have become targets for the engineering of microbial production systems of these compounds. The rhizomes of turmeric (Curcuma longa) and ginger (Zingiber officinalis) are highly regarded for medicinal and culinary purposes and are the sources of bioactive curcuminoid and gingeroid polyketides. Fast growing demand for these compounds has sparked effort to identify their biosynthetic pathways to facilitate their heterologous production. In turmeric, a collaborative diketide synthase (DCS) and PKS (CURS) pair synthesizes curcumin from feruloyl- and malonyl-CoA. Yet, bona fide genes for the biosynthesis of gingeroids in Ginger are not known. Here we report the identification of two DCS/PKS pairs in Ginger that have different activity profiles in E. coli engineered to provide feruloyl- and hexanoyl-CoA as substrates. We show that one PKS (ZoPKS2) makes 6-dehydrogingerdione (6-DHG) as its major product while the other PKS (ZoPKS1) is a curcumin synthase. We found that ZoPKS2 becomes an efficient curcumin synthase when hexanoyl-CoA is not available, making it a dual-function enzyme that can be used to easily switch heterologous productions towards either of these two valuable products. Precursor feeding studies show that the substrate promiscuity of the collaborative DCS/PKSs may be exploited to access different dehydrogingerdione derivatives, while structural models of the Ginger PKSs offer insights for future engineering of product profiles. We believe that this work will add to the type III PKS toolbox and enable the development of efficient production platforms for gingeroids.

Municipal wastewater constitutes a major reservoir of unutilized reactive nitrogen, representing a significant opportunity for biological valorization. The biopolymer cyanophycin is promising as a means of nitrogen capture and recovery, but current production strategies are not optimized for the physicochemical constraints of municipal wastewater systems. Here, we engineered the naturally competent soil bacterium Acinetobacter baylyi ADP1 ISx to synthesize cyanophycin from carbon and nitrogen sources prevalent in municipal wastewater and over a range of wastewater-relevant temperatures. To overcome the recurring problem of arginine availability limiting cyanophycin synthesis, we engineered an arginine-producing strain (AP1) which accumulated cyanophycin when grown on acetate and ammonium (19% CDW), nitrate (9% CDW), or urea (29% CDW) and without arginine supplementation. During this work, we observed that conditions associated with reduced cell fitness correlated with increased intracellular cyanophycin content. As temperature strongly influences cell growth but cannot be realistically modulated in wastewater contexts, we investigated the potential of induced fructose-auxotrophy to modulate cell growth independently from temperature. This intervention, accomplished with a single knockout (gap), expanded the effective range of cyanophycin accumulation from 12 C up to 30 C. Collectively, these results establish the relevance of arginine-producing strains for cyanophycin biosynthesis and position A. baylyi as a promising chassis for continued development under real-world wastewater conditions.

Dynamic Flux Balance Analysis (DFBA) enables simulation of microbial culture dynamics under changing environmental conditions, but remains computationally expensive for tasks such as parameter calibration and fermentation optimization when applied using genome-scale metabolic models (GEMs). To address this challenge, we introduce Dynamic Flux Vector Balancing (DFVB), a reformulation of DFBA that solves an equivalent problem using a pre-computed, sparse basis of flux solutions that reduces the dimensionality of the internal optimization problem without information loss. Notably, DFVB provides a compact, interpretable representation of flux states that can readily identify dynamically inactive pathways and enable simulation-based automatic metabolic network reduction. We showed that DFVB produces the same culture dynamics as DFBA across multiple model scales and conditions, and identifies inactive reactions more accurately than Flux Variability Analysis (FVA) when compared to transcriptomic data profiles. Furthermore, computational performance analyses demonstrated that integrating DFVB with solver warm-start strategies and model reduction enhances computational efficiency relative to DFBA, yielding up to 3-fold reductions in simulation time for large-scale metabolic models. Finally, kinetic parameter estimation of culture dynamics with DFVB in two fermentation scenarios using a large-scale yeast GEM reached equal or higher prediction fidelity and narrower confidence intervals than DFBA, indicating improved parameter identifiability and robustness. Together, these results position DFVB as a scalable, robust, and biologically coherent framework for dynamic metabolic modeling, easing the integration of GEMs for culture dynamics simulation.

There are huge variations in metabolic complexity between the different kingdoms of life. Whilst it has been shown that some simple, unicellular organisms such as E. coli direct their energetic resources towards maximising proliferation, the metabolic goals of more complex organisms are unclear. This is an especially important topic for engineered organisms, such as Chinese Hamster Ovary (CHO) cells, that have been modified to produce therapeutically relevant compounds. This metabolic goal is reflected in the objective function of a constraint-based model (CBM) and has a direct impact on the metabolic flux distribution that is predicted using Flux Balance Analysis (FBA). However, there is no broadly applicable approach to infer this objective function from experimental data, to ensure CBMs represent real growth conditions. Here, we developed SIMOFF (SIMulated annealing Objective Function Finder) to infer the most appropriate objective function from minimal experimental flux data. Our applications of SIMOFF to S. cerevisiae demonstrated that the most suitable objective function is dependent on key metabolic phenotypes, even when the same organism and conditions are being modelled. Furthermore, we demonstrated the translatability of SIMOFF through application to CHO cells, where we showed that a SIMOFF-inferred objective function improved the accuracy of gene essentiality simulations, resulting in more reliable experimental target predictions.

Biological ethanol production is important for the circular carbon economy and makes up 73% of the U.S. biological fuels market. Previously, we produced ethanol by reversing methanogenesis and capturing methane by cloning methyl-coenzyme M reductase (Mcr) from an unculturable population of anaerobic methanotrophic archaea; this process was predicated on the generation of the intermediate acetate and its conversion by the methanogenic host to ethanol. Moreover, methanogens are generally thought to be detrimental for converting acetate to ethanol and are usually intentionally inhibited. Here, we demonstrate that direct growth on acetate as the sole carbon and energy source by the methanogen Methanosarcina acetivorans C2A results in 40% of the metabolized acetate becoming ethanol and that there is 430% more ethanol produced, compared to growth on methane via Mcr. In addition, we found growth on methanol results primarily in methane generation and low levels of ethanol. Therefore, acetate may be readily converted by the methanogen M. acetivorans to ethanol at high yields.