Optimization of cell culture conditions is critically important for successful commercial production of therapeutic antibodies; however, improvements in productivity may be offset by changes in critical quality attributes. We assessed the impact of two different process conditions (high and low yield) on growth, productivity, and glycan distribution for two CHO-S clones producing adalimumab, clone 1 (DHFR clone) and clone 2 (GS clone) in fed-batch shake flask cultures. High-yield cultures showed approximately 1.6-fold and 7.4-fold increases in integral of viable cells (IVC) for clones 1 and 2, respectively, compared to the low-yield cultures. Although specific productivity was lower in the high-yield cultures, this decrease was offset by the increased IVC, yielding higher titers in both cell lines. High-yield cultures showed a marked decrease in galactosylation, marked by a decrease in G1F glycans compared with the low-yield cultures, across both clones. Man5 glycans remained unchanged in clone 1 cultures, while a slight increase was observed in the high-yield clone 2 cultures. Metabolic analysis showed significant changes in glucose utilization in high-yield cultures with substantial decreases in specific glucose uptake and lactate yield, particularly for clone 2. Calculation of nucleotide sugar fluxes revealed that the increased utilization of UDP-N-acetylglucosamine and UDP-galactose for cellular glycosylation limited their availability for monoclonal antibody (mAb) glycosylation in high IVC, high-yield cultures, consistent with the observed changes in mAb glycans.
N,N-dimethyltryptamine (DMT) is a bioactive indole alkaloid that could greatly benefit from scalable, fermentation-based production for research and pharmaceutical applications. In this study, we reconstructed a two-step bacterial pathway converting L-tryptophan to DMT via tryptamine. This involved combining a pyridoxal 5'-phosphate (PLP)-dependent tryptophan decarboxylase from the bacterium Ruminococcus gnavus (RgnTDC) with an S-adenosyl-L-methionine (SAM)-dependent N-methyltransferase from the cane toad Rhinella marina (RmNMT) in Escherichia coli. We optimised conditions for each step, determining 37 °C (pH 8.0) as the optimal condition for tryptamine production and 25 °C (pH 7.5) for DMT. While PLP supplementation did not raise tryptamine levels, methionine supplementation increased DMT levels by 2.8 times, emphasising the importance of methyl-donor supply. Co-culture and co-expression experiments showed that DMT accumulation depends on sufficient methylation capacity. Increased tryptophan availability led to tryptamine accumulation without a proportional increase in DMT formation, indicating a downstream limitation after decarboxylation. Together with the stimulatory effect of methionine supplementation, this result points to N-methylation and methyl-donor supply as key constraints in this system. In shake-flask cultures, a co-expression strain (TN1) produced 103 mg/L DMT after 48 h in complex medium without direct tryptophan supplementation. To enable growth in a defined medium, we used a workflow involving a tryptophan-enriched supernatant from a Corynebacterium glutamicum tryptophan overproducer, which supported de novo DMT formation at 16 mg/L in defined medium. These findings establish a plasmid-based platform for DMT production with E. coli and identify methyltransferase capacity as a key target for further yield improvements.
Escherichia coli strains are widely used across numerous industrial and biotechnological applications. Yet their performance varies substantially in ways that can not be anticipated from genome annotation. Because transcriptional regulatory networks (TRNs) govern cellular functions such as motility, stress responses, metabolic flexibility, and production efficiency, differences in TRN organization and use may underlie many observed phenotypic differences. To investigate TRN differences between strains, we generated a compendium of 433 matched RNA-Seq profiles for six commonly used industrial E. coli strains (BL21, C, Crooks, MG1655, W, and W3110) and applied iModulon analysis to compare the state of their TRNs under similar growth conditions. This analysis revealed that core regulatory programs with similar functions are wired differently across the strains, and that the strains engage these programs in distinct ways when exposed to the same environmental challenges. Together, these findings highlight transcriptional regulation diversity underlying phenotypic expression among industrial E. coli strains. By providing an integrated view of TRN differences across widely used hosts, this work offers a fundamental basis for interpreting strain-specific behaviors and supports more informed approaches to strain selection and optimization.
Glutamine is an important nitrogen donor in the biosynthesis of nucleotides and several other amino acids. Proliferating cells consume high amounts of glutamine, and cell culture media contain glutamine as the most abundant amino acid. Glutamine is industrially manufactured through bacterial fermentation, which requires external supplementation with sugars as the carbon source. Using the cyanobacterium Picosynechococcus sp. PCC 7002, this study aimed to develop a method for the photosynthetic production of glutamine using CO2 as the sole carbon source. The introduction of glutamate dehydrogenase from Corynebacterium glutamicum and glutamine synthase from Saccharomyces cerevisiae increased the concentration of extracellularly released glutamine. Metabolome analysis revealed decreased intracellular citrate levels in glutamine-producing cells. To enhance citrate replenishment, metabolic engineering approaches, including l-lactate assimilation and glycogen deficiency, were examined. The introduction of pyruvate carboxylase and citrate synthase from C. glutamicum significantly increased glutamine production. After optimizing light intensity and CO2 concentration, the recombinant strain produced 1168.5 μM (170.76 mg L-1) glutamine. This study establishes metabolic engineering approaches for converting CO2 into glutamine and demonstrates that cyanobacteria are promising photosynthetic producers of glutamine.
As a major cash crop, tobacco quality improvement is constrained by the limited effectiveness of conventional approaches, including traditional breeding and agronomic regulation. This review focuses on the application of metabolic engineering to enable precise and targeted modulation of tobacco quality traits. We first analyze the key factors and underlying mechanisms governing aroma, health-related components, functional metabolites, and combustion characteristics. We then summarize recent advances in metabolic engineering strategies, including pathway rewiring, metabolic flux redistribution, enzyme engineering, and regulatory-network modulation, for optimizing the biosynthesis and accumulation of target compounds such as terpenoids, polyphenols, and alkaloids. In addition, selected integrative approaches that support metabolic regulation and quality formation are briefly discussed. Finally, we highlight current challenges and outline future research directions to facilitate more precise and efficient improvement of tobacco quality.
Etoposide, a semisynthetic derivative of podophyllotoxin originally isolated from the rhizomes of mayapple (Podophyllum peltatum) and indian Podophyllum (P. hexandrum) plants, is one of the most powerful chemotherapeutic agents used to treat various types of solid tumors and blood malignancies. Despite its clinical importance, its supply is recurrently constrained due to a heavy reliance on plant extraction, where low natural precursor abundance and increasing climate-related pressures limit production scalability. Developing alternative manufacturing routes has therefore become a major objective, though reconstruction of this complex biosynthetic pathway has long posed significant challenges, even with recent advances in synthetic biology and metabolic engineering. Yeast has emerged as a robust cellular chassis for reconstituting, either partially or entirely, plant secondary metabolite pathways, and enabling cost-effective bioproduction. Here, we established an integrated biotechnological strategy for the sustainable production of advanced etoposide intermediates using engineered yeast cell factories. By combining pathway refactoring, gene copy number optimization, and tailored co-enzyme compatibility, we established an efficient heterologous pathway converting yatein into (-)-4'-desmethyl-epipodophyllotoxin (4'dEPT) in yeast. Iterative strain engineering improved metabolic flux distribution, leading to enhanced titers and accelerated production kinetics, while process engineering proved essential to maximizing overall system performance. Finally, we also demonstrated the viability of coupling bioproduction in cell factories with downstream, semisynthetic conversion by successfully isolating bioreactor-derived 4'dEPT and converting it into etoposide. In parallel, identifying resilient plant resources that can accumulate high levels of YAT provides a complementary strategy for securing the precursor supply at scale. Overall, this report validates the concept of a hybrid etoposide production platform integrating controlled plant biomass sourcing, engineered yeast cell factories, and chemical transformation steps.
Controllable gene expression is essential in microbial biotechnology, yet most systems rely on costly external inducers that limit large-scale applicability. Here, we employed phosphate-responsive promoters controlled by the SphS-SphR phosphate-sensing two-component system as auto-inducible expression systems in cyanobacteria. Specifically, we characterized the promoters of phoA, sphX, and pstS2, which are activated under low phosphate availability and achieved induction folds of up to 13, as well as PurtA and a synthetic promoter PP i -neg, which are repressed under these conditions. Replacement of the ribosome binding site in the native promoter systems further expanded the accessible range of expression levels, with context-dependent increases or decreases depending on the promoter. By systematically adjusting the phosphate concentration and cell density, the timing of gene expression could be precisely controlled. Notably, intracellular phosphate storage during early growth enables transient buffering of external depletion, allowing promoter activation prior to growth limitation. This, in turn, enables their use in continuously growing cultures. As a proof of concept, we established a sucrose production process that autonomously transitioned from a growth phase with basal production to a production phase with a 5.5-fold higher sucrose titre, triggered by phosphate depletion during cultivation. Overall, this auto-inducible expression system expands the cyanobacterial genetic toolbox and enhances the applicability of cyanobacteria in scalable production processes.
Glycolysis is central to Clostridium thermocellum metabolism; however, strains engineered for high ethanol titer exhibit a decrease in the conversion of cellobiose to ethanol (i.e., cellobiose-to-ethanol yield), suggesting the presence of glycolytic bottlenecks. We expressed heterologous triosephosphate-isomerases (tpi), fructose-1,6-bisphosphate aldolases (fba), and glyceraldehyde-3-phosphate dehydrogenase (gapDH) genes from Thermoanaerobacterium saccharolyticum and Zymomonas mobilis, along with 26 non-phosphorylating glyceraldehyde-3-phosphate dehydrogenases (gapN) variants, to identify limiting reactions. We demonstrated functional expression and increased activity for several Fba and Tpi enzymes in the PPi-free glycolysis engineered strain of C. thermocellum (LL1711). Despite C. thermocellum's low native FBA activity compared to other industrial strains, increasing Fba or Tpi activity via heterologous expression had no significant effect on cellobiose uptake or ethanol titers in high-substrate fermentations. Furthermore, 25 of 26 tested gapN genes had toxic effects on C. thermocellum upon transformation. In conclusion, none of the tested glycolytic enzyme modifications improved product titers. These results suggest that the primary metabolic limitation is not at the FBA or TPI reactions, supporting a shift in future engineering efforts toward downstream fermentation pathways.
In times of a climate crisis caused by extreme emissions of green-house gases on a global scale, mitigation solutions need to be found. One solution is the system of carbon capture and utilization (CCU), where C1 gases, such as carbon monoxide (CO), carbon dioxide (CO2), or methane, are either redirected from industrial off-gas streams or directly air-captured. A biotechnological process for CCU is the use of Methanothermobacter marburgensis for CO2 fixation and production of value-added compounds. In this study, we focused on valine production, an amino acid important for human or feedstock nutrition. We demonstrated overproduction of valine from CO2 in M. marburgensis with temperature-induced promoters. Here, we reached a 12.9-fold increase in valine production between the OFF- and ON states of the inducible promoter with a maximal specific production rate of 14.17 mg gCDW-1 h-1 of valine in closed batch experiments. In the second approach for valine production, we overexpressed acetolactate synthase genes with resistance to allosteric valine inhibition from Methanothermobacter thermautotrophicus recombinant in M. marburgensis. We identified a strong reduction in allosteric inhibition towards valine. This resulted in specific valine productivity of up to 40 mg gCDW-1 h-1 and states the highest specific productivity on an individual amino acid in methanogens. With those findings, we expanded the toolbox for genetic modification of M. marburgensis by a thermo-inducible promoter system and applied protein engineering for enhanced production of value-added compounds to M. marburgensis. This proof of concept shows the feasibility of archaeal cell factories generation via genetic engineering for industrial production of value-added compounds with thermophilic methanogens.
2,4-Dihydroxybutyric acid (DHB) is a promising C4 platform compound for the synthesis of methionine analogues and biodegradable polymers. However, aerobic DHB production from glucose in Escherichia coli involves transient acetate overflow prior to product synthesis, which could be challenging for process scalability.Therefore, we engineered Escherichia coli K-12 MG1655 for optimized DHB production by replacing the phosphotransferase system mediated glucose uptake with the galactose permease GalP, coupled to ATP-dependent phosphorylation via endogenous glucokinase. In combination with targeted deletions of malate- and fumarate-consuming reactions, we obtained a strain with enhanced flux through the tricarboxylic acid (TCA) cycle and pentose phosphate pathway leading to improved NADPH availability and increased anaplerotic activity, as revealed by 13C metabolic flux analyses. Deletion of the mdh gene encoding for the cytosolic malate dehydrogenase further promoted DHB formation. The resulting strain achieved DHB yields up to 0.20 mol mol-1 (2.43 g L-1), a 4-fold increase compared to the wildtype background (0.05 mol mol-1, 0.60 g L-1), under aerobic conditions while suppressing acetate formation.Together, these results demonstrate that GalP-mediated glucose uptake and engineering of the TCA cycle provide a robust metabolic framework for efficient DHB biosynthesis and establish a foundation for further process and pathway development.
Methanotrophs, which utilize methane as their sole carbon and energy source, are promising platforms for valorizing methane to industrially valuable products, such as bioplastics or biofuels. However, methanotrophic activity is highly affected by the availability of metals, and therefore, a greater understanding of these effects is important for efficient conversion of methane by methanotrophs. To address this, the impact of copper on methanotrophy was investigated at a systems level via the reconstruction of a genome-scale metabolic model (GEM) for Methylosinus trichosporium OB3b. The GEM was further integrated with transcriptomic data for improved functionality, which was then employed to identify metabolic engineering strategies for increased putrescine production. Despite these efforts, the GEM framework was not successful in significantly enhancing putrescine production. Therefore, the steady-state 13C-metabolic flux analysis was conducted to estimate intracellular flux distributions and to validate the GEM functionality, revealing the indispensability of the ethylmalonyl-CoA cycle and inflexibility of flux utilization in central methane metabolism. These findings highlight key pathway constraints for methane valorization and expand the system-level understanding of copper-regulated methanotrophic metabolism.
Isoprenyl acetate, a volatile ester derived from isoprenol, is a key biosynthetic intermediate for the advanced aviation fuel candidate, 1,4-dimethylcyclooctane. Here, we engineered Pseudomonas putida KT2440 for the production of isoprenyl acetate from mixed sugar substrates. We first generated isoprenyl acetate by introducing a heterologous alcohol acetyltransferase (ATF1) and deleting three promiscuous native esterases to reduce product degradation. Then, we engineered efficient glucose and xylose co-utilization by integrating a heterologous xylose isomerase pathway and deleting global regulators crc and hexR to alleviate catabolite repression. Additionally, intracellular acetyl-CoA flux was reinforced through the expression of auxiliary carbon-conserving routes, including non-oxidative glycolysis and acetate assimilation. Culture conditions were systematically optimized by adjusting medium composition, induction, and overlay solvent to maximize product yields and titers. These cumulative efforts achieved isoprenyl acetate titers of 1.5 g/L in shake flasks and 1.9 g/L in fed-batch bioreactor cultures from mixed sugars, corresponding to a yield of 0.067 g/g of total sugar consumed. Our work demonstrates the potential of P. putida as a robust microbial chassis for scalable biosynthesis of ester-based biofuels from lignocellulosic feedstocks.
Aromatics have many important applications in modern society but are traditionally produced in non-sustainable processes from fossil resources. Whole-cell biocatalysis bears great potential to provide a variety of aromatics from renewable carbon sources, thereby offering a more sustainable alternative. In this context, chorismate, the end product of the shikimate pathway, is an important biosynthetic hub compound that serves as precursor of a multitude of industrially relevant aromatics. Here, we screened several pathways for chorismate-derived bioproduction of five different mono- and dihydroxybenzoates in tyrosine-overproducing Pseudomonas taiwanensis GRC3Δ5-TYR1. Subsequently, twelve different modifications targeting the bifunctional chorismate mutase/prephenate dehydratase PheA were screened to reduce flux from chorismate to phenylalanine and tyrosine, thereby further enhancing the production of 2-hydroxy- and 2,3-dihydroxybenzoate without causing an auxotrophy. An auxotrophic ΔpheA strain served as benchmark control. Most promising modifications were subsequently also evaluated for 3-hydroxy-, 4-hydroxy- and 2,5-dihydroxybenzoate production demonstrating increased yields. Replacing the native pheA gene with the unmodified homolog from Escherichia coli was the most beneficial, enabling an increased production of up to 38.2% when combined with attTn7::P14g-SmCH-IV. With this modification, the highest production was achieved for 4-hydroxybenzoate resulting in titers of 3.59 mM and a yield of 20.9% (Cmol/Cmol) from glucose. However, the impact of the respective pheA modification varies with the applied production module, further emphasizing the strong interplay with the production host’s metabolism.
Dimethyl terephthalate (DMT) serves as the precursor in the production of polyethylene terephthalate, polytrimethylene terephthalate, and polybutylene terephthalate. The widespread use of DMT in the polymer industry and its ubiquitous existence in end products raise alarms about its potential harm to humans and animals. DMT can enter the environment through the degradation of polymers and their end products, and cause endocrine disruption, oxidative stress, and an elevated risk of cancer. In recent years, DMT has also gained renewed interest in its potential for plastic recycling and upcycling. In this study, we identified two strains of Rhodococcus that possess DMT-degrading capabilities and utilized transcriptomic analysis and gene knockout to elucidate the mechanisms of DMT degradation. R. opacus PD630 and R. jostii RPET were found to convert up to 1 g/L DMT into mono-methyl terephthalate (MMT). A putative DMTase (RS34275) was identified for this conversion. R. jostii RPET also demonstrates the ability to convert DMT into MMT and to utilize MMT for its cellular growth via the terephthalate pathway. A putative MMTase (RS21885) as the sole enzyme was identified for the conversion of MMT into terephthalate in the RPET strain. In addition, we successfully produced lycopene and lipids from an engineered RPET strain using DMT as a substrate. Our findings will facilitate future DMT bioremediation and bio-upcycling of DMT-associated plastics, enabling the production of value-added products.
Phosphofructokinase (Pfk), a key regulatory enzyme in glycolysis, is composed of Pfk1 and Pfk2 subunits in Saccharomyces cerevisiae. However, the distinct roles of these subunits in central carbon metabolism remain unclear. Here, we examined the metabolic consequences of deleting PFK1 or PFK2. The pfk2Δ strain exhibited more severe defects than pfk1Δ. Its maximum specific growth rate was reduced by approximately 54 % in pfk2Δ and by about 15 % in pfk1Δ, both relative to the reference strain. Ethanol production decreased by 36 % and 82 % in pfk1Δ strain and pfk2Δ strain, respectively, relative to the reference strain. Both deletion strains accumulated higher acetate levels compared to the reference strain, increasing by 25.4 % in the pfk1Δ strain and 82 % in the pfk2Δ strain. Flux balance analysis (FBA) revealed a markedly increased carbon flux to the tricarboxylic acid cycle (TCA) in the pfk2Δ strain, with respiration-associated carbon flux elevated 1.5-fold compared to the pfk1Δ strain. Consistently, transcriptomic profiling showed significant upregulation of respiration-related genes in the pfk2Δ strain compared to the reference strain. Notably, deletion of PFK2 enhanced acetyl-CoA-derived product formation, with free fatty acid (FFA) titers increasing from 412 mg L-1 to 517 mg L-1 (a 33.3 % increase). These findings establish PFK2 as a key regulatory node redirecting carbon flux from fermentation toward respiration and biosynthesis, offering new opportunities for metabolic engineering of acetyl-CoA-derived products.
Thermophilic methanogens of the genus Methanothermobacter are established biocatalysts in power-to-gas applications, converting H 2 and CO 2 into CH 4 through the process of methanogenesis. Further expanding this platform for the bioproduction of value-added compounds (power-to-x) has the potential to increase the economic viability of such processes. This requires a genetic toolset that enables the controlled expression of recombinant pathways. Here, we report the fully autotrophic inducible recombinant bioproduction of acetoin from H 2 and CO 2 in Methanothermobacter thermautotrophicus ΔH. To facilitate inducible gene expression, we implemented an anhydrotetracycline (aTc)-inducible promoter system, expanding our available set of promoters. The aTc-inducible system enabled controlled expression of a codon-optimized acetoin-production operon comprising the acetolactate synthase- and acetolactate decarboxylase-encoding genes from Streptococcus thermophilus . Batch cultivation at 42°C demonstrated aTc-dependent acetoin formation, yielding up to 0.45 ± 0.08 mM acetoin. Fed-batch bioreactor experiments confirmed growth-coupled, recombinant acetoin production, while eliminating the non-specific acetoin accumulation that we observed during non-growth phases in batch cultivation. Continuous cultivation in a chemostat resulted in stable acetoin production rates of 1.28 ± 0.07 µmol L⁻¹ h⁻¹ at 42°C. Elevated temperatures led to reduced acetoin production, suggesting diminished activity or thermal instability of the heterologous enzymes. This study demonstrates the feasibility of value-added bioproduction in Methanothermobacter and establishes an inducible expression system suitable for pathway engineering in thermophilic methanogens. Together with genome-scale modeling and emerging enzyme engineering strategies, these results lay the foundation for developing robust, CH 4 -co-producing power-to-x bioprocesses with Methanothermobacter species.
Clostridium thermocellum is a promising host for consolidated bioprocessing due to its ability to directly ferment cellulose into fuels and chemicals. However, natural product formation in this organism is limited. Here, we report engineering C. thermocellum for the production of 2,3-butanediol (23BD), a valuable industrial chemical. We functionally expressed a thermophilic 23BD pathway in this organism resulting in a 23BD titer of 19.7 mM from cellulose, representing a metabolic yield of 24%. We used a cell-free systems biology approach to identify limiting steps in the 23BD pathway, revealing that exogenous 23BD dehydrogenase (BDH) activity was essential for production, while native acetolactate synthase (ALS) and acetolactate decarboxylase (ALDC) activities were present but limiting in the parent strain. This approach also revealed redox balance limitations. We demonstrated that this improved understanding of redox balance limitations could be used to increase 23BD titer in vivo, showing that adding acetate could be used to increase 23BD yield. This work establishes a foundation for developing C. thermocellum into a robust platform for 23BD production directly from cellulose and highlights the utility of cell-free systems for guiding metabolic engineering in non-model organisms.
Optimization of flux distribution in central carbon metabolism is important to improve the microbial productivity. As the number of precursors required for synthesis differs for each target compound, optimal flux distribution also varies. A library of mutant strains with diverse flux distributions can aid in optimal strain screening. Therefore, in this study, we aimed to construct a library of Escherichia coli strains with stepwise changes in flux distribution by introducing mutations into the ribosome-binding sites of key enzyme genes on its chromosome. We focused on the flux ratios at the glucose-6-phosphate and acetyl-CoA branch points to enhance mevalonate production. Mutations were introduced into the ribosome-binding sites of pgi and gltA to vary the flux ratios of the two pathway branches. Furthermore, a combinatorial repression library comprising 16 strains was constructed by varying pgi and gltA expression at four levels, and a plasmid containing mevalonate synthesis genes was introduced into each strain. Batch cultures were performed to obtain strains with mevalonate titers and yields 2.4- and 3.4-fold higher than those of the parent strain. Overall, our combinatorial suppression library of pgi and gltA facilitated the effective identification of mutants with optimal metabolism for mevalonate production.
Heterologous compound production is a complex trait since the native metabolic fluxes supplying the precursors, redox power, and energy are under multilevel cellular regulation. Improving complex traits using targeted engineering needs combinatorially charting the complex genetic underpinnings. While this is laborious, adaptive laboratory evolution (ALE) has been used to improve many traits of microbial strains that are of application relevance such as tolerance of harsh conditions and nutrient utilization. However, in contrast to such traits, heterologous production can seldom be intuitively coupled with cellular fitness.Here, a novel method EvolveXGA was developed for genome-scale metabolic model guided design of strategies combining chemical environments and genetic engineering of the metabolic network to allow ALE of desired traits. Adaptive evolution of traits occurs when the co-variance between the traits and fitness involves a genetic dependency like a flux coupling would indicate. Thus, combinations of chemical environments and metabolic network structures were searched using a genetic algorithm to identify those that render desired traits (i.e., sets of metabolic fluxes) flux-coupled with fitness. The search was performed for the production of 29 heterologous compounds in yeast Saccharomyces cerevisiae. Strategies for coupling the production routes of 13 compounds with fitness were found with four metabolic reaction knock outs and three components in the chemical environment. In addition, strategies for fitness-coupling native fluxes involved in the production was found for the remaining compounds. In addition, a model-guided strategy was implemented for fitness-coupling of heterologous glycolic acid (GA) synthesis in S. cerevisiae via oxaloacetase, oxalyl-CoA synthetase, and oxalyl-CoA reductase (i.e., oxalate pathway). ALE was performed and evolved populations and isolated clones were characterized using whole-genome sequencing and quantitative metabolite analysis. Three out of six isolates had better GA yield from glucose than a non-optimized control strain expressing the oxalate pathway and glyoxylate reductase.EvolveXGA generalizes metabolic model-guided design of strategies to couple production routes with cell fitness. The strategies bring optimizing heterologous production in engineered microbial cells in the realm of ALE. Slow and expensive strain optimization is a major hinder of novel processes using engineered microbial cells reaching industrial realization. Thus, EvolveXGA contributes to biotechnological solutions for the brighter future.
Many cardiac pathologies are characterised by increased stiffness of the myocardium, due to excess deposition of extracellular matrix (ECM) proteins and structural remodelling, impacting the behaviour of cardiomyocytes (CMs). Metabolism of CMs shifts in cardiac pathologies, with the healthy heart primarily utilising fatty acids as its source of energy production, whilst the diseased heart switches to utilise glucose. The shift in metabolic source with stiffness of the myocardium has not been investigated. To investigate the effect of ECM stiffnesses on iPSC-CM metabolism, iPSC-CMs were cultured on polydimethylsiloxane (PDMS) substrates of healthy and fibrotic stiffness (20 kPa and 130 kPa respectively) and plastic. Cellular metabolism of iPSC-CMs was assessed through isotope-labelled mass spectrometry with central carbon tracing as well as real-time cellular bioenergetics using extracellular flux analysis. Key metabolic genes were investigated at transcript and protein level, with proteomics analysis conducted to identify protein profiles on substrate stiffnesses. Mass spectrometry data revealed greater utilisation of glucose in iPSC-CMs cultured on plastic compared to softer PDMS substrates, indicating greater glycolytic activity. Extracellular flux analysis demonstrated greater lactic acid efflux from iPSC-CMs cultured on plastic substrates, reflective of increased glycolytic flux and a shift towards aerobic glycolysis as the primary source of ATP synthesis. This study revealed culture of iPSC-CMs on traditional cell culture plastics or glass coverslips displaying pathological metabolism, highlighting the use of physiological substrates for metabolic investigation.
