Cell-free multi-enzyme cascades enable precise biosynthesis, but using them for cost-efficient bioproduction remains challenging. This issue can be addressed through model-based process optimization using kinetic models. However, the predictive power of kinetic models is often limited by structural and parametric uncertainties. In this work, we present MoRSel, a novel method that tackles structural uncertainties through automated refinement and selection of kinetic models. MoRSel starts with a kinetic core model, a library of potential model changes, and available experimental data. The algorithm iteratively expands the model to achieve an optimal balance between fit quality and model complexity according to information-theoretic criteria.We applied MoRSel to a kinetic model of a cell-free enzyme cascade for producing the nucleotide sugar UDP-GalNAc. An initial core model showed significant inconsistencies with measured experimental data. By providing candidates for potential stoichiometric and regulatory changes in the model, MoRSel identified a model that more accurately captured the observed behavior. We then used this model to inform a recently introduced robust (ensemble-based) process optimization strategy that accounts for parametric uncertainty. We addressed two economically relevant problems. First, we determined the optimal initial substrate and enzyme concentrations to maximize the product titer, resulting in a 45 % increase in product titer and yield in a validation experiment. In a second optimization, we minimized the enzyme load and tested the computed optimal enzyme concentrations in another validation experiment. With enzyme costs reduced by 53 %, the relative titer and yield could be improved by 80 % and 75 %, respectively, with small reductions in absolute titer and yield.Overall, MoRSel advances the development of predictive models for complex biochemical systems, with particular relevance for the robust optimization of cell-free bioproduction processes.
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.
