Ergothioneine (EGT) is a bioactive, rare variant of histidine with many applications in the medical, pharmaceutical, and food fields. Therefore, we aimed to investigate in this study the impact of genomic and physicochemical factors on EGT production by the industrial filamentous fungus Aspergillus oryzae. Firstly, to facilitate efficient EGT production, we analyzed the subcellular localization of the three EGT biosynthetic enzymes present in A. oryzae. During screening for the most potent producer of EGT among bioengineered constructed transformants, the strain EgtACO overexpressing both AoegtA and AoegtC showed promising EGT production in DPY medium. Five days of incubation was the optimum period, and CZYP medium was the optimum medium for EGT production. Co-cultivation with the nisin Z-producing Lactococcus lactis JCM 7638 yielded EGT production equivalent to that of the EgtACO strain alone. Having broad-spectrum antimicrobial activity without suppressing growth of the EgtACO strain suggested that bacteriocin may help reduce the risk of contamination during long-term cultivation. Moreover, supplementing the production medium with L-methionine or zinc sulfate improved EGT production (1468.5 or 1565mg/L, respectively). Furthermore, repeated inoculation of the producer strain EgtACO and incubation in blue light were the optimum conditions for EGT production (1895 mg/L). Finally, we achieved cost-effective EGT production using A. oryzae strain EgtACO under the optimal culture conditions using agricultural wastes: potato peel and sweet potato peel (293 and 308mg/L, respectively).
Riboswitches are RNA regulatory elements that sense small molecules and regulate gene expression through ligand-induced conformational changes in an aptamer domain. They enable synthetic biology applications such as biosensing and gene circuit design. Accurate prediction of riboswitch-ligand binding affinity, quantified by the dissociation constant (Kd), is essential for rational riboswitch engineering. We present a neuro-symbolic framework in which LLM-derived embeddings encode riboswitch sequences, secondary structures, and ligand representations into a unified representation that captures riboswitch-ligand interaction context, while domain-informed rules encode explicit biochemical priors. Despite limited labeled data, this framework achieves strong predictive performance and improved data efficiency. It predicts min-max log-scaled pKd values and supports binary classification of binding strength, particularly by mitigating systematic overestimation of affinity. More broadly, the results suggest that LLM-based neuro-symbolic representations provide an effective route for modeling riboswitch-ligand interactions under data-limited conditions.
Hairy root cultures offer a sustainable alternative to field-grown plants for the production of bioactive compounds, but their translation to industrial-scale processes is limited by the challenges of high-volume cultivation and downstream processing. In the current study, a large-scale production pipeline for basil (Ocimum basilicum) hairy roots, a rich source of specialized bioactive metabolites, is described. The production of hairy root biomass was optimized by developing a robust and scalable cultivation protocol, enabling consistent and reproducible growth in bioreactor systems with working volumes of up to 1000 L. The entire production pipeline from initial Petri dish cultures to industrial-scale bioreactors was established by adopting a systematic, stepwise methodology, ensuring process reliability and scalability. A customized bioreactor with controlled aeration maintained biomass integrity and metabolic stability during long-term axenic cultivation, yielding 96.4 kg of biomass fresh weight within 84 days. Complementary solvent-based extraction and pulsed electric field processing allowed the recovery of metabolites with a broad range of polarities. Untargeted metabolomics revealed a chemically diverse profile comprising primary metabolites, phenolic acids, flavonoids, terpenoids and triterpenoids with relevance for cosmetic applications. These results demonstrate the feasibility of industrial-scale basil hairy root cultures and support their use as a source of plant-derived cosmetic ingredients.
Rice is a major cereal crop for global food and nutritional security and a key target for genetic improvement. CRISPR/Cas9 enables precise genetic modification in crops, but mutation screening remains a technical and economic barrier to broader genome-editing applications. Although several detection methods are available, some require labor-intensive procedures, specialized equipment, high costs, or limited sensitivity to specific mutation types. High-resolution melting (HRM) analysis is an established approach for screening CRISPR/Cas-induced mutations in plants, including rice. Here, we evaluated an adapted HRM workflow combining conventional SYBR Green-based qPCR chemistry with downstream computational analysis to detect CRISPR/Cas9-induced mutations at three rice loci: OsMADS26, OsRAC1, and OsNRT1.1b. The workflow detected insertions, deletions, and base substitutions. Across the three loci, the 1% edited-DNA mixtures showed a slight observable deviation from the wild-type melting profile under the conditions evaluated, although this should not be interpreted as a validated detection threshold. Although the assessment of heterozygous samples was limited by their availability, the results support the potential applicability of the approach for individual sample analysis and expanded sample screening. A customizable R script complemented visual analysis by evaluating melting temperature (Tm) and GCP-derived dissimilarity, supporting sample classification. By combining standard SYBR Green chemistry with an adaptable analysis workflow, the method offers an alternative to dedicated HRM reagents and proprietary platforms. This approach provides a practical and potentially lower-cost option for mutation screening at the evaluated rice loci and may be adapted and validated for other targets and plant species.
Drought severely limits crop productivity, and the processes that support yield stability in modern maize heterotic hybrids remain insufficiently characterized. We evaluated two maize elite F1 hybrids, H.393 and H.166, under prolonged moderate water deficit in greenhouse and field conditions to identify genotype-specific mechanisms of drought resistance. These two hybrids previously selected in the Steppe zone of Ukraine, yielded 5.75-8.57 t/ha in droughts of 2022-2024, with no significant yield differences between the hybrids. Water stress reduced plant height, contents of chlorophylls, carotenoids, and several minerals in both hybrids demonstrated contrast adaptive strategies to drought. H.393 exhibited a metabolic response, including strong maintenance of leaf water status, activation of guaiacol peroxidase, and distinct shifts in Na and Mg homeostasis revealing drought-avoidance strategy with metabolic adjustment. In contrast, H.166 preserved stable PSII activity, sustained photochemical performance, high PIABS values, great proline accumulation, and elevated concentrations of Mn, Cu, and Zn in leaves manifested adaptive drought-tolerance strategy. Three-factor ANOVA confirmed significant contributions of genotype, water regime and stress duration to the development of most traits, revealing divergent physiological trajectories underlying similar agronomic outcomes. These findings demonstrate that comparable yield stability can arise from fundamentally different mechanisms of drought resistance and highlight the importance of integrating physiological, biochemical, and photochemical markers into breeding programs. Such insight supports more targeted maize improvement and the development of maize hybrids with predictable resilience to increasingly variable climatic conditions.
Programmed cell death (PCD) is a fundamental biological process required for tissue homeostasis and the removal of damaged or infected cells. Apoptosis, one of the most studied PCD modalities, is routinely assessed using the Annexin V/propidium iodide (PI) assay. However, the conventional Annexin V/PI protocol frequently produces false-positive necrotic signals due to membrane damage caused during cell harvesting and handling. This artifact compromises the metrological accuracy of necrosis quantification, limiting assay standardization in cancer research and potentially leading to the premature exclusion of promising anticancer compounds during in vitro screening. To address this issue, we developed a modified Annexin V/PI protocol designed to improve the reliability of necrosis detection. In the optimized approach, PI is added directly to the culture medium prior to cell harvesting, allowing uptake exclusively in cells with truly compromised membrane integrity. This strategy effectively reduces handling-induced artifacts and provides a more accurate evaluation of the membrane-compromised population. Precise discrimination between apoptotic and necrotic cells is essential for standardized cellular assays and for the accurate interpretation of drug-induced cytotoxic effects. Our revised protocol enhances accuracy, reproducibility, and robustness of apoptosis assessment, particularly in fragile cell systems, while remaining fully compatible with commercially available Annexin V/PI detection kits.
Biopharmaceuticals, particularly monoclonal antibodies (mAbs), are a rapidly expanding class of therapeutics and have benefitted from high-throughput screening (HTS) strategies for protein engineering. Obtaining such complex biopharmaceuticals from plants might be advantageous because transient expression can yield grams of product within a week. For HTS, this system was scaled down as plant cell pack (PCP) technology to microtiter plate format and automated on a liquid-handling station, but is limited by manual cloning and protein purification. Therefore, we first generated a modular vector library comprising 15 pTRAc backbone constructs differing in regulatory gene expression and protein targeting elements, enabling rapid integration of any target gene via restriction-ligation cloning. Building on this, we developed an automated, seamless cloning workflow prior to transient expression and integrated a magnetic bead-based downstream purification process for single-chain fragment variable (scFv) mAbs. This automated workflow enables sequential cloning, expression, and purification of up to 375 protein variants simultaneously, each with 10 PCP replicates, within 9 days on a single platform. In contrast, manual processing allows only 15 variants with 10 replicates in the same period — a 25-fold increase in throughput, while costs decreased by 30%, from 79 € to 61 € per variant. Using a recombinant immunotoxin against acute myeloid leukemia based on the anti-CD64 H22 scFv as case study, a recovery of 63% and purity > 95% were obtained, suitable for in vitro assays. Thus, integrating our cloning and purification workflows into the PCP platform establishes an automated pipeline that accelerates early biopharmaceutical development.
Spent mammalian cell culture media are commonly discarded despite having residual nutrients that could be repurposed for microbial biomanufacturing. Fibroblast growth factor 2 (FGF2) is a major cost contributor in serum-free cell culture media, motivating the development of economical production strategies. Here, we investigated the use of fortified spent cell culture media for FGF2 production using Lactococcus lactis, a GRAS microorganism, and integrated statistical optimization, kinetic modeling, and continuous bioprocessing. A Box-Behnken design identified 10 g/L glucose, 35 °C cultivation temperature, and 100 ng/mL nisin as optimal conditions for enhancing intracellular and secreted FGF2 production. Subsequently, kinetic model comprising of ordinary differential equations was developed to describe temporal profiles of intracellular & secreted FGF2, glucose, lactate, and biomass. The model integrated Monod-type kinetics for growth and substrate consumption, and Luedeking-Piret kinetics to account for FGF2 production. Leveraging this model, we demonstrated that model could guide selection of dilution rate to maximize FGF2 production. To advance toward continuous manufacturing, chemostat cultivation was coupled with depth and crossflow filtration to enable real-time cell separation and FGF2 concentration. The concentrated FGF2 demonstrated comparable bioactivity to commercial FGF2 in proliferating Anguilla japonica (Japanese eel) pre-adipocytic cells. Overall, our study demonstrated utilization of design of experiments (DoE) alongside kinetic modelling to optimize bioprocess conditions, achieving FGF2 production of 16 mg/L and 396 µg/L titres for intracellular and secreted forms, respectively. The circular bioprocess, use of predictive modelling and integration of continuous upstream-downstream operation provides an opportunity for growth factor manufacturing from L. lactis.
Loss-of-function mutations in the gene encoding progranulin (PGRN) are a common cause of frontotemporal dementia, leading to markedly decreased PGRN levels. A potential therapeutic strategy is therefore to increase extracellular PGRN by blocking sortilin-mediated PGRN clearance. Here, we describe the systematic design and optimization of small biparatopic sortilin-binding proteins based on the non-immunoglobulin affibody scaffold. Two anti-sortilin affibody molecules were genetically fused into a panel of heterodimeric constructs exploring multiple domain orientations, linker lengths, and helix truncations. In total, ten distinct dimer variants were generated and evaluated for binding and functional activity. Optimization of domain arrangement and truncations yielded constructs exhibiting subnanomolar sortilin affinities, corresponding to up to an approximately 45-fold improvement over the strongest monomeric affibody and pronounced avidity effects. In a PGRN clearance assay, the lead 18.6-kDa dimer efficiently increased extracellular PGRN levels with an EC50 value of 0.32 nM and produced substantially greater PGRN fold changes than monomeric constructs. This PGRN elevation was accompanied by marked reductions in both cell surface and total sortilin levels, consistent with effective receptor blockade and modulation. Together, these results demonstrate how systematic combination and geometric optimization of two individually moderate-affinity binders can generate a highly potent biparatopic inhibitor, as well as illustrating the versatility of compact and modular affibody molecules as building blocks in therapeutic protein design. Given the growing amount of evidence implicating sortilin in neurodegeneration, inflammation, and cancer, biparatopic affibody-based inhibitors may enable exploration of this pathway in diverse biological contexts, both as research tools and as starting points for drug development.
We established a platform for reconstructing the lycopene pathway by combinatorial co-expression of five constituent enzymes using a crude Escherichia coli cell-free protein synthesis (CFPS) system. DNA templates were prepared in a modular format, and their concentrations were used as quantitative inputs to manipulate expression levels. Co-expression from mixed templates reconstructed a functional lycopene pathway directly in vitro and replaced the conventional mix-and-match of separately expressed enzymes. Using a plate-based colorimetric assay, we showed that lycopene production is highly sensitive to both enzyme identity and template dosage, reflecting resource competition, endogenous activities in the lysate, and protein solubility. One-factor-at-a-time and homolog-swap experiments revealed basic design rules but also emphasized that the results were small and biased compared to possible co-expression design space. To explore this space more efficiently, we combined CFPS experiments with a hybrid active learning (HAL) workflow that ranks candidate DNA combinations based on model-guided criteria. HAL-guided rounds suggested high-performing and even simplified lycopene pathway configurations, indicating that CFPS co-expression together with HAL can serve as a practical platform to design and refine metabolic pathways before refactoring them into cells.
A major challenge in developing effective photocatalysts lies in engineering the efficient coupling of one-electron photochemistry with the multi-electron requirements of chemical transformations. Here we demonstrate biohybrid assemblies that achieve this key performance requirement by storing photoenergized electrons on multiple heme cofactors within the MtrC enzyme which catalyzes azo dye reduction. The biohybrid assemblies were created by site-selective labeling of MtrC with a Ru(II) (bipyridine)3 photosensitizer dye. Photocatalytic azo dye reduction and decoloration occurred when these assemblies were irradiated in the presence of a sacrificial electron donor. Our Ru(II) (bipyridine)3-MtrC biohybrid assemblies operate in a manner analogous to Ru(II) (bipyridine)3-sensitized TiO2 in the sense that photoenergized electrons accumulate in the MtrC heme chain rather than in the TiO2 conduction band prior to driving reductive chemical transformations. We anticipate that decoration of the photosensitized MtrC protein with electrocatalysts (natural or synthetic) will enable the Ru(II) (bipyridine)3-MtrC assemblies to drive a wide range of light-driven reductive transformations. Thus, MtrC provides a natural alternative to TiO2 materials for which the production and disposal present significant environmental and energy impacts.
Cyprinid herpesvirus-3 (CyHV-3) is highly contagious and lethal to cyprinid fish, necessitating development of sustainable vaccination strategies. Our study explores duckweed as platform for vaccine production and as vehicle for oral administration which is considered most suitable for mass vaccination of fish. Using a deconstructed potato virus X-based transient expression system, serologically recognizable recombinant CyHV-3 antigens (ORF25, ORF81, ORF136, ORF72) and fusion proteins (ORF136::T2A::ORF72, ORF25::T2A::ORF81) were accumulated in the duckweed Landoltia punctata. The specific immune response of fish on immunization by the freeze-dried antigen-expressing duckweed biomass was proved by the serum neutralization test. Uptake of green fluorescent protein fused to the adjuvant cholera non-toxic subunit B (CTB::GFP) from duckweed biomass by fish intestinal cells was demonstrated after intubation of fish with CTB::GFP-expressing biomass. Therefore, transient expression of recombinant proteins in duckweed is a promising tool for antigen production and can facilitate the development of oral vaccines for veterinary application.
Alzheimer's disease is associated with the aggregation of amyloid-β42 (Aβ42) into species of varying sizes, with intermediate oligomers being the most neurotoxic. We recently reported that amyloid precursor protein inhibitor (APPI), a Kunitz-type protein, and a cyclic peptide derived from its β-domain reduced Aβ42-mediated neurotoxicity, the former by reducing Aβ42 aggregation and formation of toxic Aβ42 oligomers, and the latter by promoting Aβ42 aggregation to form fibrils rather than the neurotoxic Aβ42 oligomers. To address the question of whether these two inhibition mechanisms are controlled by the structure or the amino acid sequence of the protein/peptide, we exploited three Kunitz-type proteins, bikunin, bovine pancreatic trypsin inhibitor (BPTI) and tissue factor pathway inhibitor (TFPI) - chosen for their similar β-strand-rich structures but different sequences to one another and to APPI - and also short peptides that mimic their β-domains, in either cyclic or linear conformation. In-vitro studies showed that the formation of Aβ42 aggregates was reduced by the three Kunitz-type proteins and by their derived cyclic peptides, but not by the linear counterparts of the cyclic peptides. In SH-SY5Y neuroblastoma cells, the Kunitz-type proteins and the cyclic (but not the linear) peptides reduced the intracellular and extracellular accumulation of Aβ42 aggregates, respectively. Both the Kunitz-type proteins and the cyclic peptides inhibited Aβ42-induced mitochondrial membrane depolarization and reduced Aβ42-mediated apoptosis and cell death. Overall, this study thus reveals the potential of the β-hairpin structure, whether as a segment within the Kunitz-type proteins or isolated as a cyclic peptide, to interact with Aβ42, thereby reducing Aβ42 aggregation and hence its neurotoxicity.
Komagataella phaffii (syn. Pichia pastoris) is a methylotrophic yeast widely established as host for recombinant protein production and increasingly used as cell factory for C1-based metabolites. Nevertheless, synthetic biology tools for regulating protein expression in this host remain limited, relying largely on promoters of genes from carbon metabolism. Riboswitches, i.e. mRNA elements that regulate translation or splicing via ligand-dependent folding, offer a complementary layer of regulation, yet remain underused in K. phaffii synthetic biology. In the methylotrophic yeast Ogataea polymorpha, an intron within the DUR31 gene acts as a thiamine pyrophosphate (TPP) riboswitch, downregulating gene expression through alternative splicing, and retaining a premature stop codon in response to exogenous thiamine. We evaluated this TPP riboswitch in K. phaffii as a tool to modulate expression from the glycolytic GAP promoter. Using EGFP and destabilised UBIYΔkGFP* as reporters in combination with flow cytometry, we show that the orthogonal riboswitch is functional in K. phaffii. Like many other described riboswitches, the system exhibits substantial basal (leaky) expression and cannot be considered a tight molecular on/off switch. RT-PCR confirmed the presence of both spliced and unspliced transcripts in the cells regardless of external thiamine supplementation. Unexpectedly, inserting an additional exon upstream of the riboswitch intron abolished detectable downstream protein production, indicating that splicing efficiency is strongly influenced by the local sequence context of the splice sites.
The combination of the Designed Ankyrin Repeat Protein (DARPin) scaffold with the selection and evolution technology of Ribosome Display has generated a wide range of specific binders to hundreds of target proteins. The binders have been used in a very diverse set of applications, from research through diagnostics to therapy. In this article, we focus on the high-throughput (HT) methods that we have developed for this purpose, which have been applied in many selections for research applications, but have never been explained in detail. We describe here the HT methods used to select for many targets in parallel, permitting the use of even different buffers and components, as well as some of the downstream characterization. To illustrate the workflow, we summarize the properties of some of the binders so obtained and their applications as well as the structures of the DARPin/target complexes. Together, these HT methods establish a robust framework for the rapid discovery and characterization of DARPins across a broad spectrum of targets.
Hydrolytic host cell proteins (HCPs) from Chinese hamster ovary (CHO) cells, particularly lipoprotein lipase (LPL), pose a persistent challenge in biopharmaceutical manufacturing due to their degrading activity against polysorbate (PS), a commonly used surfactant in therapeutic protein formulations. While downstream purification effectively removes many HCPs, LPL remains difficult to eliminate and upstream cultivation conditions influencing its abundance are poorly understood. Here, we demonstrate that CHO cultivation medium composition is a key-contributor of extracellular LPL levels and PS degradation activity. Using a fluorescence micelle assay, recombinant LPL expression systems and live-cell LPL staining, we show that medium-dependent differences in PS degradation are not attributable to transcription, translation, secretion or proteolytic processing. Instead, LPL-cell surface interactions, probably mediated primarily by heparan sulfate proteoglycans, appear to contribute to LPL retention and release. Experimental medium A promoted strong surface binding and low levels of LPL in the supernatant, whereas experimental medium B enhanced LPL release, correlating with increased PS degradation. Heparin displacement and rapid medium-switch experiments confirmed that these interactions are dynamic, reversible and sensitive to the physicochemical environment. Our findings suggest that the charged components of the culture medium modulate LPL electrostatic binding to the cell surface, thereby controlling its extracellular abundance. From a bioprocessing perspective, these results suggest a novel factor that affects hydrolytic HCP burden by cell culture medium composition and cell surface retention mechanisms. Collectively, this study provides mechanistic insights into LPL regulation in CHO cultures and supports a framework for controlling problematic HCPs to improve the NBE production process.
UDP-GlcNAc and UDP-GalNAc are well-demanded nucleotide sugars and serve as precursors for the enzymatic synthesis of N- and O-glycans as well as glycosaminoglycans. Although the enzymatic synthesis cascade has been extensively studied, limitations remain for an efficient large-scale production. Unfavorable inhibitions by intermediate accumulation, precipitation of the byproduct phosphate with the needed cofactor magnesium, and efficient enzyme dosage still pose significant challenges. These challenges were investigated in an enzyme cascade including N-acetyl-hexosamine kinase from Bifidobacterium longum (BlNahK), UDP-N-acetylgalactosamine diphosphorylase from Homo sapiens (HsAGX1), and inorganic pyrophosphatase from Pasteurella multocida (PmPpA) for UDP-GlcNAc and UDP-GalNAc synthesis. ATP supply started from AMP and polyphosphate by polyphosphate kinase from Cytophaga hutchinsonii (ChPPK), and UTP was generated from UMP using cytidine/uridine monophosphate kinase from Escherichia coli (EcCMPK) and cytidine/uridine diphosphate kinase from Saccharomyces cerevisiae (ScCDPK). To reach a high productivity, enzyme cascade parameters were determined and optimized using Multiplex capillary electrophoresis. By kinetic modelling of the enzyme cascade and fine-tuning of the uncoupled UTP/ATP generation/regeneration system as well as observing the magnesium polyphosphate interplay with enzyme activity we were able to scale the production up to a molarity of 100 mM in 200 mL, yielding 10 g of UDP-GlcNAc with a conversion of 82.4% in 48 h and 12 g of UDP-GalNAc with full conversion in 29 h. The key limitations for successful scale-up, namely the promiscuity of BlNahK towards UTP and interplay of magnesium ions with phosphate species, were overcome. Overall, the presented insights are expected to be transferable to similar enzyme cascades.
Phytoceramides are essential sphingolipids that support skin barrier integrity and hydration, making them valuable for cosmetic and pharmaceutical applications. However, their intricate structures and low natural abundance pose significant challenges for scalable production. Here, we present an integrated metabolic engineering and lipidomics study aimed at enhancing phytoceramide production in Saccharomyces cerevisiae. We implemented three strategies: (i) overexpression of SUR2 (sphinganine C4-hydroxylase) to boost phytosphingolipid formation; (ii) deletion of SCS7 (ceramide α-hydroxylase) to redirect flux toward non-hydroxylated phytoceramides; and (iii) overexpression of ISC1 (inositol phosphosphingolipid phospholipase) to recycle complex sphingolipids into ceramide pools. SUR2 overexpression showed the highest transcript levels, whereas lipidomics revealed that scs7Δ produced the highest phytoceramide enrichment with a 15-fold increase in phytoceramide abundance relative to the wild type. In terms of relative abundance within the quantified ceramide pool, phytoceramides increased from 5% in wild type to 46% in the SUR2-OE strain and 75% in the scs7Δ strain. The combined scs7Δ SUR2-OE strain did not exhibit additive metabolic effects on the lipid profile. The presence of residual hydroxylated ceramides indicated intrinsic regulatory constraints, aligning with the bypass mechanism proposed here whereby ceramide synthases can use pre-hydroxylated acyl-CoA. Importantly, this work contributes a comprehensive lipidomic profiling of S. cerevisiae, enabling clear discrimination between engineered and wild type strains and identification of genotypes exerting the greatest impact on phytoceramide accumulation. This approach advances sphingolipid pathway modulation and positions S. cerevisiae as a valuable model for studying phytoceramide-focused remodeling.
Komagataella phaffii is a promising cell factory that can use CO2 derived methanol, a sustainable carbon (C1) source, for chemical production. Introducing the ß-alanine biosynthetic pathway alongside a NADP+-dependent formate dehydrogenase (FDH) in K. phaffii enables the production of the platform chemical 3-hydroxypropionic acid (3-HP). Co-feeding of formate and methanol (MeOH) was systematically explored to enhance cellular reducing power and improve 3-HP biosynthesis. Implementing a pulsed formate strategy alongside MeOH resulted in up to a 20.7% increase in 3-HP per gram of MeOH (Yg3-HP gMeOH-1) compared to MeOH alone in shake flask cultivations. This co-feeding strategy likely enhanced NADPH availability through formate oxidation via the introduced NADP⁺-dependent FDH, thereby improving redox balance, as supported by simulation studies based on the K. phaffii genome-scale metabolic model. Similar improvements were demonstrated in repeated batch cultivations at 1-L bioreactor scale, where a formate pulse every 4-hour led to a 25.8% increase in Yg3-HP gMeOH-1 and a 41% increase in volumetric productivity over the control without formate (only MeOH). In addition to the feeding strategy, pH regulation also played a crucial mechanistic role: strict pH control at 5 inhibited growth, due to the predominance of undissociated formic acid, whereas allowing the pH to rise to 6-7 favoured dissociation and supported higher productivity. These findings elucidate the pH dependent nature of formate assimilation and highlight the potential of coupling MeOH and formate co-utilization with dynamic feeding and pH strategies to enhance bioproduction in K. phaffii.
End-to-end continuous biopharmaceutical manufacturing extends process intensification from improved standalone unit operations to integrated process trains designed for long-duration operation with minimal pooling. This review synthesizes recent progress across upstream and downstream processing and frames end-to-end continuity as a system-level integration and interface-engineering problem in which throughput, residence-time behavior, and quality are jointly managed. Upstream perfusion sustains high viable cell density and productivity, but it tightens constraints on critical process parameters and demands robust monitoring to deliver a consistent feed. In downstream processing, continuous capture and polishing, continuous conditioning, and continuous-compatible viral clearance steps are increasingly feasible. However, long-duration robustness, cyclic dynamics, and sensitivity to feed variability remain central considerations. Across integration case studies, the dominant barrier is not missing unit operations but unresolved interface mismatches, especially between steady upstream harvest and cyclic downstream operations, that drive surge and hold requirements unless engineered out through residence-time and cycle-time synchronization, in-line conditioning, and long-duration filtration performance management. In addition, process analytical technologies and digital twins underpin real-time observability and control, while ultra scale-down and high-throughput development workflows accelerate operating-window definition and generate data for model calibration and control design. Overall, practical end-to-end continuity depends on coordinated advances in interface engineering, platform-level integration, and credible validation strategies for extended continuous operation.
