Production Platform Research Articles

Screening of novel β-carotene hydroxylases for the production of β-cryptoxanthin and zeaxanthin and the impact of enzyme localization and crowding on their production in Yarrowia lipolytica.

Zeaxanthin, a vital dietary carotenoid, is naturally synthesized by plants, microalgae, and certain microorganisms. Large-scale zeaxanthin production can be achieved through plant extraction, chemical synthesis, or microbial fermentation. The environmental and health implications of the first two methods have made microbial fermentation an appealing alternative for natural zeaxanthin production despite the challenges in scaling up the bioprocess. An intermediate between β-carotene and zeaxanthin, β-cryptoxanthin, is found only in specific fruits and vegetables and has several important functions for human health. The low concentration of β-cryptoxanthin in these sources results in low extraction yields, making biotechnological production a promising alternative for achieving higher yields. Currently, there is no industrially relevant microbial fermentation process for β-cryptoxanthin production, primarily due to the lack of identified enzymes that specifically convert β-carotene to β-cryptoxanthin without further conversion to zeaxanthin. In this study, we used genetic engineering to leverage the oleaginous yeast Yarrowia lipolytica as a bio-factory for zeaxanthin and β-cryptoxanthin production. We screened 22 β-carotene hydroxylases and identified eight novel enzymes with β-carotene hydroxylating activity: six producing zeaxanthin and two producing only β-cryptoxanthin. By introducing the β-carotene hydroxylase from the bacterium Chondromyces crocatus (CcBCH), a β-cryptoxanthin titer of 24 ± 6mg/L was achieved, representing the highest reported titer of sole β-cryptoxanthin in Y. lipolytica to date. By targeting zeaxanthin-producing β-carotene hydroxylase to the endoplasmic reticulum and peroxisomes, we increased the production of zeaxanthin by 54% and 66%, respectively, compared to untargeted enzyme. The highest zeaxanthin titer of 412 ± 34mg/L was achieved by targeting β-carotene hydroxylases to peroxisomes. In addition, by constructing multienzyme scaffold-free complexes with short peptide tags RIDD and RIAD, we observed a 39% increase in the zeaxanthin titer and a 28% increase in the conversion rate compared to the strain expressing unmodified enzyme. The zeaxanthin titers obtained in this study are not the highest reported; however, our goal was to demonstrate that specific approaches can enhance both titer and conversion rate, rather than to achieve the maximum titer. These findings underscore the potential of Y. lipolytica as a promising platform for carotenoid production and provide a foundation for future research, where further optimization is required to maximize production.

Product Screening in Live stream: An Equilibrium Strategy Analysis Considering Streamer Product Selection and Platform Inspection

Product Screening in Live stream: An Equilibrium Strategy Analysis Considering Streamer Product Selection and Platform Inspection

High-Level Production of a Recombinant Protein in Nicotiana benthamiana Leaves Through Transient Expression Using a Double Terminator

Various bio-based recombinant proteins have been produced for industrial, medical, and research purposes. Plants are potential platforms for recombinant protein production because of several advantages. Therefore, establishing a system with high target gene expression to compensate for the low protein yield of plant systems is crucial. In particular, selecting and combining strong terminators is essential because the expression of target genes can be substantially enhanced. Here, we aimed to quantify the enhancement in the fluorescence intensity of the turbo green fluorescence protein (tGFP) caused by the best double-terminator combinations compared to that of the control vector using agroinfiltration in Nicotiana benthamiana leaves. tGFP fluorescence increased by 4.1-fold in leaf samples infiltrated with a vector containing a double terminator and markedly increased by a maximum of 23.7-fold when co-infiltrated with the geminiviral vector and P19 compared to that in constructs containing an octopine synthase terminator. Polyadenylation site analysis in leaf tissues expressing single or dual terminators showed that the first terminator influenced the polyadenylation site determination of the second terminator, resulting in different polyadenylation sites compared with when the terminator is located first. The combination of the high-expression terminators and geminiviral vectors can increase the production of target proteins.

China as data coloniser? rethinking cultural production, cultural mediation, and consumer agency on Kenyan and Chinese e-commerce platforms

Has China become a neo-colonizer, exporting its cultural and economic power to the world based on its agenda of building soft power? Existing scholarship on neocolonialism and data colonialism largely focuses on how China's infrastructural expansion and increasingly platformised cultural sectors can achieve its ambitious platformised cultural sectors overseas. Yet, how China's cultural power is manifested, negotiated, or resisted in people's daily lives in a South–South setting remains under-researched and under-theorised. This article uses everyday fashion in Kenya as a case study to investigate China's cultural and economic power expansion in the Global South. We examined how cultural differences are negotiated and mediated on two Chinese(-invested) e-commerce platforms. Through focus groups and platform walkthrough method, our findings serve to enrich existing theories of cultural production–platform relationships applicable in the study of various cultural and creative sectors, to offer new understandings of how symbolic, sociopolitical and cultural meanings of fashion are constructed through divergent platform affordances, and to reveal the various forms of cultural negotiations and resistance in different contexts, multiplying our frames of reference.

Establishing the green algae Chlamydomonas incerta as a platform for recombinant protein production.

ABSTRACTChlamydomonas incerta, a genetically close relative of the model green algaChlamydomonas reinhardtii, shows significant potential as a host for recombinant protein expression. Because of the close genetic relationship betweenC. incertaandC. reinhardtii, this species offers an additional reference point for advancing our understanding of photosynthetic organisms, and also provides a potential new candidate for biotechnological applications. This study investigates C. incerta’s capacity to express three recombinant proteins: the fluorescent protein mCherry, the hemicellulose-degrading enzyme xylanase, and the plastic-degrading enzyme PHL7. We have also examined the capacity to target protein expression to various cellular compartments in this alga, including the cytosol, secretory pathway, cytoplasmic membrane, and cell wall. When compared directly withC. reinhardtii,C. incertaexhibited a distinct but notable capacity for recombinant protein production. Cellular transformation with a vector encoding mCherry revealed thatC. incertaproduced approximately 3.5 times higher fluorescence levels and a 3.7-fold increase in immunoblot intensity compared toC. reinhardtii. For xylanase expression and secretion, bothC. incertaandC. reinhardtiishowed similar secretion capacities and enzymatic activities, with comparable xylan degradation rates, highlighting the industrial applicability of xylanase expression in microalgae. Finally,C. incertashowed comparable PHL7 activity levels toC. reinhardtii, as demonstrated by the in vitro degradation of a polyester polyurethane suspension, Impranil® DLN. Finally, we also explored the potential of cellular fusion for the generation of genetic hybrids betweenC. incertaandC. reinhardtiias a means to enhance phenotypic diversity and augment genetic variation. We were able to generate genetic fusion that could exchange both the recombinant protein genes, as well as associated selectable marker genes into recombinant offspring. These findings emphasizeC. incerta’s potential as a robust platform for recombinant protein production, and as a powerful tool for gaining a better understanding of microalgal biology.GRAPHICAL ABSTRACT

Elevated endoplasmic reticulum pH is associated with high growth and bisAb aggregation in CHO cells.

Chinese hamster ovary (CHO) bioprocesses, the dominant platform for therapeutic protein production, are increasingly used to produce complex multispecific proteins. Product quantity and quality are affected by intracellular conditions, but these are challenging to measure and often overlooked during process optimization studies. pH is known to impact quality attributes like protein aggregation across upstream and downstream processes, yet the effects of intracellular pH on cell culture performance are largely unknown. Recently, advances in protein biosensors have enabled investigations of intracellular environments with high spatiotemporal resolution. In this study, we integrated a fluorescent pH-sensitive biosensor into a bispecifc (bisAb)-producing cell line to investigate changes in endoplasmic reticulum pH (pHER). We then investigated how changes in lactate metabolism impacted pHER, cellular redox, and product quality in fed-batch and perfusion bioreactors. Our data show pHER rapidly increased during exponential growth to a maximum of pH 7.7, followed by a sharp drop in the stationary phase in all perfusion and fed-batch conditions. pHER decline in the stationary phase was driven by an apparent loss of cellular pH regulation that occurred despite differences in redox profiles. Finally, we found protein aggregate levels correlated most closely with pHER which provides new insights into product aggregate formation in CHO processes. An improved understanding of the intracellular changes impacting bioprocesses can ultimately help guide media optimizations, improve bioprocess control strategies, or provide new targets for cell engineering.

Development of an Intelligent Coal Production and Operation Platform Based on a Real-Time Data Warehouse and AI Model

Smart mining solutions currently suffer from inadequate big data support and insufficient AI applications. The main reason for these limitations is the absence of a comprehensive industrial internet cloud platform tailored for the coal industry, which restricts resource integration. This paper presents the development of an innovative platform designed to enhance safety, operational efficiency, and automation in fully mechanized coal mining in China. This platform integrates cloud edge computing, real-time data processing, and AI-driven analytics to improve decision-making and maintenance strategies. Several AI models have been developed for the proactive maintenance of comprehensive mining face equipment, including early warnings for periodic weighting and the detection of common faults such as those in the shearer, hydraulic support, and conveyor. The platform leverages large-scale knowledge graph models and Graph Retrieval-Augmented Generation (GraphRAG) technology to build structured knowledge graphs. This facilitates intelligent Q&A capabilities and precise fault diagnosis, thereby enhancing system responsiveness and improving the accuracy of fault resolution. The practical process of implementing such a platform primarily based on open-source components is summarized in this paper.

Purification of T7 RNA polymerase for large scale production of mRNA vaccines and therapeutics

The COVID-19 Pandemic has revolutionized the field of mRNA vaccine technology, highlighting its utility and rapidly expanding potential, propelling it to the forefront of future vaccines & therapeutics for cryptic diseases. T7 RNA Polymerase (T7 RNAP) is a high-fidelity enzyme used for the synthesis of single-stranded mRNA from DNA templates using In vitro transcription (IVT). T7 RNAP is now the mainstay for the development of mRNA vaccines and therapeutics, accounting for 34 % of the overall raw material cost for mRNA manufacturing. Although small-scale purification processes for T7 RNAP exist, scalable process platforms and comprehensive analytical characterization are still lacking. Moreover, to address global raw material supply chain issues and to facilitate the efficient and large-scale production of mRNA vaccines & therapeutics, here we report a three-step purification process platform, yielding ∼ 85–100 mg/L of T7 RNAP from E. coli fermentation harvest. Also, to demonstrate its high-level purity and structural integrity, we have characterized it using orthogonal analytical techniques. Furthermore, when evaluated in an IVT system, this highly pure T7 RNAP showed activity that was comparable to the commercial T7 RNAP in producing integral RNA (10 kb) from a linearized plasmid DNA template. Collectively, these findings establish a purification process platform for the commercial production of T7 RNAP enzyme to support the future development of mRNA-based vaccines and therapeutics.

An efficient C-glycoside production platform enabled by rationally tuning the chemoselectivity of glycosyltransferases

Despite the broad potential applications of C-glycosides, facile synthetic methods remain scarce. Transforming glycosyltransferases with promiscuous or natural O-specific chemoselectivity to C-glycosyltransferases is challenging. Here, we employ rational directed evolution of the glycosyltransferase MiCGT to generate MiCGT-QDP and MiCGT-ATD mutants which either enhance C-glycosylation or switch to O-glycosylation, respectively. Structural analysis and computational simulations reveal that substrate binding mode govern C-/O-glycosylation selectivity. Notably, directed evolution and mechanism analysis pinpoint the crucial residues dictating the binding mode, enabling the rational design of four enzymes with superior non-inherent chemoselectivity, despite limited sequence homology. Moreover, our best mutants undergo testing with 34 substrates, demonstrating superb chemoselectivities, regioselectivities, and activities. Remarkably, three C-glycosides and an O-glycoside are produced on a gram scale, demonstrating practical utility. This work establishes a highly selective platform for diverse glycosides, and offers a practical strategy for creating various types of glycosylation platforms to access pharmaceutically and medicinally interesting products.

Beyond Anaerobic Digestion: New Perspectives for the Development of a Biorefinery Platform for the Simultaneous Production of Medium-Chain Fatty Acids by Chain Elongation and Biogas from Food Wastes

Beyond Anaerobic Digestion: New Perspectives for the Development of a Biorefinery Platform for the Simultaneous Production of Medium-Chain Fatty Acids by Chain Elongation and Biogas from Food Wastes

Operational long-term management of a salt cavern for industry targeted green H2 production

Hydrogen and its derivatives such as ammonia are increasingly important for reducing carbon emissions in sectors with limited alternatives. Large production systems are generally preferred to reduce costs and for those, advanced management is a cornerstone to the project economic viability. The present work explores the development of an Energy Management System for an industrial low-emission ammonia production platform. The latter is connected to the electrical grid and consists of 1.5 GW of renewable production units, around 500 MW of electrolyzers and storage facilities including especially a salt cavern for H2 storage. The long-term management of the salt cavern is decisive to enhance the profitability while satisfying industry related constraints such as the carbon content of the produced H2. The literature lacks in addressing both aspects simultaneously. Methodological approaches to tackle the long-term management and industrial constraints are proposed and compared. For the considered platform, it is shown that a strategy based on precalculated cost functions in the long term horizon allows relative gains of 2.5% with respect to a state-of-the-art short term management leading to an annual cost saving of approximately $6.2 million in the platform operational expenses. This translates to a reduction of the gap between the state-of-the-art and the optimal a posteriori solution by about 82%.

Coordination of interests of participants in digital commodity platforms (marketplaces): legal problems

The article examines problems that have emerged in practice related to the interaction of marketplace owners with sellers, buyers and other participants when concluding and executing contracts for the sale of goods on digital commodity platforms. The concepts of a digital platform, marketplace, and information aggregator, which have not received an unambiguous solution in legal acts, are defined, the legal status of the owner of the marketplace is investigated, and a negative assessment of the state of legal regulation of relations developing on digital product platforms (marketplaces) is given. A mechanism for legal regulation of the relations under consideration is proposed, which allows not to limit the development of marketplaces and at the same time ensure the coordination of the interests of marketplace owners as entities with significant market power with participants in transactions on digital commodity platforms.

Point-of-care manufacturing of anti-CD19 CAR-T cells using a closed production platform: Experiences of an academic in Thailand

Point-of-care manufacturing of anti-CD19 CAR-T cells using a closed production platform: Experiences of an academic in Thailand

Engineering hierarchical TiO2/ZnIn2S4 hybrid heterostructure with synergistic interfaces: A dual-functional S-scheme photocatalyst for efficient CO2 reduction and norfloxacin degradation

The development of multifunctional photocatalysts that can harness solar energy for both environmental remediation and energy generation is a considerable challenge in achieving sustainable development. This study introduces a dual-functional S-scheme hybrid catalyst comprising hierarchical 3D flower-like ZnIn2S4 (ZIS) microspheres embedded with TiO2 (TO) nanoparticles, synthesized via a facile in situ hydrothermal process. The unique structural and compositional synergy between ZIS and TO leverages their complementary photocatalytic properties, facilitating efficient solar energy utilization, increasing specific surface area, and enhancing CO2 adsorption capabilities. The S-scheme mechanism, confirmed by in situ-irradiated X-ray photoelectron spectroscopy and electron spin resonance spectroscopy, underlying this system promotes effective separation of photoexcited charge carriers while preserving the intrinsic strong reducibility of ZIS and high oxidizability of TO. These beneficial properties of the TO/ZIS hybrid provide a robust platform for CH4 production (75.6 μmol h−1 g−1) through selective (99.6 %) CO2 reduction over competing water reduction and achieve exceptional degradation and mineralization of the persistent antibiotic norfloxacin in water, addressing both energy and environmental challenges. Furthermore, the hybrid catalyst exhibits significant stability and recyclability, maintaining high catalytic performance across multiple cycles, thereby underscoring its practical viability. This work offers a promising blueprint for developing multifunctional photocatalysts capable of addressing critical global challenges, demonstrating the potential of hierarchical nanostructures and S-scheme charge transfer mechanisms.

Aeroacoustic analysis of flow induced resonance in a gas pipeline

Flow induced noise and vibration in the pipework of gas production platforms can lead to fatigue failures of attached small-bore pipes, and this can constrain the operating conditions of the platform. Typically such problems comprise a source of turbulence in the flow, an aeroacoustic feedback mechanism due to acoustic resonances inside the pipework, and mechanical resonances which amplify the resulting vibration. The problem described here occurred in the gas metering system of the platform, with the source located in the header which comprised a bifurcation into two metering lines and a number of sharp bends. Because of the cost of rectifying the problem an in-depth study was carried out to analyse the flow and induct acoustic conditions. The predictions will support recommendations for an efficient redesign of the entire system to control the problem at source. Various levels of CFD modelling (RANS and URANS) were carried out using the Openfoam code to predict the exact location and mechanism of the vortex shedding. The acoustic response of the complex ductwork was then modelled using the ACTRAN code to understand the feedback mechanism which led to resonance and high levels of response.

Advances in microalgae-based lutein production and extraction: enhancing bioavailability and applications in health and industry

BackgroundNatural lutein, a bioactive xanthophyll, is widely used in pharmaceuticals, nutraceuticals, aquaculture, and cosmetics due to its broad health benefits, especially in protecting ocular health and chronic and acute syndromes. Microalgae have become a preferred sustainable platform for high-value lutein production, surpassing marigold petals in content (∼2 mg g−1) and consistency. MethodsThis review highlights advancements in upstream lutein production and downstream extraction techniques, focusing on scalable and sustainable bioprocesses. Under optimal conditions, microalgae cultivation in open and closed reactors for 2–4 weeks is examined. Comparative yields among studies are analyzed, detailing which technologies and their combinations offer superior yields for commercial production. Significant FindingsMicroalgae demonstrate high lutein content (∼10–17 mg g−1) and productivity (2–7 mg L−1d−1). The review identifies key advancements in smart delivery systems and bioavailability enhancement strategies, which are novel aspects of lutein research. The review aims to expand lutein distribution and its health benefits by addressing production challenges, market potential, and commercial opportunities. Aligning with Sustainable Development Goals (SDGs), the review promotes good health (SDG 3), economic growth (SDG 8), industry innovation (SDG 9), and climate action (SDG 13).

Efficient enhancement of the antimicrobial activity of Chlamydomonas reinhardtii extract by transgene expression and molecular modification using ionizing radiation

BackgroundIonizing radiation has been used for mutagenesis or material modification. The potential to use microalgae as a platform for antimicrobial production has been reported, but little work has been done to advance it beyond characterization to biotechnology. This study explored two different applications of ionizing radiation as a metabolic remodeler and a molecular modifier to enhance the antimicrobial activity of total protein and solvent extracts of Chlamydomonas reinhardtii cells.ResultsFirst, highly efficient transgenic C. reinhardtii strains expressing the plant-derived antimicrobial peptides, AtPR1 or AtTHI2.1, were developed using the radiation-inducible promoter, CrRPA70Ap. Low transgene expression was significantly improved through X-irradiation (12–50 Gy), with peak activity observed within 2 h. Protein extracts from these strains after X-irradiation showed enhanced antimicrobial activity against the prokaryotic bacterium, Pseudomonas syringae, and the eukaryotic fungus, Cryptococcus neoformans. In addition, X-irradiation (12 Gy) increased the growth and biomass of the transgenic strains. Second, C. reinhardtii cell extracts in ethanol were γ-irradiated (5–20 kGy), leading to molecular modifications and increased antimicrobial activity against the phytopathogenic bacteria, P. syringae and Burkholderia glumae, in a dose-dependent manner. These changes were associated with alterations in fatty acid composition. When both transgenic expression of antimicrobial peptides and molecular modification of bioactive substances were applied, the antimicrobial activity of C. reinhardtii cell extracts was further enhanced to some extent.ConclusionOverall, these findings suggest that ionizing radiation can significantly enhance the antimicrobial potential of C. reinhardtii through efficient transgene expression and molecular modification of bioactive substances, making it a valuable source of natural antimicrobial agents. Ionizing radiation can act not only as a metabolic remodeler of transgene expression in microalgae but also as a molecular modifier of the bioactive substances.

A Hybrid Data-Driven Optimization and Decision-Making Approach for a Digital Twin Environment: Towards Customizing Production Platforms

A Hybrid Data-Driven Optimization and Decision-Making Approach for a Digital Twin Environment: Towards Customizing Production Platforms

Synthetic recovery of Yada Yada virus expands insect-specific alphavirus knowledge and facilitates production of chimeric viruses

Few insect-specific alphaviruses (ISA) have been discovered, with even fewer culturable to facilitate full characterisation. Here, we report the recovery of an infectious clone of Yada Yada virus (YYV)—a virus previously only detected by metagenomic sequencing of mosquito homogenates. Using the infectious clone, we confirmed the inability of YYV to replicate in vertebrate cells in vitro, with replication limited to only Aedes mosquito-derived cell lines. We further produced and characterised the first monoclonal antibodies (mAbs) to ISAs. Through successful replacement of the structural proteins of YYV with those of other ISAs, Eilat virus, Agua Salud (ASALV), Taï Forest (TALV) and Mwinilunga alphaviruses (MWAV), we established that a replication block for in vitro culture of TALV and MWAV in mosquito cells does not exist at virus entry. Unexpectedly, ASALV structural proteins were recognised by cross-reactive mAbs made to chikungunya (CHIKV) and Ross River viruses (RRV), suggesting a potential antigenic link between ASALV and pathogenic alphaviruses. The YYV genetic backbone was also investigated to generate chimeras displaying the structural proteins of various pathogenic vertebrate-infecting alphaviruses including CHIKV, RRV, Barmah Forest, Sindbis and Mayaro viruses. These chimeras retained the antigenic properties of the parental viruses and did not replicate in vertebrate cells, demonstrating the potential of the YYV platform for vaccine and diagnostic antigen production.

Evaluation of Recombinant Antibody Production Efficiency in CHO Cells with Sleeping Beauty Transposon Vector System

Chinese hamster ovary (CHO) mammalian cell lines are widely used as cell platforms in biopharmaceutical productions. Different transfection systems are employed for the integration of the target gene cassette into the cell genome and have limitations, such as (i) the integration region in the genome, (ii) the size of the target cassette, and (iii) long selection periods for stable expression. Transposon systems can be utilized to overcome the limitations mentioned in the efficient production of commercially significant recombinant proteins. This study aims to demonstrate the differences in production potential and selection periods by using a specially designed vector system for random genome integration in CHODG44 DHFR -/- cells and the Sleeping Beauty (SB) transposon system. In this context, the optimal transfer ratio between the donor and the helper plasmid was determined for the most efficient co-transfection in the SB transposon system. According to the results, the pools obtained using the SB transposon system had titers ranging from 1300 to 2600 mg/L in 13-day fed-batch studies, while the pool obtained using the random transfer system had a titer of 0.056 mg/L. Additionally, stable cell pools obtained using the transposon system underwent selection in a short period of 52 days, compared to over 100 days for the pool obtained through random transfer. Considering all these results together, it is demonstrated that stable CHO pools obtained using the optimal SB transposon system can achieve high-efficiency monoclonal antibody production in a short period, making it an optimal production platform in the biopharmaceutical field.