Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Ancestral sequence reconstruction (ASR) offers a revolutionary approach to resurrect functional proteins, yet its potential in transporter engineering remains underexplored. Here, we pioneered the application of ASR to reconstructing ancestral xylose transporters, addressing the persistent challenge of glucose-mediated inhibition of xylose uptake in Saccharomyces cerevisiae during xylose co-fermentation. Through rigorous ASR analysis, we reconstructed ancestral xylose transporters (Xt) and selected two candidates—Xt3 (approximately 140 million years old) and Xt7 (approximately 40 million years old)—based on their phylogenetic positioning, degree of sequence divergence from extant homologs, and predicted structural integrity. Functional characterization demonstrated that both Xt3 and Xt7 significantly enhance xylose uptake efficiency and mitigate glucose-induced repression. In fermentation experiments with mixed sugars (40 g/L xylose and 40 g/L glucose) within 72 h, recombinant S. cerevisiae expressing Xt3 achieved 22.75 g/L xylose consumption, surpassing the benchmark N326F Xltr1p (16.22 g/L) by 40.27% and outperforming Xt7 (21.36 g/L) by 6.51%, highlighting Xt3 as the most efficient transporter. Molecular docking suggested a potentially more favorable binding mode for xylose in the ancestral transporters (binding affinity: −3.68 kcal/mol for Xt3 vs. −3.15 kcal/mol for N326F Xltr1p ). Molecular dynamics simulations further demonstrated that the ancestral transporters formed complexes with xylose that exhibited faster convergence to a stable state and maintained significantly greater conformational stability throughout the simulation compared to the N326F Xltr1p complex. These computational insights provide a plausible structural basis for their enhanced performance. This work contributes to the advancement of lignocellulosic biorefinery technology and provides a practical reference for resurrecting other valuable proteins using ASR’. The online version contains supplementary material available at 10.1186/s40643-025-00995-1. Keywords: Ancestral sequence reconstruction, Xylose transporter, Glucose/xylose co-utilization, Saccharomyces cerevisiae

Candidates:
A. Ancestral sequence reconstruction (ASR) offers a revolutionary approach to resurrect functional proteins, yet its potential in transporter engineering remains underexplored.
B. Functional characterization demonstrated that both Xt3 and Xt7 significantly enhance xylose uptake efficiency and mitigate glucose-induced repression.
C. Through rigorous ASR analysis, we reconstructed ancestral xylose transporters (Xt) and selected two candidates—Xt4 (approximately 140 million years old) and Xt7 (approximately 40 million years old)—based on their phylogenetic positioning, degree of sequence divergence from extant homologs, and predicted structural integrity.
D. Functional characterization demonstrated that both Xt4 and Xt7 significantly enhance xylose uptake efficiency and mitigate glucose-induced repression.
E. The evidence does not state that ancestral sequence reconstruction (ASR) offers a revolutionary approach to resurrect functional proteins, yet its potential in transporter engineering remains underexplored.
F. The evidence does not state that here, we pioneered the application of ASR to reconstructing ancestral xylose transporters, addressing the persistent challenge of glucose-mediated inhibition of xylose uptake in Saccharomyces cerevisiae during xylose co-fermentation.
G. Here, we pioneered the application of ASR to reconstructing ancestral xylose transporters, addressing the persistent challenge of glucose-mediated inhibition of xylose uptake in Saccharomyces cerevisiae during xylose co-fermentation.
H. Through rigorous ASR analysis, we reconstructed ancestral xylose transporters (Xt) and selected two candidates—Xt3 (approximately 140 million years old) and Xt7 (approximately 40 million years old)—based on their phylogenetic positioning, degree of sequence divergence from extant homologs, and predicted structural integrity.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Natural products (NPs) and their analogues have long underpinned therapies in humans, animals, and plants health, yet, discovering truly novel scaffolds remains a formidable challenge, even with the enormous diversity offered. Over the last two decades, breakthroughs in bioinformatics, cheminformatics, advanced analytical methods, synthetic biology toolkits, and optimized microbial culture have surmounted many of the bottlenecks that stalled NP research in the 1990s and 2000s. Researchers now deploy innovative extraction and purification protocols alongside high-throughput dereplication tools to fish trace metabolites out of complex matrices. These combined approaches not only enable the discovery and rigorous characterization of biosynthesized metabolites, bio-transformed analogues and new chemical entities but also allow precise tuning of biosynthetic gene clusters (BGCs) and culture conditions- modulation and optimization, dramatically improving yield, scalability, and cost-efficiency. Several of these newly unearthed compounds exhibit unique bioactivities that directly inspire drug-development programs against metabolic disorders, cancer drug resistance, and infectious diseases. In this review, we present an up-to-date, concise roadmap of natural product discovery (NPD), majorly covering strategies for awakening silent BGCs, genome mining, and late-stage diversification systems, and we discuss the current limitations and perspectives of rational NPD. Keywords: Natural products, Culturing modulation, Unexplored reservoirs, Genome mining, Natural product diversification

Candidates:
A. The evidence does not state that natural products (NPs) and their analogues have long underpinned therapies in humans, animals, and plants health, yet, discovering truly novel scaffolds remains a formidable challenge, even with the enormous diversity offered.
B. The evidence does not state that these combined approaches not only enable the discovery and rigorous characterization of biosynthesized metabolites, bio-transformed analogues and new chemical entities but also allow precise tuning of biosynthetic gene clusters (BGCs) and culture conditions- modulation and optimization, dramatically improving yield, scalability, and cost-efficiency.
C. The evidence does not state that researchers now deploy innovative extraction and purification protocols alongside high-throughput dereplication tools to fish trace metabolites out of complex matrices.
D. Over the last two decades, breakthroughs in bioinformatics, cheminformatics, advanced analytical methods, synthetic biology toolkits, and optimized microbial culture have surmounted many of the bottlenecks that stalled NP research in the 1990s and 2000s.
E. Over the last two decades, breakthroughs in bioinformatics, cheminformatics, advanced analytical methods, synthetic biology toolkits, and optimized microbial culture have surmounted many of the bottlenecks that stalled NP research in the 1991s and 2000s.
F. Researchers now deploy innovative extraction and purification protocols alongside high-throughput dereplication tools to fish trace metabolites out of complex matrices.
G. Natural products (NPs) and their analogues have long underpinned therapies in humans, animals, and plants health, yet, discovering truly novel scaffolds remains a formidable challenge, even with the enormous diversity offered.
H. These combined approaches not only enable the discovery and rigorous characterization of biosynthesized metabolites, bio-transformed analogues and new chemical entities but also allow precise tuning of biosynthetic gene clusters (BGCs) and culture conditions- modulation and optimization, dramatically improving yield, scalability, and cost-efficiency.


Prompt:
Classify every fixed-vocabulary term as present or absent in the evidence. Vocabulary: bioreactor, cell culture, fermentation, chromatography, purification, filtration, viability, titer, potency, sterility, viral vector, gene therapy, cell therapy, monoclonal antibody, recombinant protein, dissolved oxygen. Return JSON with exactly two alphabetized keys: {"absent":[...],"present":[...]}. Preserve vocabulary order within each list. JSON whitespace is ignored.

Evidence:
High-yield influenza virus production is essential for efficient vaccine manufacturing to support global demands. Using Madin-Darby canine kidney (MDCK) cells to produce influenza viruses is an attractive alternative to the conventional method of manufacturing vaccines using embryonated eggs. MDCK cells exhibit heterogeneity which can impact viral yields. However, the factors driving the variation between MDCK cells are not fully understood. Utilizing an untargeted liquid chromatography-mass spectrometry lipidomic approach, we investigated two proprietary MDCK clones (C59 and C113) provided by Sartorius (Germany) that differ in biochemical and viral production properties and examined their lipid profiles and dynamics upon influenza A virus (IAV) infection between 24 and 72 h. C113, a high-yield clone, displayed elevated levels across all lipid classes, aside from ether lipids compared to C59, a clone with superior growth properties. IAV infection in clone C59 and C113 displayed key differences, specifically triacylglycerols. Analysis of progeny virions from C59 and C113 clones revealed subtle differences with a positive correlation in lipid profile ( R 2 = 0.77), suggesting similar lipid raft domains between clones. Overall, these findings highlight specific cellular lipid signatures associated with high-yield production and demonstrate the value of integrating lipidomics methods into biomanufacturing pipelines, providing complimentary quality assurance markers. The online version contains supplementary material available at 10.1038/s41598-025-33499-1. Subject terms: Biochemistry, Biological techniques, Biotechnology, Microbiology


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
2-Hydroxy-4-Methoxybenzaldehyde (2H4MB) is a valuable aromatic compound with applications in flavour, fragrance, and pharmaceuticals. Because of its endangered status and root-specific accumulation, its production in native plants is restricted. In order to increase 2H4MB yield, this study emphasises recent developments in metabolic engineering, synthetic biology, in vitro culture methods, and AI-assisted route prediction. This review discussed about how CRISPR-based genome editing can be used to modify important biosynthetic genes and regulatory components, as well as how predictive machine learning techniques can be used to improve production conditions. Inadequate genetic resources, poorly understood biosynthetic pathways, and a dearth of reliable transformation systems are among the present constraints. The work highlights the importance of using integrative plant biotechnology techniques to fully realise the industrial and medicinal potential of this underutilised chemical.

Candidates:
A. Because of its endangered status and root-specific accumulation, its production in native plants is restricted.
B. This review discussed about how CRISPR-based genome editing cannot be used to modify important biosynthetic genes and regulatory components, as well as how predictive machine learning techniques can be used to improve production conditions.
C. In order to increase 3H4MB yield, this study emphasises recent developments in metabolic engineering, synthetic biology, in vitro culture methods, and AI-assisted route prediction.
D. 3-Hydroxy-4-Methoxybenzaldehyde (2H4MB) is a valuable aromatic compound with applications in flavour, fragrance, and pharmaceuticals.
E. 2-Hydroxy-4-Methoxybenzaldehyde (2H4MB) is a valuable aromatic compound with applications in flavour, fragrance, and pharmaceuticals.
F. This review discussed about how CRISPR-based genome editing can be used to modify important biosynthetic genes and regulatory components, as well as how predictive machine learning techniques can be used to improve production conditions.
G. In order to increase 2H4MB yield, this study emphasises recent developments in metabolic engineering, synthetic biology, in vitro culture methods, and AI-assisted route prediction.
H. Because of its endangered status and root-specific accumulation, its production in native plants is not restricted.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
The exploration and multiscale manufacturing in outer space hold vital significance Chemical and biological nano/micro/meso-scale manufacturing offer strategies to address challenges Emerging advances encompass novel manufacturing technologies and resource utilization strategies across orbital space stations, the Moon, Mars, and asteroids Emerging technologies like synthetic biology and artificial intelligence are discussed Key innovations, cross-disciplinary applications, and limitations are highlighted Keywords: In-space manufacturing, Biomanufacturing, Chemical manufacturing, Long-term space mission, In-situ resource utilization

Candidates:
A. The exploration and multiscale manufacturing in outer space hold vital significance Chemical and biological nano/micro/meso-scale manufacturing offer strategies to address challenges Emerging advances encompass novel manufacturing technologies and resource utilization strategies across orbital space stations, the Moon, Mars, and asteroids Emerging technologies like synthetic biology and artificial intelligence are not discussed Key innovations, cross-disciplinary applications, and limitations are highlighted Keywords: In-space manufacturing, Biomanufacturing, Chemical manufacturing, Long-term space mission, In-situ resource utilization
B. The exploration and multiscale manufacturing in outer space hold vital significance Chemical and biological nano/micro/meso-scale manufacturing offer strategies to address challenges Emerging advances encompass novel manufacturing technologies and resource utilization strategies across orbital space stations, the Moon, Mars, and asteroids Emerging technologies like synthetic biology and artificial intelligence are discussed Key innovations, cross-disciplinary applications, and limitations are highlighted Keywords: In-space manufacturing, Biomanufacturing, Chemical manufacturing, Long-term space mission, In-situ resource utilization


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Bone fractures represent a significant global healthcare burden. Although fractures typically heal on their own, some fail to regenerate properly, leading to nonunion, a condition that causes prolonged disability, morbidity, and mortality. The challenge of treating nonunion fractures is further complicated in patients with underlying bone disorders where systemic and local factors impair bone healing. Traditional treatment approaches, including autografts, allografts, xenografts, and synthetic biomaterials, face limitations such as donor site pain, immune rejection, and insufficient mechanical strength, underscoring the need for alternative strategies. Biologic therapies have emerged as promising tools to enhance bone regeneration by leveraging the body’s natural healing processes. This review explores the critical role of conventional and emerging biologics in fracture healing. We categorize biologic therapies into protein-based treatments, gene and transcript therapies, small molecules, peptides, and cell-based therapies, highlighting their mechanisms of action, advantages, and clinical relevance. Finally, we examine the potential applications of biologics in treating fractures associated with bone disorders such as osteoporosis, osteogenesis imperfecta, rickets, osteomalacia, Paget’s disease, and bone tumors. By integrating biologic therapies with existing biomaterial-based strategies, these innovative approaches have the potential to transform clinical management and improve outcomes for patients with difficult-to-heal fractures. Subject terms: Bone, Pathogenesis

Candidates:
A. Traditional treatment approaches, including autografts, allografts, xenografts, and synthetic biomaterials, face limitations such as donor site pain, immune rejection, and insufficient mechanical strength, underscoring the need for alternative strategies.
B. The evidence does not state that traditional treatment approaches, including autografts, allografts, xenografts, and synthetic biomaterials, face limitations such as donor site pain, immune rejection, and insufficient mechanical strength, underscoring the need for alternative strategies.
C. The evidence does not state that although fractures typically heal on their own, some fail to regenerate properly, leading to nonunion, a condition that causes prolonged disability, morbidity, and mortality.
D. The challenge of treating nonunion fractures is not further complicated in patients with underlying bone disorders where systemic and local factors impair bone healing.
E. Bone fractures represent a significant global healthcare burden.
F. The challenge of treating nonunion fractures is further complicated in patients with underlying bone disorders where systemic and local factors impair bone healing.
G. The evidence does not state that bone fractures represent a significant global healthcare burden.
H. Although fractures typically heal on their own, some fail to regenerate properly, leading to nonunion, a condition that causes prolonged disability, morbidity, and mortality.


Prompt:
Classify every fixed-vocabulary term as present or absent in the evidence. Vocabulary: bioreactor, cell culture, fermentation, chromatography, purification, filtration, viability, titer, potency, sterility, viral vector, gene therapy, cell therapy, monoclonal antibody, recombinant protein, dissolved oxygen. Return JSON with exactly two alphabetized keys: {"absent":[...],"present":[...]}. Preserve vocabulary order within each list. JSON whitespace is ignored.

Evidence:
The production of odd-chain fatty acids (OCFAs) is gaining increasing importance due to their diverse applications in food, chemical, and biofuel industries. These fatty acids, which are relatively rare in nature, can be produced from renewable carbon sources through microbial fermentation processes. This review covers the significance of OCFAs in the market and their occurrence, followed by a detailed exploration of their production in mixed and single strain cultures. Specifically, the anaerobic fermentation (AF) conditions and feedstocks used to produce short OCFAs (SOCFAs), such as propionic, valeric, and heptanoic acids are discussed. Additionally, the production of long OCFAs (LOCFAs) by single strains is focusing on yeast, bacteria, and microalgae. Novel approaches for LOCFAs generation from waste carbon sources are also reviewed. This work delves both into the manipulation of microbial communities covering bioaugmentation and process optimization for bioenrichment in open mixed cultures and genetic manipulation in single-strain systems. Finally, the potential for scalable and sustainable production of OCFAs through microbial processes is discussed, as well as the technological advances needed to optimize these pathways.


Prompt:
Some candidate values differ from the evidence by only one number. Select only the strings that appear verbatim, preserving candidate order. Return JSON using exactly this schema: {"reported_values":["candidate string"]}. Candidates: ["31%", "66%", "39%", "59%", "30%", "67%", "40%", "58%"]

Evidence:
Palm oil is the world’s most widely used vegetable oil, with a sizeable impact on the environment. As an alternative, microalgae are considered oil producers since they produce a variety of fatty acids (FA) depending on growth conditions. A collection of ten microalgae strains naturally producing oils similar in composition to palm oil was selected, and the effects of cultivation regime and varying light intensity on their growth and FA production and composition were analysed. To achieve high biomass density as well as total fatty acid (TFA) content, the optimum irradiance of 400 µmol photons m −2 s −1 in a photoautotrophic regime was determined for most of the strains. The growth rates of Scenedesmus and Desmodesmus strains in general were approximately twice as high as Chlamydomonas . The highest TFA content was found in S. obliquus CCALA 455 and D. subspicatus CCALA 467, grown photoautotrophically, reaching the values of about 66% and 58% of their dry weight, respectively. Moreover, the content of palmitic (PA), oleic (OA) and linoleic acid (LA) of about 39%, 30% and 14% of TFA, respectively, determined in D. subspicatus CCALA 467 was closest to that in palm oil (44% of PA, 39% of OA and 10% of LA). Eight of the ten microalgae strains were capable of heterotrophic growth, although their production under this regime has not been considered suitable in terms of TFA and individual FA content. • The optimum irradiance of 400 µmol photons m −2 s −1 was determined • CCALA 467 produces selected FAs in amounts close to those in palm oil • TFA content (% of dry weight) in CCALA 467 is 1.6-fold higher than in the palm The online version contains supplementary material available at 10.1007/s00253-025-13682-0. Keywords: Microalga, Biomass, Photoautotrophic and heterotrophic cultivation, Fatty acid, Palm oil


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Toxoplasmosis is a widespread zoonotic disease that poses risks to pregnant women and immunocompromised individuals. Despite considerable efforts, no licensed vaccines are currently available for humans or animals. Rational vaccine design increasingly relies on immunoinformatics approaches to identify immunodominant epitopes and key immunological features. This study aimed to characterise the Toxoplasma gondii ‐secreted protein with an altered thrombospondin repeat (TgSPATR) using immunoinformatics tools to evaluate its suitability as a vaccine candidate. A comprehensive panel of bioinformatics servers was used to predict allergenicity, solubility, antigenicity, secondary and tertiary structures, post‐translational modification (PTM) regions, and B‐ and T‐cell epitopes, followed by in silico immune simulation. TgSPATR consists of 534 amino acids with an estimated molecular weight of ∼57 kDa. The aliphatic index (65.71) and GRAVY score (–0.507) indicate moderate thermostability and an overall hydrophilic nature. Additionally, a total of 120 PTM sites were predicted, including phosphorylation, O ‐ and N ‐glycosylation, and palmitoylation sites. Secondary structure analysis (GOR IV, SOPMA, and NetSurfP‐3.0) revealed a predominance of random coils. Moreover, multiple servers (BcePred, SVMTriP, ABCpred, IEDB, ElliPro, and CTLpred) identified several high‐scoring B‐ and T‐cell epitopes capable of binding MHC class I and II molecules. According to SAVES v6.1, 74.1% and 93.8% of the residues of the initial and refined 3D models were located in favoured regions, indicating improved structural quality after refinement. In line with this, the ERRAT score also increased from 89.557 to 95.046. TgSPATR was predicted to be immunogenic and non‐allergenic. Finally, virtual immune simulation using the C‐ImmSim server showed that TgSPATR can elicit both humoral and cellular immune responses following three injections. This study provides foundational evidence that TgSPATR possesses key immunogenic properties and may serve as a promising vaccine candidate against acute and chronic toxoplasmosis. Nonetheless, wet‐lab experiments are required to validate these computational findings. Keywords: bioinformatics, in silico, secreted protein with an altered thrombospondin repeat (SPATR), Toxoplasma gondii , vaccine

Candidates:
A. Toxoplasmosis is a widespread zoonotic disease that poses risks to pregnant women and immunocompromised individuals.
B. Despite considerable efforts, no licensed vaccines are currently available for humans or animals.
C. Toxoplasmosis is not a widespread zoonotic disease that poses risks to pregnant women and immunocompromised individuals.
D. Despite considerable efforts, no licensed vaccines are not currently available for humans or animals.
E. Rational vaccine design increasingly relies on immunoinformatics approaches to identify immunodominant epitopes and key immunological features.
F. The evidence does not state that this study aimed to characterise the Toxoplasma gondii ‐secreted protein with an altered thrombospondin repeat (TgSPATR) using immunoinformatics tools to evaluate its suitability as a vaccine candidate.
G. This study aimed to characterise the Toxoplasma gondii ‐secreted protein with an altered thrombospondin repeat (TgSPATR) using immunoinformatics tools to evaluate its suitability as a vaccine candidate.
H. The evidence does not state that rational vaccine design increasingly relies on immunoinformatics approaches to identify immunodominant epitopes and key immunological features.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
To guarantee consistent quality of therapeutic proteins, the relationship between manufacturing process parameters and glycosylation profiles must be investigated and understood. The most important manufacturing step to investigate is the cell culture unit operation, where glycoprotein structure is highly dependent on raw materials, cell line genetics, and process control ranges. Because of the critical role glycosylation plays in certain drug mechanisms of action, the relationship between specific process inputs and glycosylation have been documented extensively. However, despite the extensive body of published work, general relationships between different cell culture conditions and glycosylation profiles remain fragmented across diverse studies, hindering systematic analysis and data-driven decision-making. To better elucidate these general relationships from published research, we introduce an innovative framework that leverages text mining and knowledge graph technologies to automatically extract, integrate, and visualize complex relationships from scientific literature, enabling actionable insights for biopharmaceutical process (bioprocess) development. Our methodology centers on the design and development of a specialized text-mining pipeline to extract and quantify relationships between cell culture conditions (raw materials, cell line genetics, and process control ranges) and glycosylation profiles from unstructured scientific literature. To enhance precision, we implement a dual normalization strategy: 1) dictionary-based concept standardization to reconcile term variants, and 2) ontological classification to organize entities into hierarchically structured categories. These curated relationships are then systematically integrated into a knowledge graph, which not only captures direct parameter-outcome associations but also reveals higher-order indirect connection through graph, providing a comprehensive view of bioprocess interactions. We present an intuitive web-based interface that enables researchers to dynamically explore and visualize complex bioprocess relationships through interactive queries. The system demonstrates robust performance with an 88% F1-score in relation extraction, effectively revealing hidden relationships between process parameters and glycan attributes. By combining scalable knowledge graph technology with interpretable analytics, our solution empowers pharmaceutical researchers to optimize therapeutic glycan profiles and accelerate manufacturing process development. This advancement represents a significant step forward in data-driven bioprocess optimization.

Candidates:
A. The evidence does not state that because of the critical role glycosylation plays in certain drug mechanisms of action, the relationship between specific process inputs and glycosylation have been documented extensively.
B. The most important manufacturing step to investigate is the cell culture unit operation, where glycoprotein structure is highly dependent on raw materials, cell line genetics, and process control ranges.
C. The evidence does not state that however, despite the extensive body of published work, general relationships between different cell culture conditions and glycosylation profiles remain fragmented across diverse studies, hindering systematic analysis and data-driven decision-making.
D. The evidence does not state that to guarantee consistent quality of therapeutic proteins, the relationship between manufacturing process parameters and glycosylation profiles must be investigated and understood.
E. To guarantee consistent quality of therapeutic proteins, the relationship between manufacturing process parameters and glycosylation profiles must be investigated and understood.
F. The most important manufacturing step to investigate is not the cell culture unit operation, where glycoprotein structure is highly dependent on raw materials, cell line genetics, and process control ranges.
G. Because of the critical role glycosylation plays in certain drug mechanisms of action, the relationship between specific process inputs and glycosylation have been documented extensively.
H. However, despite the extensive body of published work, general relationships between different cell culture conditions and glycosylation profiles remain fragmented across diverse studies, hindering systematic analysis and data-driven decision-making.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Secretory protein production by microbial hosts simplifies product recovery and is therefore preferred over intracellular production. Efficient secretion of heterologous proteins by bacteria requires the identification of optimal signal peptides (SPs), a step that often limits process development. Using Corynebacterium glutamicum as a model host, we established a modular cloning system enabling rapid assembly of expression plasmids for secretory protein production. Screening a library of 30 individually cloned endogenous SPs with a fungal cutinase as target protein demonstrated that several native SPs achieved substantially higher secretion levels than the widely used Bacillus subtilis NprE reference SP. To accelerate SP discovery, we developed a one‐pot approach in which C. glutamicum was directly transformed with a single modular cloning mixture containing all 30 SPs. Combined with the AutoBioTech high‐throughput platform for cultivation, harvesting, and protein quantification, this strategy enabled screening of several hundred clones in parallel. Superior SPs were rapidly identified not only for cutinase but also for four polyethylene terephthalate hydrolases (PETases). This streamlined workflow significantly reduces time and cost for selecting effective SPs and provides a versatile platform for advancing secretory protein production in C. glutamicum .

Candidates:
A. Efficient secretion of heterologous proteins by bacteria requires the identification of optimal signal peptides (SPs), a step that often limits process development.
B. Screening a library of 30 individually cloned endogenous SPs with a fungal cutinase as target protein demonstrated that several native SPs achieved substantially higher secretion levels than the widely used Bacillus subtilis NprE reference SP.
C. Screening a library of 31 individually cloned endogenous SPs with a fungal cutinase as target protein demonstrated that several native SPs achieved substantially higher secretion levels than the widely used Bacillus subtilis NprE reference SP.
D. Using Corynebacterium glutamicum as a model host, we established a modular cloning system enabling rapid assembly of expression plasmids for secretory protein production.
E. The evidence does not state that efficient secretion of heterologous proteins by bacteria requires the identification of optimal signal peptides (SPs), a step that often limits process development.
F. The evidence does not state that using Corynebacterium glutamicum as a model host, we established a modular cloning system enabling rapid assembly of expression plasmids for secretory protein production.
G. Secretory protein production by microbial hosts simplifies product recovery and is therefore preferred over intracellular production.
H. Secretory protein production by microbial hosts simplifies product recovery and is not therefore preferred over intracellular production.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Antimicrobial resistance is a growing global health crisis, responsible for nearly five million deaths annually and projected to double by 2050 as conventional antibiotics fail against multidrug-resistant pathogens. AMR is aggravated by antibiotic misuse, weak regulations, inadequate prevention, high treatment costs, and the limited discovery of new antimicrobials. In this context, antimicrobial peptides, including natural, synthetic, and computationally designed variants, have emerged as promising alternatives. AMPs display broad-spectrum antibacterial, antifungal, antiviral, antiparasitic, antibiofilm, and immunomodulatory activities, with a lower tendency to induce resistance. Their mechanisms include membrane disruption, intracellular targeting, immune modulation, and selective binding to negatively charged microbial membranes. Structural features such as α-helices, β-sheets, cyclic motifs, and post-translational modifications enhance potency and specificity. Recent advances in chemical modification, recombinant expression systems, nanotechnology, and AI-driven computational approaches have improved AMP stability, bioavailability, and therapeutic efficacy. Synthetic derivatives like innate defense regulators and conjugated AMPs further enhance immunomodulatory properties and reduce toxicity, while combination therapies increase effectiveness. Challenges remain, including degradation, short half-life, production costs, and microbial defenses such as biofilms and efflux pumps. Nevertheless, high-throughput sequencing and screening, structural biology, and structure–activity relationship studies continue to accelerate AMP development, positioning them as vital next-generation therapeutics against AMR.

Candidates:
A. Antimicrobial resistance is a growing global health crisis, responsible for nearly five million deaths annually and projected to double by 2051 as conventional antibiotics fail against multidrug-resistant pathogens.
B. AMR is aggravated by antibiotic misuse, weak regulations, inadequate prevention, high treatment costs, and the limited discovery of new antimicrobials.
C. The evidence does not state that in this context, antimicrobial peptides, including natural, synthetic, and computationally designed variants, have emerged as promising alternatives.
D. In this context, antimicrobial peptides, including natural, synthetic, and computationally designed variants, have emerged as promising alternatives.
E. The evidence does not state that aMPs display broad-spectrum antibacterial, antifungal, antiviral, antiparasitic, antibiofilm, and immunomodulatory activities, with a lower tendency to induce resistance.
F. AMR is not aggravated by antibiotic misuse, weak regulations, inadequate prevention, high treatment costs, and the limited discovery of new antimicrobials.
G. AMPs display broad-spectrum antibacterial, antifungal, antiviral, antiparasitic, antibiofilm, and immunomodulatory activities, with a lower tendency to induce resistance.
H. Antimicrobial resistance is a growing global health crisis, responsible for nearly five million deaths annually and projected to double by 2050 as conventional antibiotics fail against multidrug-resistant pathogens.


Prompt:
Classify every fixed-vocabulary term as present or absent in the evidence. Vocabulary: bioreactor, cell culture, fermentation, chromatography, purification, filtration, viability, titer, potency, sterility, viral vector, gene therapy, cell therapy, monoclonal antibody, recombinant protein, dissolved oxygen. Return JSON with exactly two alphabetized keys: {"absent":[...],"present":[...]}. Preserve vocabulary order within each list. JSON whitespace is ignored.

Evidence:
Hemophilia is an inherited disorder characterized by impaired blood clotting caused by mutations in the genes responsible for producing coagulation factor (F) VIII (hemophilia A, HA) or FIX (hemophilia B, HB). Current treatment primarily relies on replacement therapy, involving frequent and costly infusions of FVIII or FIX concentrates. While effective, these treatments come with the risk of developing neutralizing antibodies (inhibitors) against the infused factor. In recent years, non‐factor replacement therapies have emerged as innovative treatment options, offering enhanced efficacy especially for patients with inhibitors. Despite their advantages, these approaches still fall short of providing a definitive, long‐term cure. Since hemophilia is a monogenic disease, it presents an excellent opportunity for cell and gene therapy approaches aimed at achieving durable treatment and potentially a cure. Over the past three decades, remarkable advancements have been made in hemophilia gene therapy, culminating in the approval of Valoctocogene roxaparvovec (ROCTAVIAN, AAV‐FVIII) and Etranacogene dezaparvovec (HEMGENIX, AAV‐FIX) for patients with severe HA and HB, respectively. Nevertheless, gene therapy poses questions regarding its long‐term efficacy and safety. This review synthesizes findings from clinical trials, addresses persistent challenges in hemophilia gene therapy, and underscores the biological constraints and limitations inherent to viral vector‐based approaches.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
The A-Cell Case Study published by the “Alliance of Regenerative Medicine” illustrates how Quality-by Design can be applied to the manufacturing of Advanced Therapeutical Medicinal Products (ATMPs), using Chimeric Antigen Receptor (CAR)-T cell therapy as a ‘model’ process. However, no emphasis is given to different degrees of automation in this study. CAR-T cell therapies have been developed for various forms of leukemia, such as Acute Lymphoblastic Leukemia (ALL) or Non Hodgkin-Lymphoma (NHL). As more CAR-T cell therapies reach market approval and are being considered as first- or second line treatments, the economic efficiency and scalability of the chosen production modality become increasingly critical. Currently, academic and industrial manufacturers employ a range of approaches, from fully manual and open processing to closed and automated systems. New technologies, investments and cleanroom space requirements must be considered to assess economic and spatial efficiency in cell therapy manufacturing. This study analyses the costs and space requirements of different production modalities for autologous CAR-T cell production. The analysis shows that a higher degree of automation can reduce manufacturing costs by lowering personnel costs, cleanroom grade requirements and spatial footprint. It emphasizes the importance of maximizing cleanroom efficiency to support the scalable production of cell therapies as clinical demand grows. These results underscore the need for both industry and academia to consider automated production as a strategic approach to optimize resource use in CAR-T cell manufacturing.

Candidates:
A. The evidence does not state that cAR-T cell therapies have been developed for various forms of leukemia, such as Acute Lymphoblastic Leukemia (ALL) or Non Hodgkin-Lymphoma (NHL).
B. However, no emphasis is not given to different degrees of automation in this study.
C. As more CAR-T cell therapies reach market approval and are being considered as first- or second line treatments, the economic efficiency and scalability of the chosen production modality become increasingly critical.
D. CAR-T cell therapies have been developed for various forms of leukemia, such as Acute Lymphoblastic Leukemia (ALL) or Non Hodgkin-Lymphoma (NHL).
E. As more CAR-T cell therapies reach market approval and are not being considered as first- or second line treatments, the economic efficiency and scalability of the chosen production modality become increasingly critical.
F. However, no emphasis is given to different degrees of automation in this study.
G. The A-Cell Case Study published by the “Alliance of Regenerative Medicine” illustrates how Quality-by Design cannot be applied to the manufacturing of Advanced Therapeutical Medicinal Products (ATMPs), using Chimeric Antigen Receptor (CAR)-T cell therapy as a ‘model’ process.
H. The A-Cell Case Study published by the “Alliance of Regenerative Medicine” illustrates how Quality-by Design can be applied to the manufacturing of Advanced Therapeutical Medicinal Products (ATMPs), using Chimeric Antigen Receptor (CAR)-T cell therapy as a ‘model’ process.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Escherichia coli ( E. coli ) has long served as a versatile workhorse for recombinant protein production. As synthetic biology expands the demand for coordinated expression of multiple genes, co-expression systems in E. coli have evolved from basic dual-gene constructs to programmable, polygenic expression platforms. This review critically examines the major strategies enabling multigene co-expression in E. coli, including internal ribosome entry sites (IRES), 2A self-cleaving peptides, dual-promoter cassettes, multicistronic operons, and multi-plasmid configurations. We highlight the mechanistic principles, design trade-offs, and regulatory bottlenecks associated with each approach, such as translational imbalance, inclusion body formation, and plasmid compatibility. Real-world applications in metabolic engineering, complex protein assembly, and biomanufacturing are analyzed to demonstrate the functional advantages of these systems. Finally, we explore emerging programmable toolkits that integrate modular architecture, expression modeling, and AI-assisted design, paving the way for next-generation synthetic expression control in microbial chassis. This review offers a comprehensive and strategic roadmap for researchers engineering multi-gene systems in E. coli and beyond.

Candidates:
A. This review critically examines the major strategies enabling multigene co-expression in E.
B. coli ) has long served as a versatile workhorse for recombinant protein production.
C. The evidence does not state that coli have evolved from basic dual-gene constructs to programmable, polygenic expression platforms.
D. The evidence does not state that as synthetic biology expands the demand for coordinated expression of multiple genes, co-expression systems in E.
E. As synthetic biology expands the demand for coordinated expression of multiple genes, co-expression systems in E.
F. The evidence does not state that this review critically examines the major strategies enabling multigene co-expression in E.
G. coli have evolved from basic dual-gene constructs to programmable, polygenic expression platforms.
H. The evidence does not state that coli ) has long served as a versatile workhorse for recombinant protein production.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Whey, a by‐product of the cheese manufacturing industry, represents one of the most abundant and polluting effluents in the global food industry. Despite traditionally being underutilized and often discarded, its rich nutrient profile, particularly protein and lactose, has increasingly sparked an interest in its value within biotechnological processes. This review analyses the potential of whey as a sustainable substrate for the microbial production of value‐added bioproducts, focussing on L‐threonine production as a strategic case study, while addressing the environmental impact of inadequate disposal and current utilization strategies. A comparative analysis with other agroindustrial waste demonstrates whey’s competitive advantages in terms of composition, cost‐effectiveness and sustainability metrics. Furthermore, L‐threonine biological and industrial importance, and the most relevant advances in metabolic engineering, optimized fermentation and emerging tools such as optogenetics and machine learning are discussed, as they facilitate enhanced L‐threonine yields through the creation of robust, high‐producing strains. Technoeconomic analysis at pilot scale (33.8 tons/year) indicates that whey‐based production offers a comparative cost advantage of 7.4% over glucose‐based processes (20.55 USD/kg vs. 22.20 USD/kg). While absolute costs at pilot scale exceed current industrial market prices (1.31–1.66 USD/kg)—reflecting typical scale effects—the demonstrated comparative advantage and substantial environmental benefits (waste valorization, elimination of disposal costs and circular economy alignment) position whey‐based L‐threonine production as a strategic biorefinery opportunity with significant potential for industrial‐scale implementation. This cost benefit is primarily driven by the lower market price of whey compared to commercial glucose substrates, which compensates for the slightly higher downstream processing costs (5.90 vs. 5.40 USD/kg) required for complex matrices. Downstream processing considerations, including recovery, purity requirements and economic viability, are comprehensively addressed. This review concludes that whey, far from being merely a pollutant, has the characteristics required to become an asset for biotechnology. Utilizing whey as a culture medium for L‐threonine production by E. coli in bioreactors not only offers a solution to mitigate a significant environmental issue but also opens a path for the cost‐effective, sustainable production of a globally high‐demand amino acid. Whey represents a strategic biorefinery platform with potential for industrial‐scale implementation. Continued research and development in this area are fundamental to fully realizing this potential.

Candidates:
A. The evidence does not state that despite traditionally being underutilized and often discarded, its rich nutrient profile, particularly protein and lactose, has increasingly sparked an interest in its value within biotechnological processes.
B. Whey, a by‐product of the cheese manufacturing industry, represents one of the most abundant and polluting effluents in the global food industry.
C. The evidence does not state that a comparative analysis with other agroindustrial waste demonstrates whey’s competitive advantages in terms of composition, cost‐effectiveness and sustainability metrics.
D. Despite traditionally being underutilized and often discarded, its rich nutrient profile, particularly protein and lactose, has increasingly sparked an interest in its value within biotechnological processes.
E. The evidence does not state that whey, a by‐product of the cheese manufacturing industry, represents one of the most abundant and polluting effluents in the global food industry.
F. The evidence does not state that this review analyses the potential of whey as a sustainable substrate for the microbial production of value‐added bioproducts, focussing on L‐threonine production as a strategic case study, while addressing the environmental impact of inadequate disposal and current utilization strategies.
G. This review analyses the potential of whey as a sustainable substrate for the microbial production of value‐added bioproducts, focussing on L‐threonine production as a strategic case study, while addressing the environmental impact of inadequate disposal and current utilization strategies.
H. A comparative analysis with other agroindustrial waste demonstrates whey’s competitive advantages in terms of composition, cost‐effectiveness and sustainability metrics.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Yarrowia lipolytica, a versatile and oleaginous yeast, has garnered significant attention as a promising microbial chassis for producing a vast array of important metabolic products, including trace minerals, vitamins, amino acids, protein and peptides, carbohydrates, and single-cell oil (SCO), primarily in the form of saturated high-value lipids like cocoa-butter equivalents and mono-unsaturated fatty acids (MUFAs). The US FDA has designated Y. lipolytica as a “safe-to-use organism” and given it GRAS (generally regarded as safe) classification for the synthesis of EPA, citric acid, and erythritol. Co-culturing multiple interspecies microorganisms together has proven to be a feasible and comparatively more efficient strategy than monoculture to target the degradation of waste components as a substrate and boost the production of significant metabolites. In recent years, a great deal of research has been devoted to exploring the potential of this host for the biosynthesis of valuable compounds from a wide variety of strategies. Despite ongoing efforts to improve our understanding of xylose metabolism in this yeast, there has been a notable lack of research focused specifically on the biosynthesis of natural products using xylose as a precursor, which is the second most abundant sugar in lignocellulosic biomass. This review also explores recent advances in the genetic modification of Y. lipolytica to enhance its ability to assimilate xylose and produce various secondary metabolites by using xylose as a substrate.

Candidates:
A. The evidence does not state that lipolytica as a “safe-to-use organism” and given it GRAS (generally regarded as safe) classification for the synthesis of EPA, citric acid, and erythritol.
B. Yarrowia lipolytica, a versatile and oleaginous yeast, has garnered significant attention as a promising microbial chassis for producing a vast array of important metabolic products, including trace minerals, vitamins, amino acids, protein and peptides, carbohydrates, and single-cell oil (SCO), primarily in the form of saturated high-value lipids like cocoa-butter equivalents and mono-unsaturated fatty acids (MUFAs).
C. lipolytica as a “safe-to-use organism” and given it GRAS (generally regarded as safe) classification for the synthesis of EPA, citric acid, and erythritol.
D. Co-culturing multiple interspecies microorganisms together has proven to be a feasible and comparatively more efficient strategy than monoculture to target the degradation of waste components as a substrate and boost the production of significant metabolites.
E. The evidence does not state that co-culturing multiple interspecies microorganisms together has proven to be a feasible and comparatively more efficient strategy than monoculture to target the degradation of waste components as a substrate and boost the production of significant metabolites.
F. In recent years, a great deal of research has been devoted to exploring the potential of this host for the biosynthesis of valuable compounds from a wide variety of strategies.
G. The evidence does not state that yarrowia lipolytica, a versatile and oleaginous yeast, has garnered significant attention as a promising microbial chassis for producing a vast array of important metabolic products, including trace minerals, vitamins, amino acids, protein and peptides, carbohydrates, and single-cell oil (SCO), primarily in the form of saturated high-value lipids like cocoa-butter equivalents and mono-unsaturated fatty acids (MUFAs).
H. The evidence does not state that in recent years, a great deal of research has been devoted to exploring the potential of this host for the biosynthesis of valuable compounds from a wide variety of strategies.


Prompt:
Some candidate values differ from the evidence by only one number. Select only the strings that appear verbatim, preserving candidate order. Return JSON using exactly this schema: {"reported_values":["candidate string"]}. Candidates: ["2 L", "91%", "3 L", "251 mL", "90%", "250 mL"]

Evidence:
The emergence of allogeneic, universal chimeric antigen receptor (CAR) T cell therapies requires intensified and scalable manufacturing workflows supported by representative scale-down models (SDMs) to enable efficient process development and future large-scale production of off-the-shelf therapies. Here, we present a 7-day CAR-T cell expansion process intensified via perfusion of serum-free medium in a 2 L Univessel® Single-Use stirred-tank bioreactor (STR), consistently achieving 30 × 10 6 cells/mL, corresponding to 113 ± 7 anti-CD19 CAR-T doses per batch. Parallel runs in 250 mL Ambr® 250 STRs conducted at equivalent volumetric power input ( P/V ) of ∼8.78 W/m 3 demonstrated comparable process performance and final product quality, with univariate and multivariate analyses of cell growth, phenotype, cytotoxicity, and cytokine secretion validating the Ambr® 250 as a predictive SDM for the 2 L process. Integrating capacitance sensing in the 2 L STR enabled robust monitoring of viable cell concentrations in real-time, with strong correlation to offline measurements (R 2 = 0.98). For downstream processing, the Ksep® 400 was used to automate CAR-T cell harvesting, concentration, and washing at the 2 L scale, achieving >90% product recovery and nine-fold volume reduction without impacting product quality attributes compared to manual methods. This study establishes a scalable CAR-T manufacturing workflow supported by a predictive SDM, providing an efficient platform for process development and scale-up to enable future large-scale production of allogeneic CAR-T cell therapies.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Microalgae and cyanobacteria are emerging as sustainable alternatives to chemical fertilizers and pesticides, offering nutrient recycling, stress mitigation, and environmental restoration within the framework of circular bioeconomy. This review synthesizes recent advances in the utilization of cyanobacteria and green microalgae as biofertilizers, biostimulants, and biopesticides, emphasizing their physiological mechanisms and agronomic potential. Microalgae and cyanobacteria can fix atmospheric nitrogen, solubilize phosphorus, and supply essential micronutrients through exopolysaccharides, organic acids, and siderophores, thereby improving soil fertility and structure. Their metabolites, including phytohormones, amino acids, and antioxidants, stimulate seed germination, root growth, nutrient uptake, and tolerance to abiotic stresses such as drought and salinity. Moreover, allelochemicals and antimicrobial compounds from microalgae can suppress plant pathogens and reduce pesticide dependence. Integrating microalgae cultivation with wastewater and flue gas utilization promotes nutrient recycling and CO 2 sequestration, further enhancing environmental sustainability. However, large-scale application remains limited by biomass production costs, inconsistent performance under field conditions, and regulatory uncertainty. Overall, microalgae-based fertilizers and biostimulants hold great promise for sustainable crop production and soil health improvement. Future research should focus on low-cost cultivation and harvesting technologies, field scale validation, and standardized product formulations to accelerate the transition toward climate smart and resource sustainable agriculture.

Candidates:
A. Microalgae and cyanobacteria cannot fix atmospheric nitrogen, solubilize phosphorus, and supply essential micronutrients through exopolysaccharides, organic acids, and siderophores, thereby improving soil fertility and structure.
B. Microalgae and cyanobacteria can fix atmospheric nitrogen, solubilize phosphorus, and supply essential micronutrients through exopolysaccharides, organic acids, and siderophores, thereby improving soil fertility and structure.
C. Their metabolites, including phytohormones, amino acids, and antioxidants, stimulate seed germination, root growth, nutrient uptake, and tolerance to abiotic stresses such as drought and salinity.
D. The evidence does not state that their metabolites, including phytohormones, amino acids, and antioxidants, stimulate seed germination, root growth, nutrient uptake, and tolerance to abiotic stresses such as drought and salinity.
E. The evidence does not state that this review synthesizes recent advances in the utilization of cyanobacteria and green microalgae as biofertilizers, biostimulants, and biopesticides, emphasizing their physiological mechanisms and agronomic potential.
F. Microalgae and cyanobacteria are emerging as sustainable alternatives to chemical fertilizers and pesticides, offering nutrient recycling, stress mitigation, and environmental restoration within the framework of circular bioeconomy.
G. Microalgae and cyanobacteria are not emerging as sustainable alternatives to chemical fertilizers and pesticides, offering nutrient recycling, stress mitigation, and environmental restoration within the framework of circular bioeconomy.
H. This review synthesizes recent advances in the utilization of cyanobacteria and green microalgae as biofertilizers, biostimulants, and biopesticides, emphasizing their physiological mechanisms and agronomic potential.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
L-( +)-Tartaric acid is a valuable organic acid with broad applications in the food, pharmaceutical, and chemical industries. Its eco-friendly synthesis typically relies on the enzymatic hydrolysis of cis -epoxysuccinate (CES) catalyzed by cis -epoxysuccinate hydrolases (CESHs), but conventional single-batch processes suffer from low space–time yields and poor continuity. To address these challenges, we devised two complementary fed-batch strategies to simplify the enzyme–product separation by exploiting differences in their solubilities. Strategy A employs carrier-free cross-linking immobilization of whole cells using 0.02% glutaraldehyde and 0.1% polyethylenimine. In this system, both the substrate sodium cis -epoxysuccinate (CESNa) and the product sodium L-( +)-tartrate remain soluble, while the enzyme is retained in the insoluble cell matrix. Under fed-batch operation, this configuration achieves a space–time yield of 150 g L −1 h −1 . Strategy B uses cell-free extract of CESH to hydrolyze calcium cis -epoxysuccinate (CESCa) with inherently low solubility. Here, the enzyme is fully soluble but the L-( +)-tartrate formed precipitates as an insoluble calcium salt, allowing easy separation of the product from the reaction mixture. This approach overcomes potential substrate inhibition and minimizes sodium-ion discharge, delivering a space–time yield of 136 g L −1 h −1 and a specific productivity of 484 g product /g catalyst . Both the soluble-product/insoluble-enzyme system (A) and the insoluble-product/soluble-enzyme system (B) represent effective strategies to streamline downstream processing and markedly enhance productivity. Together, they offer a viable route to scalable and cost-effective industrial production of L-( +)-tartaric acid.

Candidates:
A. To address these challenges, we devised two complementary fed-batch strategies to simplify the enzyme–product separation by exploiting differences in their solubilities.
B. Strategy A employs carrier-free cross-linking immobilization of whole cells using 1.02% glutaraldehyde and 0.1% polyethylenimine.
C. L-( +)-Tartaric acid is not a valuable organic acid with broad applications in the food, pharmaceutical, and chemical industries.
D. The evidence does not state that its eco-friendly synthesis typically relies on the enzymatic hydrolysis of cis -epoxysuccinate (CES) catalyzed by cis -epoxysuccinate hydrolases (CESHs), but conventional single-batch processes suffer from low space–time yields and poor continuity.
E. L-( +)-Tartaric acid is a valuable organic acid with broad applications in the food, pharmaceutical, and chemical industries.
F. Its eco-friendly synthesis typically relies on the enzymatic hydrolysis of cis -epoxysuccinate (CES) catalyzed by cis -epoxysuccinate hydrolases (CESHs), but conventional single-batch processes suffer from low space–time yields and poor continuity.
G. Strategy A employs carrier-free cross-linking immobilization of whole cells using 0.02% glutaraldehyde and 0.1% polyethylenimine.
H. The evidence does not state that to address these challenges, we devised two complementary fed-batch strategies to simplify the enzyme–product separation by exploiting differences in their solubilities.


Prompt:
Some candidate values differ from the evidence by only one number. Select only the strings that appear verbatim, preserving candidate order. Return JSON using exactly this schema: {"reported_values":["candidate string"]}. Candidates: ["2.18 g/L", "3.18 g/L"]

Evidence:
D-Pantothenic acid (DPA), also known as vitamin B 5 , is a water-soluble organic acid, widely applied in foods, feeds, cosmetics, and medicines. Although numerous and rapidly developing cell factories have been established for DPA biosynthesis, there has been no report of any attempts to engineer Yarrowia lipolytica to synthesize DPA. To explore further possibilities in DPA biosynthesis, we tried to employ systematic metabolic engineering strategies to identify and break the potential bottlenecks in DPA biosynthesis by Y. lipolytica . By improving the rate-limiting steps of the DPA biosynthesis pathway, weakening the strongly competitive pathways, and enhancing the multiple cofactor supplies, a robust Y. lipolytica cell factory for DPA biosynthesis was successfully constructed. Consequently, the resulting strain DPA34 produced 2.18 g/L DPA in a 5-L bioreactor, representing the first report of DPA production to date in Y. lipolytica. This work is believed to facilitate the development of Y. lipolytica for sustainable manufacturing of vitamin B 5 and its derivatives. The online version contains supplementary material available at 10.1186/s40643-026-01009-4.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Heparin, mainly used as an anticoagulant, has also shown potential in the treatment of diseases such as inflammation and cancer. Currently, heparin is mainly extracted from the intestinal mucosa of pigs. However, due to concerns about disease transmission and contamination associated with animal-derived products, biomanufacturing techniques have been explored as alternative production methods. Through enzyme engineering, metabolic engineering, and synthetic biology approaches, the heparin biosynthetic pathways have been systematically optimized. The main biomanufacturing techniques include in vivo/in vitro combination strategy (microbial heparosan fermentation followed by chemoenzymatic modification) and de novo biosynthesis. This article comprehensively discusses the latest advancements, challenges, and future perspectives of these heparin biomanufacturing techniques. Keywords: Bioengineered heparin, Heparosan fermentation, Chemoenzymatic modification, De novo biosynthesis, Synthetic biology

Candidates:
A. Currently, heparin is mainly extracted from the intestinal mucosa of pigs.
B. The evidence does not state that however, due to concerns about disease transmission and contamination associated with animal-derived products, biomanufacturing techniques have been explored as alternative production methods.
C. The evidence does not state that through enzyme engineering, metabolic engineering, and synthetic biology approaches, the heparin biosynthetic pathways have been systematically optimized.
D. The evidence does not state that heparin, mainly used as an anticoagulant, has also shown potential in the treatment of diseases such as inflammation and cancer.
E. Through enzyme engineering, metabolic engineering, and synthetic biology approaches, the heparin biosynthetic pathways have been systematically optimized.
F. Heparin, mainly used as an anticoagulant, has also shown potential in the treatment of diseases such as inflammation and cancer.
G. Currently, heparin is not mainly extracted from the intestinal mucosa of pigs.
H. However, due to concerns about disease transmission and contamination associated with animal-derived products, biomanufacturing techniques have been explored as alternative production methods.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Umbilical cord blood (UCB) is an attractive source of natural killer (NK) cells for the development of allogeneic ‘off-the-shelf’ cancer immunotherapies. This is due to the relatively high proportion of highly proliferative NK cells compared to adult peripheral blood (APB), a low risk of graft-versus-host disease and ease of procurement. However, due to the limited starting volume of UCB and naïve phenotype of isolated cells, ex vivo NK cell expansion and activation is essential to generate clinically relevant doses of cells with potent anti-tumor activity. Furthermore, intrinsic variability in both in vitro and clinical performance of NK cells from different UCB units (CBUs) has been reported. To better characterize this variability, we measured UCB NK cell ex vivo fold expansion, phenotype and cytotoxic potential using a basic expansion system. We then used these results to identify characteristics related to superior performance, enabling the optimization, selection and processing of CBUs for the manufacture of NK cells as therapies at a larger scale. Our results revealed that despite wide inter-donor variability in performance between CBUs, a priori selection could be used to identify units likely to show high expansion and/or cytotoxicity. We observed that decreased time between UCB collection and CD3 - UCB mononuclear cell (CBMC) isolation was associated with significantly higher NK fold expansion (n=13; p<0.05). Furthermore, a cryopreservation step following early isolation and prior to expansion, significantly increased the expansion potential of the isolated NK cells (p<0.05), thus providing an opportunity for pre-selection and parallel culture of multiple optimal units. Finally, the NK cells from CBUs collected from caesarean sections had statistically significantly increased proliferative potential compared to those from vaginal deliveries (n=13; p<0.05). In conclusion, early isolation and cryopreservation of CD3 - CBMCs from caesarean section CBUs offer an optimal starting material for use in UCB-derived NK cell immunotherapies, providing superior ex vivo performance and enabling batch testing to selectively expand cells from CBUs with the greatest potential.

Candidates:
A. The evidence does not state that furthermore, intrinsic variability in both in vitro and clinical performance of NK cells from different UCB units (CBUs) has been reported.
B. This is due to the relatively high proportion of highly proliferative NK cells compared to adult peripheral blood (APB), a low risk of graft-versus-host disease and ease of procurement.
C. Furthermore, intrinsic variability in both in vitro and clinical performance of NK cells from different UCB units (CBUs) has been reported.
D. However, due to the limited starting volume of UCB and naïve phenotype of isolated cells, ex vivo NK cell expansion and activation is not essential to generate clinically relevant doses of cells with potent anti-tumor activity.
E. This is not due to the relatively high proportion of highly proliferative NK cells compared to adult peripheral blood (APB), a low risk of graft-versus-host disease and ease of procurement.
F. Umbilical cord blood (UCB) is not an attractive source of natural killer (NK) cells for the development of allogeneic ‘off-the-shelf’ cancer immunotherapies.
G. However, due to the limited starting volume of UCB and naïve phenotype of isolated cells, ex vivo NK cell expansion and activation is essential to generate clinically relevant doses of cells with potent anti-tumor activity.
H. Umbilical cord blood (UCB) is an attractive source of natural killer (NK) cells for the development of allogeneic ‘off-the-shelf’ cancer immunotherapies.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Codon optimization is widely used to improve heterologous gene expression in Escherichia coli . However, many existing methods focus primarily on maximizing the codon adaptation index (CAI) and neglect broader aspects of biological context. In this study, we present ColiFormer, a transformer-based codon optimization framework fine-tuned on 3676 high-expression E. coli genes curated from the NCBI database. Built on the CodonTransformer BigBird architecture, ColiFormer employs self-attention mechanisms and a mathematical optimization method (the augmented Lagrangian approach) to balance multiple biological objectives simultaneously, including CAI, GC content, tRNA adaptation index (tAI), RNA stability, and minimization of negative cis-regulatory elements. Based on in silico evaluations on 37,053 native E. coli genes and 80 recombinant protein targets commonly used in industrial studies, ColiFormer demonstrated significant improvements in CAI and tAI values, maintained GC content within biologically optimal ranges, and reduced inhibitory cis-regulatory motifs compared with established codon optimization approaches, while maintaining competitive runtime performance. These results represent computational predictions derived from standard in silico metrics; future experimental work is anticipated to validate these computational predictions in vivo. ColiFormer has been released as an open-source tool alongside the benchmark datasets used in this study.

Candidates:
A. coli genes curated from the NCBI database.
B. Codon optimization is widely used to improve heterologous gene expression in Escherichia coli .
C. In this study, we present ColiFormer, a transformer-based codon optimization framework fine-tuned on 3676 high-expression E.
D. In this study, we present ColiFormer, a transformer-based codon optimization framework fine-tuned on 3677 high-expression E.
E. However, many existing methods focus primarily on maximizing the codon adaptation index (CAI) and neglect broader aspects of biological context.
F. Codon optimization is not widely used to improve heterologous gene expression in Escherichia coli .
G. The evidence does not state that however, many existing methods focus primarily on maximizing the codon adaptation index (CAI) and neglect broader aspects of biological context.
H. The evidence does not state that coli genes curated from the NCBI database.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Syringic acid (SA) is a natural derivative of syringaldehyde (SD), derived from lignin depolymerization. Its application in the food industry focuses on the properties of natural functional ingredients; it is mainly used as a food antioxidant and food preservative, but can also be used as an ingredient to enhance food flavor and functional foods. This compound exhibits a remarkable spectrum of biological activities, including potent antioxidant, anti-inflammatory, neuroprotective, hypoglycemic, detoxifying, and anti-cancer effects, positioning it as a highly promising candidate for pharmaceutical and nutraceutical applications. In this study, suitable sites were first screened through homologous sequence alignment, and a variant of aryl-alcohol oxidase (CgAAO) with high efficiency in catalyzing the conversion of SD to SA was obtained via site-directed mutagenesis. A deep eutectic solvent (DES) system based on choline chloride/urea (ChCl/UR) in water was developed to enhance SA production. Additionally, key parameters of the biological reaction were optimized, including temperature, pH, metal ions, as well as the type and dosage of DES. The optimal performance was achieved using recombinant E. coli pRSFDuet-CgAAO-Y335F whole-cell biocatalysts, yielding 75% and producing 0.75 g/L SA in 100 mM KPB buffer (pH 7.0) containing 5 wt% ChCl/UR and 1 mM Fe 3+ . This study established a novel biosynthetic pathway for SA that was efficient, mild, green, and environmentally friendly.

Candidates:
A. Its application in the food industry focuses on the properties of natural functional ingredients; it is mainly used as a food antioxidant and food preservative, but can also be used as an ingredient to enhance food flavor and functional foods.
B. In this study, suitable sites were first screened through homologous sequence alignment, and a variant of aryl-alcohol oxidase (CgAAO) with high efficiency in catalyzing the conversion of SD to SA was obtained via site-directed mutagenesis.
C. Syringic acid (SA) is not a natural derivative of syringaldehyde (SD), derived from lignin depolymerization.
D. This compound exhibits a remarkable spectrum of biological activities, including potent antioxidant, anti-inflammatory, neuroprotective, hypoglycemic, detoxifying, and anti-cancer effects, positioning it as a highly promising candidate for pharmaceutical and nutraceutical applications.
E. In this study, suitable sites were first screened through homologous sequence alignment, and a variant of aryl-alcohol oxidase (CgAAO) with high efficiency in catalyzing the conversion of SD to SA was not obtained via site-directed mutagenesis.
F. Its application in the food industry focuses on the properties of natural functional ingredients; it is not mainly used as a food antioxidant and food preservative, but can also be used as an ingredient to enhance food flavor and functional foods.
G. Syringic acid (SA) is a natural derivative of syringaldehyde (SD), derived from lignin depolymerization.
H. The evidence does not state that this compound exhibits a remarkable spectrum of biological activities, including potent antioxidant, anti-inflammatory, neuroprotective, hypoglycemic, detoxifying, and anti-cancer effects, positioning it as a highly promising candidate for pharmaceutical and nutraceutical applications.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Enzyme technology, characterized by high efficiency, environmental compatibility, and precise controllability, has become a pivotal biocatalytic approach for quality enhancement and nutritional improvement in modern food industries. This review summarizes recent advances and underlying mechanisms of enzyme applications in food processing optimization, nutritional enhancement, and functional food development. In terms of process optimization, enzymes such as transglutaminase, laccase, and peroxidase enhance protein crosslinking, thereby markedly improving the texture and stability of dairy products, meat products, and plant-based protein systems. Proteases and lipases play essential roles in flavor development, maturation, and modulation of sensory attributes. From a nutritional perspective, enzymatic hydrolysis significantly improves the bioavailability of proteins, minerals, and dietary fibers, while simultaneously degrading antinutritional factors and harmful compounds, including phytic acid, tannins, food allergens, and acrylamide, thus contributing to improved food safety and nutritional balance. With respect to functional innovation, enzyme-directed production of bioactive peptides has demonstrated notable antihypertensive, antioxidant, and immunomodulatory activities. In addition, enzymatic synthesis of functional oligosaccharides and rare sugars, glycosylation-based modification of polyphenols, and enzyme-assisted extraction of plant bioactive compounds provide novel strategies and technological support for the development of functional foods. Owing to their high specificity and eco-friendly nature, enzyme technologies are driving food and nutrition sciences toward more precise, personalized, and sustainable development pathways. Despite these advances, critical research gaps remain, particularly in the limited mechanistic understanding of enzyme behavior in complex food matrices, the insufficient integration of multi-omics data with enzymatic process design, and the challenges associated with translating laboratory-scale enzymatic strategies into robust, data-driven, and scalable industrial applications.

Candidates:
A. In terms of process optimization, enzymes such as transglutaminase, laccase, and peroxidase enhance protein crosslinking, thereby markedly improving the texture and stability of dairy products, meat products, and plant-based protein systems.
B. Enzyme technology, characterized by high efficiency, environmental compatibility, and precise controllability, has become a pivotal biocatalytic approach for quality enhancement and nutritional improvement in modern food industries.
C. The evidence does not state that enzyme technology, characterized by high efficiency, environmental compatibility, and precise controllability, has become a pivotal biocatalytic approach for quality enhancement and nutritional improvement in modern food industries.
D. Proteases and lipases play essential roles in flavor development, maturation, and modulation of sensory attributes.
E. The evidence does not state that this review summarizes recent advances and underlying mechanisms of enzyme applications in food processing optimization, nutritional enhancement, and functional food development.
F. This review summarizes recent advances and underlying mechanisms of enzyme applications in food processing optimization, nutritional enhancement, and functional food development.
G. The evidence does not state that proteases and lipases play essential roles in flavor development, maturation, and modulation of sensory attributes.
H. The evidence does not state that in terms of process optimization, enzymes such as transglutaminase, laccase, and peroxidase enhance protein crosslinking, thereby markedly improving the texture and stability of dairy products, meat products, and plant-based protein systems.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
The metal-binding periplasmic protein CusF has been proposed as a bifunctional tag that enhances the solubility of recombinant proteins and enables purification using Cu affinity chromatography. However, evidence for its performance remains limited to a few model proteins. Here, we evaluated CusF as a solubility tag for two heterologous proteins: a putative poly(A)-polymerase from Enterococcus faecalis (Efa PAP) and the red fluorescent protein mCherry. The proteins were fused to CusF, expressed in E. coli BL21 (DE3) pLysS and Rosetta 2 (DE3) strains, and assessed for solubility and IMAC binding. Native Efa PAP was completely insoluble under all tested conditions, and fusion to CusF did not improve its solubility. Similarly, CusF–mCherry accumulated predominantly in the insoluble fraction, with only trace amounts detectable in soluble lysates. Soluble CusF–mCherry did not bind Cu 2+ -charged IMAC resin, while moderate binding to Ni 2+ -charged resin was attributable to the vector-encoded His tag rather than CusF. These results indicate that CusF does not universally enhance protein solubility and may not consistently bind Cu-based IMAC resin. Our findings expand empirical knowledge of solubility tag performance and emphasize the necessity of testing multiple tags to identify optimal strategies for recombinant protein production.

Candidates:
A. The evidence does not state that here, we evaluated CusF as a solubility tag for two heterologous proteins: a putative poly(A)-polymerase from Enterococcus faecalis (Efa PAP) and the red fluorescent protein mCherry.
B. The evidence does not state that the metal-binding periplasmic protein CusF has been proposed as a bifunctional tag that enhances the solubility of recombinant proteins and enables purification using Cu affinity chromatography.
C. The evidence does not state that however, evidence for its performance remains limited to a few model proteins.
D. However, evidence for its performance remains limited to a few model proteins.
E. The metal-binding periplasmic protein CusF has been proposed as a bifunctional tag that enhances the solubility of recombinant proteins and enables purification using Cu affinity chromatography.
F. The proteins were fused to CusF, expressed in E.
G. The proteins were not fused to CusF, expressed in E.
H. Here, we evaluated CusF as a solubility tag for two heterologous proteins: a putative poly(A)-polymerase from Enterococcus faecalis (Efa PAP) and the red fluorescent protein mCherry.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Phycobiliproteins are recognized as potential bioactive compounds and described as highly valued natural products for industrial and biotechnological applications. Moreover, they have been observed to possess antioxidant, anticancer/antineoplastic, and anti-inflammatory activities. Therefore, the search for new methods of their extraction and isolation is still ongoing. Foam fractionation, a bubble separation technique that allows amphiphilic molecules to be separated from their aqueous solutions, is a promising but understudied method. The process may be carried out both under mild conditions that are suitable for proteins and also for diluted solutions. This paper presents the results of applying the foam fractionation process to concentrate and separate phycobiliproteins. Allo- and C-phycocyanin from a thermophilic Synechococcus PCC 6715 strain were used in extract form after biomass cultivation and disintegration. Two ways of running the process were investigated: batch mode and continuous mode, the latter of which has not been reported in the literature previously. The results indicate that the method can be applied on a larger scale, as the outcomes of the continuous mode processes were comparable to those of the batch mode. Moreover, the results indicate that the process provides, to a certain extent, the opportunity of separating phycobiliproteins from each other.

Candidates:
A. Phycobiliproteins are recognized as potential bioactive compounds and described as highly valued natural products for industrial and biotechnological applications.
B. Foam fractionation, a bubble separation technique that allows amphiphilic molecules to be separated from their aqueous solutions, is not a promising but understudied method.
C. Foam fractionation, a bubble separation technique that allows amphiphilic molecules to be separated from their aqueous solutions, is a promising but understudied method.
D. The evidence does not state that moreover, they have been observed to possess antioxidant, anticancer/antineoplastic, and anti-inflammatory activities.
E. Phycobiliproteins are not recognized as potential bioactive compounds and described as highly valued natural products for industrial and biotechnological applications.
F. Therefore, the search for new methods of their extraction and isolation is still ongoing.
G. Moreover, they have been observed to possess antioxidant, anticancer/antineoplastic, and anti-inflammatory activities.
H. Therefore, the search for new methods of their extraction and isolation is not still ongoing.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Xylanases (EC 3.2.1.8) are value-added enzymes essential for biomass deconstruction and are widely used in the pulp and paper, food, feed, and biofuel sectors. This review provides a comprehensive analysis of the current state and future prospects of xylanase research and application. It begins by examining the structural diversity of xylan substrates and the corresponding classification of xylanase enzymes, their catalytic mechanisms, and methods for their functional study, such as inhibitor analysis. The discussion then covers the challenges and methods involved in the purification of xylanases from complex biological mixtures. While natural microbial sources (fungi and bacteria) remain important, the limitations of wild-type (WT) strains for industrial production are highlighted. The review assesses the most common recombinant production systems, including Escherichia coli , Bacillus subtilis , and Komagataella phaffii , comparing their advantages for high-yield enzyme production. Finally, the paper focuses on protein engineering strategies as powerful tools for enhancing key enzyme properties (thermostability, specific activity, and pH tolerance). By integrating fundamental knowledge with applied technological approaches, this review underscores the critical role of xylanases in industrial biotechnology and identifies future research directions for their optimization.

Candidates:
A. The evidence does not state that this review provides a comprehensive analysis of the current state and future prospects of xylanase research and application.
B. The evidence does not state that it begins by examining the structural diversity of xylan substrates and the corresponding classification of xylanase enzymes, their catalytic mechanisms, and methods for their functional study, such as inhibitor analysis.
C. This review provides a comprehensive analysis of the current state and future prospects of xylanase research and application.
D. Xylanases (EC 3.2.1.8) are value-added enzymes essential for biomass deconstruction and are widely used in the pulp and paper, food, feed, and biofuel sectors.
E. The discussion then covers the challenges and methods involved in the purification of xylanases from complex biological mixtures.
F. The evidence does not state that the discussion then covers the challenges and methods involved in the purification of xylanases from complex biological mixtures.
G. It begins by examining the structural diversity of xylan substrates and the corresponding classification of xylanase enzymes, their catalytic mechanisms, and methods for their functional study, such as inhibitor analysis.
H. Xylanases (EC 4.2.1.8) are value-added enzymes essential for biomass deconstruction and are widely used in the pulp and paper, food, feed, and biofuel sectors.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
Astaxanthin, derived from Haematococcus pluvialis , is a potent antioxidant with significant therapeutic potential. However, its large-scale commercialization is hindered by the “thick-wall challenge”, a phenomenon where the stress conditions required for astaxanthin accumulation also trigger the formation of resistant secondary cell walls. This challenge complicates extraction and reduces bioaccessibility, thereby increasing production costs. Recent advancements have focused on uncoupling astaxanthin biosynthesis from cell wall reinforcement, utilizing metabolic engineering and strain selection to reduce wall formation while maintaining high yields. Furthermore, green extraction techniques, such as electrotechnologies and ionic liquids, are being explored to improve efficiency and environmental sustainability. This review synthesizes these innovations, including biorefinery systems that maximize biomass valorization, and discusses emerging clinical applications. We highlight the challenges in bridging the gap between laboratory successes and clinical translation, and suggest future directions for resolving the thick-wall challenge, advancing astaxanthin production, and expanding its therapeutic uses in nutraceuticals and pharmaceuticals.

Candidates:
A. The evidence does not state that recent advancements have focused on uncoupling astaxanthin biosynthesis from cell wall reinforcement, utilizing metabolic engineering and strain selection to reduce wall formation while maintaining high yields.
B. However, its large-scale commercialization is hindered by the “thick-wall challenge”, a phenomenon where the stress conditions required for astaxanthin accumulation also trigger the formation of resistant secondary cell walls.
C. However, its large-scale commercialization is not hindered by the “thick-wall challenge”, a phenomenon where the stress conditions required for astaxanthin accumulation also trigger the formation of resistant secondary cell walls.
D. Recent advancements have focused on uncoupling astaxanthin biosynthesis from cell wall reinforcement, utilizing metabolic engineering and strain selection to reduce wall formation while maintaining high yields.
E. This challenge complicates extraction and reduces bioaccessibility, thereby increasing production costs.
F. Astaxanthin, derived from Haematococcus pluvialis , is not a potent antioxidant with significant therapeutic potential.
G. The evidence does not state that this challenge complicates extraction and reduces bioaccessibility, thereby increasing production costs.
H. Astaxanthin, derived from Haematococcus pluvialis , is a potent antioxidant with significant therapeutic potential.


Prompt:
Identify every candidate statement reproduced exactly from the evidence. Return JSON using exactly this schema: {"supported":["A"]}. Preserve candidate order. JSON whitespace is ignored.

Evidence:
The growing concern over plastic pollution and the widespread presence of micro- and nanoplastics has renewed interest in polyhydroxybutyrate (PHB) as a biodegradable alternative; however, its industrial deployment remains constrained by costly recovery operations with a high environmental burden. This study examines how PHB biosynthesis and intracellular organization, physicochemical properties, and the characteristics of the producing microorganism influence the performance of conventional recovery routes, including extraction with organic solvents, alkaline/oxidative chemical digestion, and enzymatic–physical schemes coupled with mechanical disruption. Based on this foundation, quantitative data are analyzed for PHB content in bacteria, mixed microbial cultures, cyanobacteria, and microalgae, along with extraction yields, polymer purity, and solvent recyclability in processes employing chlorine-free solvents, green solvents, and hydrophobic natural deep eutectic solvents (NaDESs) formulated with terpenes and organic acids. The analysis integrates mechanistic perspectives on NaDES–cell and NaDES–PHB interactions with solvent design criteria, biorefinery configurations, and preliminary evidence from technoeconomic and life cycle assessments. The findings identify NaDES as an up-and-coming platform capable of reconciling biopolymer quality with the principles of green chemistry while delineating critical gaps in recovery efficiency, viscosity management, solvent recycling, and pilot-scale validation.

Candidates:
A. This study examines how PHB biosynthesis and intracellular organization, physicochemical properties, and the characteristics of the producing microorganism influence the performance of conventional recovery routes, including extraction with organic solvents, alkaline/oxidative chemical digestion, and enzymatic–physical schemes coupled with mechanical disruption.
B. Based on this foundation, quantitative data are not analyzed for PHB content in bacteria, mixed microbial cultures, cyanobacteria, and microalgae, along with extraction yields, polymer purity, and solvent recyclability in processes employing chlorine-free solvents, green solvents, and hydrophobic natural deep eutectic solvents (NaDESs) formulated with terpenes and organic acids.
C. The evidence does not state that the analysis integrates mechanistic perspectives on NaDES–cell and NaDES–PHB interactions with solvent design criteria, biorefinery configurations, and preliminary evidence from technoeconomic and life cycle assessments.
D. The analysis integrates mechanistic perspectives on NaDES–cell and NaDES–PHB interactions with solvent design criteria, biorefinery configurations, and preliminary evidence from technoeconomic and life cycle assessments.
E. The evidence does not state that this study examines how PHB biosynthesis and intracellular organization, physicochemical properties, and the characteristics of the producing microorganism influence the performance of conventional recovery routes, including extraction with organic solvents, alkaline/oxidative chemical digestion, and enzymatic–physical schemes coupled with mechanical disruption.
F. The growing concern over plastic pollution and the widespread presence of micro- and nanoplastics has renewed interest in polyhydroxybutyrate (PHB) as a biodegradable alternative; however, its industrial deployment remains constrained by costly recovery operations with a high environmental burden.
G. Based on this foundation, quantitative data are analyzed for PHB content in bacteria, mixed microbial cultures, cyanobacteria, and microalgae, along with extraction yields, polymer purity, and solvent recyclability in processes employing chlorine-free solvents, green solvents, and hydrophobic natural deep eutectic solvents (NaDESs) formulated with terpenes and organic acids.
H. The evidence does not state that the growing concern over plastic pollution and the widespread presence of micro- and nanoplastics has renewed interest in polyhydroxybutyrate (PHB) as a biodegradable alternative; however, its industrial deployment remains constrained by costly recovery operations with a high environmental burden.