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Metabolic engineering of Candida yeasts for biotechnological applications.

Candida yeasts represent a versatile yet underexploited platform for industrial biotechnology. These yeasts utilize a remarkably broad range of carbon sources, particularly for hydrophobic carbon sources, coupled with robust growth and diverse biosynthetic capacities, making them promising hosts for sustainable production of chemicals, fuels, and proteins. Despite these advantages, industrial deployment of Candida species has been hindered by concerns regarding opportunistic pathogenicity and the historical lack of efficient genetic manipulation tools, leading to a substantial gap between metabolic potential and practical utilization. Recent advances in functional genomics, genome editing, and systems metabolic engineering are rapidly overcoming these barriers, enabling more precise and efficient strain development. In this review, we systematically summarize recent progress in the metabolic engineering of Candida species as microbial cell factories, with particular emphasis on expanding genetic toolkits, utilizting renewable and non-conventional carbon sources, and biosynthesizing high-value compounds. In addition, we propose a biosafety-oriented classification framework to support their safe industrial deployment. Finally, we discuss current challenges and emerging opportunities, emphasizing that the synergy of synthetic biology and artificial intelligence-driven design holds the key to unlocking the biotechnological potential of Candida yeasts.

Candida

Thermophilic bacteria of the Arabian Gulf and their emerging biotechnological applications: current insights and future prospects.

Thermophilic bacteria represent a powerful class of extremophiles whose ability to thrive at elevated temperatures makes them indispensable to modern biotechnology. The Arabian Gulf characterized by extreme heat, geothermal systems, hot springs, oil reservoirs, and hypersaline habitats hosts a rich yet understudied reservoir of these organisms. This review consolidates current insights into the diversity, ecological niches, and biotechnological relevance of thermophilic bacteria isolated from the region. Dominant genera such as Bacillus, Geobacillus, Thermus, Anoxybacillus, and Brevibacillus exhibit remarkable physiological and molecular strategies that enable survival under intense thermal, saline, and pH stress. Their capacity to produce thermostable enzymes, including proteases, amylases, lipases, cellulases, and DNA polymerases, positions them as high-value contributors to sectors spanning bioenergy, pharmaceuticals, food processing, agriculture, and environmental remediation. Beyond enzyme production, emerging applications such as antimicrobial compound discovery, hydrocarbon bioremediation, wastewater treatment, and sustainable bioprocessing highlight the region's untapped biotechnological potential. However, systematic exploration remains limited, hindered by sparse isolation efforts, incomplete physiological profiling, and a lack of genomic and omics-driven studies. The review underscores the need for integrated approaches that merge classical microbiology with advanced molecular and systems-level tools. Collectively, thermophilic bacteria from the Arabian Gulf constitute a promising yet underutilized biological resource poised to drive sustainable industrial innovation and environmental solutions.

Arabian Gulf

Genomic features, metabolism, and biotechnological applications of Candida tropicalis and other non-albicans Candida species.

The production of bio-based products by yeasts from agroindustrial byproducts is a key strategy for advancing circular bioeconomy. While Saccharomyces species remain the predominant industrial yeasts, their limited ability to assimilate lactose, pentoses, and glycerol, as well as their sensitivity to lignocellulose-derived inhibitors, restricts their efficient application in bioprocesses based on using industrial byproducts as fermentation media. In contrast, several non-albicans Candida species exhibit broad substrate utilization capacities and enhanced tolerance to industrial stresses, making them attractive candidates for the bioconversion of agroindustrial residues. This review critically examines recent advances in the genomic, metabolic, and physiological characterization of promising non-albicans Candida species, including Candida tropicalis, Candida parapsilosis, Candida viswanathii, Candida sojae, and Candida maltosa. Emphasis is given to genome-scale metabolic models, carbon assimilation pathways, stress-response mechanisms, and metabolic engineering approaches aiming at the production of value-added compounds. By identifying current achievements, knowledge gaps, and biotechnological bottlenecks, this review highlights the potential of these yeasts as emerging platforms for sustainable bioprocesses within a circular bioeconomy framework.

Biotechnology

Cross-Kingdom Siderophores: Biosynthesis, Ecology, and Biotechnological Applications.

Microbial siderophores are high-affinity iron-binding compounds which are produced by bacteria, fungi, and actinomycetes to obtain iron and survive and interact with different species in an iron-deficient environment. While the conventional research on siderophore systems deals mainly with the study within the same taxa, modern researchers have increased their inclination toward cross-kingdom integration of siderophore behavior and their impact on host-associated environments. This can be largely attributed to differences in biosynthetic gene clusters, receptor systems, and regulatory networks, which produce distinct genotype-to-phenotype results determining microbial cooperation and competition. Current advancements in genomic research, together with omics studies like transcriptomics, proteomics, and metabolomics, have created newer insights into how siderophores function. However, the present literature evidences multiple major gaps in multi-omics data because the link between genomes and metabolomes remains weak due to inconsistent regulatory data sets and failure in identifying producer-consumer relationships in polymicrobial systems. Additionally, major constraints like molecular instability, delivery system limitations, host toxicity, limitations in upscaling, and regulatory issues delimit the use of siderophores in medical treatment, agricultural practices, and environmental biotechnology. This review aims to bridge the existing knowledge about siderophore biochemistry, biosynthesis, ecological functions, and genetic regulation across kingdoms while integrating multi-omics outlook with translational considerations. Thus, by connecting molecular mechanisms with evolutionary cross-talk, this study aims to provide a system-level framework in the world of siderophore-mediated iron uptake and therefore shapes future directions in emerging fields of microbial engineering, precision therapies, and sustainable biotechnology.

Fur regulation

Systematic Optimization Enables Near-Perfect In Vitro Transformation Efficiencies for Spirodela polyrhiza (Greater Duckweed).

The in vitro transformation of plants, or the delivery of foreign genetic material that is incorporated into their genomes, represents a powerful tool both for elucidating genotype-phenotype relationships and for generating plant cultivars which have desirable traits for agriculture and/or biotechnological applications. However, outside of a few model species, the processes involved in transformation are often inefficient and can take months to perform for many plant species, with several bottlenecks occurring at the different stages of calli induction, genetic transfection, and plant regeneration. While duckweeds - aquatic monocots whose species include some of the smallest and fastest-growing flowering plants on the planet - have distinguished themselves with several emerging biotechnological applications, they too are the subject of conflicting reports regarding their transformation potential and ability to be genetically manipulated. Here, we synthesized and optimized the protocols for in vitro transformation of duckweed Spirodela polyrhiza (Greater Duckweed) from start-to-finish: achieving >90% - 100% efficiencies for each of calli induction; transient and stable genetic transformation; visual marker-free selection of transformants; and regeneration of genetically modified plants with stable transgene expression for over 100 generations - and which in S. polyrhiza can be achieved over the course of weeks instead of months. The integrated, streamlined approaches for all stages of in vitro transformation overcome many bottlenecks and can help to pave the way for high-throughput functional genomics studies and synthetic biology applications in this biotechnologically-important species.

CRISPR/Cas9

The snakehead retrovirus promoter functions independently of the 3'ORF protein and its products are maternally inherited in transgenic zebrafish.

The exogenous snakehead retrovirus (SnRV) is an unclassified member of the Orthoretrovirinae subfamily, discovered in cell lines derived from several fish species. SnRV resembles complex lentiviruses and potentially encodes accessory proteins, including the product of the 3' open reading frame (3'ORF). The 3'ORF protein was suggested to function as a transactivator of transcription (Tat). Here, we constructed an infectious molecular clone for SnRV and tested the effects of 3'ORF mutations on SnRV transcription. Although replacing 3'ORF with foreign sequences strongly reduced virus expression and production, an out-of-frame point mutation in 3'ORF had only a minimal effect on SnRV replication. This latter result suggests that the 3'ORF protein does not function as Tat and that SnRV transcription is largely independent of the product of this ORF. We also show that in vitro, the SnRV promoter is versatile and robustly functioning in both fish and mammalian cultured cells. Finally, the SnRV promoter was transiently active in injected zebrafish embryos as early as the blastula stage. In transgenic zebrafish, this promoter drives enhanced expression in sensory organs and gonads, and its generated products are maternally inherited. Considering these characteristics, the SnRV promoter emerges as a promising candidate for developing versatile expression vectors applicable to research and biotechnological applications.

Animals

Temporal proteomic analysis reveals a three-phase adaptation strategy in Phytophthora cinnamomi during salinity stress.

Phytophthora cinnamomi, a highly invasive hemibiotrophic oomycete, threatens global agriculture, forestry, and native ecosystems. Although drought and temperature effects on P. cinnamomi-host interactions are well studied, current knowledge of abiotic stress responses in P. cinnamomi remains largely centered on infection and phytopathology, with limited molecular insight into the pathogen's direct response to salinity independent of its host. To address this gap, we combined growth assays, time-resolved proteomics, and network analysis to define how P. cinnamomi responds and adapts to salinity exposure. Growth assays showed that NaCl-modified agar enhanced mycelial expansion in a concentration-dependent manner, with 100 mM NaCl significantly increasing growth at 48, 72, and 96 h compared with controls, while 50 mM NaCl remained comparable to control conditions. Temporal proteomic analysis of 100 mM NaCl treatment at 0, 1, 6, 12, and 24 h post treatment revealed dynamic shifts in protein abundance. Early induction of ROS (Reactive Oxygen Species)-detoxifying enzymes, including glutathione S-transferases and peroxidases, was consistent with ROS-specific staining assays. Network analysis identified modules enriched for redox regulation, ATP generation, ion transport, and translational control, highlighting multi-layered adaptation to elevated NaCl levels. Notably, clusters of conserved hypothetical proteins were strongly upregulated, indicating unexplored stress tolerance components in Phytophthora species. Here, we propose that P. cinnamomi rapidly activates a three-phase strategy involving metabolism readjustments, redox defenses, and cellular structure alterations under salinity conditions. With increasing soil salinization due to climate change, our study provides first mechanistic insights into P. cinnamomi's adaptive plasticity and ecological resilience to abiotic stress. SIGNIFICANCE: This study represents the first temporal proteomic analysis of salinity stress adaptation in Phytophthora cinnamomi, revealing a sophisticated three-phase adaptation strategy. This research fundamentally advances our understanding of how this globally destructive plant pathogen, P. cinnamomi, maintains environmental resilience. Our findings reveal proteome remodelling as a mechanistic framework for understanding stress tolerance in oomycetes, a group of microorganisms responsible for some of the world's most destructive agricultural and forest diseases. Our results show proteins involved in emergency damage control through metabolic recalibration to sustained adaptation. These findings have relevance for predicting pathogen behavior under climate change scenarios, where increasing soil salinity threatens agricultural productivity while simultaneously enhancing pathogen survival and virulence. Understanding how P. cinnamomi responds to prolonged salinity exposure may inform targeted biocontrol strategies and improve predictive models of disease pressure in salt-affected agricultural regions. The temporal analysis framework we present offers a broadly applicable approach for understanding microbial stress adaptation, with implications extending beyond plant pathology to environmental microbiology and biotechnology applications where stress tolerance is paramount.

Phytophthora

Cloning, Transformation, and Reporter Gene Analysis of the SalT Promoter in Barley (Hordeum vulgare).

Constitutive gene expression can lead to pleiotropic effects. Therefore, spatial or temporal restriction of expression via specific promoters provides a more targeted approach. This study aimed to clone the SalT promoter and analyze its activity in transgenic barley using GFP and GUS reporter genes. The T-DNA constructs carrying the SalT promoter were introduced into barley cv. Golden Promise, and transgenic plants were confirmed through PCR, hygromycin selection, and Southern hybridization. Both constructs, SalT-GFP and SalT-GUS, were transformed in barley cv. Golden Promise. Here, we characterized the expression pattern of the SalT promoter in barley and utilized it to drive the expression of reporter genes GFP and GUS. The SalT promoter was isolated from rice genomic DNA, cloned into the pNos-AB-M vector, and confirmed through PCR and restriction analysis. Subsequently, GFP and GUS genes were cloned under the SalT promoter in the same vector. The constructs were then subcloned into the p6U vector for plant expression. Agrobacterium-mediated genetic transformation of barley cultivar "Golden Promise" was conducted, resulting in successful integration of the transgenes. Callus induction, regeneration, and root formation efficiency were assessed, demonstrating the potential of the SalT promoter to drive gene expression during various stages of plant development. Molecular analyses, including PCR and Southern hybridization, confirmed the presence and integration of transgenes in the barley genome. Furthermore, GFP fluorescence and GUS staining analyses revealed strong expression of the respective genes under control of the SalT promoter in different plant tissues. This study provides insights into the application of the SalT promoter for genetic manipulation and functional characterization in barley, offering opportunities for crop improvement and biotechnological applications.

Hordeum

Exploring the interactions between algae and archaea.

Algae and archaea co-exist in diverse aquatic ecosystems and play a significant role in ecological functions and biogeochemical cycles. Compared to well-studied algal-bacterial interactions, there is a lack of information on algal-archaeal interactions and how their interactions affect their physiological fitness and nutrient cycles in either artificial cultivation systems or natural environments. The vast archaeal biodiversity, as indicated by genomic sequencing and computational approaches, has stimulated great interest in exploring uncultivated archaea to expand our knowledge of algae-archaea symbiosis. In this review, we summarize the latest studies on the diversity of algae-associated archaea and their (putative) symbiotic interactions, highlight the effects of algal-archaeal interactions on biogeochemical cycles and extend such knowledge to facilitate novel archaeal isolation and a broad range of algae-based biotechnological applications.

Algal biotechnology

Targeted multiplex gene knockouts in Lemna minor using CRISPR/Cas9.

Lemna minor (commonly known as duckweed) is a fast-growing aquatic plant recognized as a promising green bioreactor for recombinant protein production. Its rapid proliferation, high protein yield, environmental adaptability, and edibility make it highly attractive for biotechnological applications. It is essential to develop and expand genetic tools tailored to this species to maximize these advantages and further unlock its biotechnological potential. A key strategy for achieving this goal is the implementation of advanced genome editing technologies, such as the CRISPR/Cas9 system. Although multiplex CRISPR/Cas9 gene editing has previously been successfully applied in Lemna aequinoctialis, the capability of the endogenous plant tRNA processing system for multiplex editing in L. minor using the polycistronic tRNA-sgRNA (PTG)/Cas9 system has not yet been explored. In this study, a PTG construct was engineered to include four sgRNAs designed to simultaneously target two plant-specific glycosyltransferase genes: α-1,3-fucosyltransferase (FucT) and β-1,2-xylosyltransferase (XylT). As anticipated, the PTG-Cas9 system successfully induced frameshift mutations, characterized by insertions and deletions (indels), in regenerated L. minor plants derived from transformed calli. Validation via PCR and RT-PCR analysis, followed by sequencing of the target loci, confirmed the presence of indels at the target sites. Furthermore, western blot analyses utilizing antibodies specific to XylT and FucT in two homozygous lines (lines 44 and 217) revealed truncated XylT proteins in both lines. Moreover, an in-frame FucT protein was detected in line 217, whereas FucT expression was absent in line 44. This study marked the first successful demonstration of PTG-Cas9 system for multiplex genome editing in L. minor, paving the way for advanced genetic engineering in this species.

CRISPR-Cas Systems

Evolving conservation: The role of unconventional approaches to restore contemporary vertebrate populations and genomic biodiversity.

Conservation biology and restoration ecology are two essential yet distinct disciplines that address the growing challenge of biodiversity loss. Traditionally, these fields have relied on ecological principles and management practices aimed at protecting or reestablishing natural systems. The crisis is no longer just ecological; it is evolutionary and genomic. The accelerating pace of environmental change has outstripped the capacity of conventional approaches, creating a pressing need for innovative solutions. Biotechnology offers potentially transformative tools that can enhance the effectiveness and precision of both conservation and restoration efforts, especially for species where conventional conservation approaches have proved insufficient. Techniques such as genetic rescue, synthetic biology, and gene editing are increasingly being explored to address critical challenges, such as invasive species control, genetic diversity loss, and habitat fragmentation, to both invigorate endangered species and restore historical biodiversity. Despite its promise, the integration of biotechnology into conservation and restoration has raised ethical, ecological, and regulatory concerns. These include ecological unpredictability and public resistance to genetic interventions in wild populations. This perspective examines the current landscape of biotechnological applications in conservation and restoration, highlighting successful case studies, ongoing controversies, and optimism for additional progress. We argue that thoughtful, transparent integration of biotechnology that is grounded in ecological knowledge and stakeholder engagement can reconcile the goals of conservation and restoration. As ecosystems face mounting pressures, biotech-enabled strategies may prove essential for fostering resilience and ensuring long-term ecological sustainability.

Conservation of Natural Resources

Microbial decaprenoxanthin: From understanding an extremophile-derived C50 carotenoid to its bioprocessing for large-scale applications.

Decaprenoxanthin (DPXT) is an unusual bacterial C50 carotenoid that has historically received limited attention despite its well-defined structure. For decades, carotenoid research and industrial development have been dominated by C40 carotenoids, leaving longer-chain carotenoids largely overlooked. Recent discoveries, particularly from microorganisms inhabiting Antarctic and other extreme environments, have repositioned DPXT as an adaptive pigment shaped by intense environmental pressures. Its extended polyene chain and membrane-associated behavior suggest roles in membrane stabilization and protection against ultraviolet radiation and oxidative stress, features that may hold relevance for food and biotechnological applications. This review integrates historical and recent knowledge on DPXT, covering its structural characteristics, biosynthetic pathways, ecological function, and emerging technological relevance. Special attention is given to microbial sources, particularly Actinomycetota from extreme environments, and to recent advances in microbial genomics, metabolic engineering, and sustainable bioprocess development that enable the production and exploration of C50 carotenoids beyond their native extremophilic context. The analysis highlights DPXT as a representative example of stress-resilient carotenoids, with physicochemical and membrane-interacting properties that may offer advantages for future food and biotechnological systems. Although significant challenges remain in cultivation strategies, yield optimization, and downstream recovery, advances in microbial cell factories and green extraction technologies open new opportunities for valorizing C50 carotenoids. This review bridges extremophile microbiology, carotenoid biochemistry, and sustainable food innovation, positioning DPXT as an emerging molecule that may expand the functional and structural landscape of carotenoids relevant to food science.

Carotenoids

In silico analysis of metal resistance genes in Pseudomonas extremaustralis 2E-UNGS: Genomic insights and safety assessment for wastewater biotreatment.

Pseudomonas extremaustralis 2E-UNGS is a non-pathogenic strain isolated from the polluted Reconquista River basin (Buenos Aires Metropolitan Area, Argentina), with a 20-year history of study focused on its survival strategies that have enabled its application in various processes such as waste biotreatment and biosensing. Regarding bacterial-metal interactions, P. extremaustralis 2E-UNGS is capable of biosorbing Cd(II), Zn(II), and Cu(II), and biotransforming Cr(VI) to Cr(III), facilitating both the removal of these metals from aqueous systems and their use in biosensor development. The complete circular chromosome (6,372,594 bp) has been annotated in the NCBI GenBank under accession number NZ_CP091043.1. The aim of this work was to perform an in-depth exploration of the P. extremaustralis 2E-UNGS genome to support the optimization of sustainable bioprocesses within the One Health framework. To this end, the integration of experimental evidence with a detailed in silico analysis of key genes involved in metal-microorganism interactions, antibiotic resistance, and their interconnections provides valuable insights for the optimization of future biotechnological applications. Considering its antibiotic resistance profile, together with the activation of efflux pumps induced by metal stimuli-particularly observed under Zn(II) exposure-P. extremaustralis 2E-UNGS can be regarded as suitable for the design of confined bioreactor processes, minimizing the risk of potential accidental environmental releases. Therefore, modulation of gene expression emerges as a promising approach to enhance the efficiency of metal-loaded wastewater biotreatments.

Pseudomonas

Time-course transcriptome and proteomic dynamics during the de novo shoot organogenesis in Chinese fir (Cunninghamia lanceolata).

De novo shoot organogenesis (DNSO) enables plants to regenerate shoots from various explants, offering valuable opportunities for research and plant biotechnology applications. While significant progress has been made in understanding regeneration in angiosperms, the regulatory mechanisms in gymnosperms, particularly Chinese fir (Cunninghamia lanceolata), remain poorly understood, despite its importance as a key timber species in China. This study successfully established an efficient DNSO protocol for Chinese fir, identifying six distinct stages in the process through cellular-level analysis. Time-course transcriptome and proteomics analyses revealed dynamic changes in mRNA and protein levels during regeneration. Notably, proteins showed more significant alterations across a broad range of biological processes, often independent of corresponding mRNA changes. Key pathways associated with ethylene metabolism and abiotic stress responses were enriched, highlighting their critical roles in regeneration. Further experiments confirmed that moderate osmotic stress treatments (150 mm mannitol) and ethylene treatment (100 μm ACC and 5 μm AgNO3) substantially enhanced DNSO efficiency. In summary, this study uncovers the molecular mechanisms underlying Chinese fir DNSO, providing valuable insights into improving plant regeneration efficiency in this economically important species. These findings contribute to advancements in plant biotechnology and sustainable forestry practices.

Cunninghamia

Unveiling the Probiotic Properties of Lacticaseibacillus paracasei UFTM 2.9 Through Probiogenomic Analysis.

Lactic acid bacteria (LAB) comprise a group of Gram-positive bacteria with biotechnological applications. LAB, including Lacticaseibacillus spp., are recognized as potential probiotics due to their ability to confer benefits to the host. Here we employ probiogenomic and in vitro analyses to characterize the probiotic potential of Lc. paracasei UFTM 2.9, a LAB that previously demonstrated probiotic properties in vitro. The draft genome of Lc. paracasei UFTM 2.9 comprises 127 contigs, totaling 3 216 252 base pairs, with a GC content of 46.20%. The bacteria showed metabolic versatility, growing in five carbon sources. A total of 170 genes potentially associated with probiotic characteristics were identified, with functions linked to stress resistance (n = 106), adhesion (n = 12), biosynthesis of vitamins (n = 10), and others. No virulence genes or CRISPR elements were detected, and two phages were identified in Lc. paracasei UFTM 2.9. Gene clusters encoding bacteriocins were detected and confirmed in vitro. Lc. paracasei UFTM 2.9 inhibited all indicator bacteria tested (n = 12), including strains of Listeria innocua, Staphylococcus aureus, Streptococcus agalactiae, and Escherichia coli. The results indicate the potential use of Lc. paracasei UFTM 2.9 as a probiotic, considering its genetic potential to express traits of interest and survive in the gastrointestinal tract (GIT).

Probiotics

Establishment and longitudinal characterisation of a feeder-free embryonic stem-like cell line (ATES1) derived from blastomeres of the climbing perch, Anabas testudineus.

Embryonic stem (ES) cell-based platforms in non-model teleosts remain scarce, which may constrain in vitro studies in functional genomics and developmental biology in aquaculture-relevant species. Here, we report the derivation and characterisation of ATES1, an ES-like cell line derived from Anabas testudineus, a commercially important freshwater fish. ATES1 has been maintained for over 250 passages (> 1500 days) under feeder-free conditions in Leibovitz-15 medium supplemented with foetal bovine serum, fish serum, embryo extract, and human basic fibroblast growth factor (hbFGF). Proliferation was optimal at 28 °C, with both fish serum and hbFGF significantly enhancing cell growth. The cell line exhibited multiple characteristics associated with ES-like cells in vitro during early passages (approximately up to passages 25-35), including ES cell-like morphology, high self-renewal capacity (5-bromo-2'-deoxyuridine incorporation > 90% and a doubling time of 39.9 h), alkaline phosphatase activity, responsiveness to differentiation induction conditions, and expression of stemness-associated genes (sox2, klf4, sall4, nanog, myc). Immunocytochemistry confirmed Sox2 protein expression. However, real-time PCR revealed a significant decline in klf4 and sall4 expression, along with a lack of responsiveness to differentiation cues, increased heterogeneity, and the emergence of chromosomal abnormalities, suggesting compromised maintenance of ES-like properties during extended passaging. Despite these limitations, ATES1 remained continuously proliferative under feeder-free conditions and supported moderate to high (~ 45.8 ± 4.26%) non-viral transgene delivery efficiency via lipofection, suggesting its potential utility as a genetically manipulable in vitro system for future cellular and biotechnological applications in Anabas testudineus.

Animals

Coating materials enhance urochordate primary cell culture adherence.

Marine invertebrate cell cultures are a potential source for diverse biotechnological applications, given the wide range of bioactive compounds they synthesize and accumulate. Yet, the number of established marine invertebrate cell culture systems remains limited compared with those of insects and vertebrates, particularly with respect to adherent cell cultures. Here we studied the in vitro adherence of circulating blood cells from the colonial ascidian Botryllus schlosseri. Two experimental approaches were employed, seeding blood cells either alone (setup 1) or in combination with tissue fragments (setup 2), using three basal media (DMEM, DMEM/F-12, RPMI) on culture plates coated with either Poly-L/D-lysine, gelatin, collagen, or laminin. Each experiment lasted for up to 3 d. Setup 1 results reveal that Botryllus cells remain viable and can adhere to coated surfaces in all tested media. Collagen- and laminin-coated plates supported longer-term cultures, whereas Poly-D (or L)-lysine coatings were more suitable for short-term studies. Among the basal media, RPMI and DMEM/F12 most effectively supported cell attachment. Setup 2 plates consistently showed higher cell adherence compared to setup 1, suggesting that tissue-derived factors may enhance attachment. Overall, circulating Botryllus cells demonstrate the capacity for substrate adhesion in vitro, offering a foundation for the development of adherent cell cultures.

Animals

Site-specific gene integration by recombinase-mediated cassette exchange in anhydrobiotic Pv11 cells.

Pv11 cells, derived from Polypedilum vanderplanki, uniquely tolerate complete desiccation (anhydrobiosis). Although a CRISPR/Cas9-based precise integration method (CRIS-PITCh) has been developed for Pv11 cells, a CRISPR-free strategy that fixes both the genomic locus and transgene copy number has not yet been established. Here, we implement recombinase-mediated cassette exchange (RMCE) in Pv11 cells and generate master cell lines that retain anhydrobiosis following genetic engineering. We first evaluated the activity of multiple site-specific recombinases in Pv11 cells using a transient two-plasmid reporter assay. Flp, Cre, and Bxb1 recombinases all excised a test cassette and activated a green fluorescent protein (GFP) reporter, whereas phiC31 integrase mediated recombination at the DNA sequence level but did not induce reporter expression under our construct configuration. To enable genomic RMCE, we inserted an FRT/FRT3-landing pad (LP) into a previously identified genomic safe-harbor locus using CRIS-PITCh and isolated clonal master cell lines by single-cell sorting. Using the established master line, Flp-based RMCE achieved site-specific cassette exchange at the LP, producing HaloTag fluorescence and drug resistance upon successful exchange. In addition, the expected post-exchange sequence was confirmed by sequencing. We further established an all-in-one RMCE vector combining the Flp recombinase and donor cassette on a single plasmid. Together, these results demonstrate locus-defined, single-copy transgene integration in anhydrobiotic Pv11 cells via RMCE and provide a standardized, CRISPR-free workflow for routine genetic manipulation in this unique cell line. This workflow facilitates both fundamental research and applied biotechnological applications using desiccation-tolerant cells.

Anhydrobiosis