Scalable production of stem cell-derived beta-like cells is essential for advancing iPSC-based therapies for type 1 diabetes. Microcarriers offer a promising strategy for bioprocess intensification due to their high surface-tovolume ratio and compatibility with suspension bioreactors. This study evaluated whether microcarriers can support the direct differentiation of induced pluripotent stem cells (iPSC) into beta-like cells under dynamic conditions. iPSC were cultured on Cytodex-1 microcarriers in spinner flasks and differentiated using two protocols: Hogrebe et al. and Velazco-Cruz et al. Differentiation efficiency and marker expression were assessed via flow cytometry and immunohistochemistry. Due to poor differentiation outcomes, an alternative approach was tested: iPSC were first differentiated into pancreatic progenitor cells (PPCs) in 2D, enriched for GP2+ cells via MACS, and then expanded on microcarriers. Neither protocol induced beta-cell maturation on microcarriers, as shown by poor cell adhesion and absence of key markers. However, microcarriers supported robust expansion of GP2+ PPCs, with higher proliferation rates and preservation of PDX1+ and NKX6.1+ profiles compared to 2D culture. This expansion stage provides a critical intermediate for scalable alpha and beta-like cell production, facilitating largescale PPC generation for subsequent aggregation and terminal differentiation-though this was not tested in this study. This strategy offers a scalable upstream solution for producing high-quality precursor cells for islet-like cluster formation in future therapeutic applications.

Plastic pollution presents a critical environmental challenge, necessitating innovative and sustainable solutions. In this context, biodegradation using microorganisms and enzymes offers an environmentally friendly alternative. This work introduces an AI-driven framework that integrates machine learning (ML) and generative models to accelerate the discovery and design of plastic-degrading enzymes. By leveraging pre-trained protein language models and curated datasets, we developed seven ML-based binary classification models to identify enzymes targeting specific plastic substrates, achieving an average accuracy of 89%. The framework was applied to over 6,000 enzyme sequences from the RemeDB to classify enzymes targeting diverse plastics, including PET, PLA, and Nylon. Besides, generative learning strategies combined with trained classification models in this work were applied for de novo generation of PET-degrading enzymes. Structural bioinformatics validated potential candidates through in-silico analysis, highlighting differences in physicochemical properties between generated and experimentally validated enzymes. Moreover, generated sequences exhibited lower molecular weights and higher aliphatic indices, features that may enhance interactions with hydrophobic plastic substrates. These findings highlight the utility of AI-based approaches in enzyme discovery, providing a scalable and efficient tool for addressing plastic pollution. Future work will focus on experimental validation of promising candidates and further refinement of generative strategies to optimize enzymatic performance. ### Competing Interest Statement The authors have declared no competing interest.

Microorganisms are a valuable source of pharmaceutically active chemicals, serving as scaffolds for synthesis as well as lead structures. Investigating novel biomes frequently yields intriguing chemistry; the Atacama Desert in Chile is one such example. This study reports the isolation of a new reduced anthracycline-related compound from the Atacama Desert-derived bacterium Saccharothrix S26. Structural characterisation was achieved by one-dimensional and two-dimensional NMR, HR-LCMS, and X-ray crystallography. The compound was tested against the ESKAPE pathogens, bovine mastitis-related pathogens, and the fungal strain Cryptococcus neoformans, but no antimicrobial activity was observed.

In recent years, therapies based on regenerative medicine have gained wide interest owing to their potential in organ transplantation and in the treatment of multiple diseases. These cell-based therapies rely mainly on the therapeutic potential of stem cells as a result of their self-renewal, differentiation, capacities and immunosuppressive properties. Adipose tissue contains a large number of mesenchymal stem cells compared to other tissues and is easily accessible through noninvasive techniques; hence the primary recovery of adipose-derived mesenchymal stem cells (ASC) has become a critical step. Aqueous two-phase systems (ATPS) have shown promise as a separation technique for mesenchymal stem cells (MSC) as a result of their scalability, cost-effectiveness and compatibility. In this study, we investigated the partitioning behavior of the stromal vascular fraction cells, including ASC, in different ATPS compositions [polyethylene glycol, dextran (DEX), Ficoll and Ucon). Among the three ATPS tested, most of the ASC consistently migrates to the top phase as indicated by immunostaining studies for CD73, CD90 and CD105 stem cell markers. Nevertheless, the Ficoll-DEX system retrieves the greatest quantity of ASC recovered in the top phase (89% of total ASC), whereas the Ucon-DEX system effectively eliminates the most contaminating cells (38%). Our findings provide insights into the conditions under which ASC and contaminant cells concentrate in opposite phases in different ATPS, which can contribute to the development of efficient and scalable separation systems for MSC in regenerative medicine and tissue engineering applications. (c) 2024 Society of Chemical Industry (SCI).

This study was designed to recover representative culturable actinomycetes from the Atacama Desert, and to detect their ability to promote plant growth under drought conditions. Environmental samples were taken from three Atacama Desert habitats, namely, from the Aguas Calientes, Lomas Bayas and Yungay core regions. With one exception higher actinomycete counts were obtained when isolation media were inoculated with mineral particles than with corresponding aliquots of serial dilution. Comparative 16S rRNA gene sequencing showed that representative isolates belonged to thirteen genera including putative novel Blastococcus, Kocuria, Micromonospora, Pseudonocardia, Rhodococcus and Streptomyces species. Representative isolates produced indole-3-acetic acid, siderophore and solubilized phosphate as well as displaying an ability to grow under drought conditions. In conclusion, the current findings open up exciting prospects for the promising potential of actinomycetes from the Atacama Desert to be used as bioinoculants to promote plant growth in arid and semi-arid biomes.

Two novel Micromonospora strains, STR1-7T and STR1S-6T, were isolated from the rhizosphere of a Parastrephia quadrangularis plant growing in the Salar de Tara region of the Atacama Desert, Chile. Chemotaxonomic, cultural and phenotypic features confirmed that the isolates belonged to the genus Micromonospora. They grew from 20 to 37 °C, from pH7 to 8 and in the presence of up to 3 %, w/v NaCl. The isolates formed distinct branches in Micromonospora gene trees based on 16S rRNA gene sequences and on a multi-locus sequence analysis of conserved house-keeping genes. A phylogenomic tree generated from the draft genomes of the isolates and their closest phylogenetic neighbours showed that isolate STR1-7T is most closely related to Micromonospora orduensis S2509T, and isolate STR1S-6 T forms a distinct branch that is most closely related to 12 validly named Micromonospora species, including Micromonospora saelicesensis the earliest proposed member of the group. The isolates were separated from one another and from their closest phylogenomic neighbours using a combination of chemotaxonomic, genomic and phenotypic features, and by low average nucleotide index and digital DNA-DNA hybridization values. Consequently, it is proposed that isolates STR1-7T and STR1S-6T be recognized as representing new species in the genus Micromonospora, namely as Micromonospora parastrephiae sp. nov. and Micromonospora tarensis sp. nov.; the type strains are STR1-7T (=CECT 9665T=LMG 30768T) and STR1S-6T (=CECT 9666T=LMG 30770T), respectively. Genome mining showed that the isolates have the capacity to produce novel specialized metabolites, notably antibiotics and compounds that promote plant growth, as well as a broad-range of stress-related genes that provide an insight into how they cope with harsh abiotic conditions that prevail in high-altitude Atacama Desert soils.

Bacteria belonging to the phylum Actinobacteria are a very good source of antibiotics, and indeed dominate the current clinical antibiotic space. This paper reports Mutactimycin AP, a new compound belonging to an anthracycline-type family of antibiotics, isolated from a Saccharothrix sp. This actinobacterial strain was isolated from the rhizosphere of lupine plants growing in the extreme hyper-arid Atacama Desert. Structural characterization was carried out using electrospray ionization-mass spectrometry (ESI-MS) and NMR spectroscopy in combination with molecular modelling. The compound was tested against the ESKAPE pathogens, where it showed activity against MRSA and five strains associated with bovine mastitis, where it showed activity against Enterococcus pseudoavium and Staphylycoccus Aureus subsp. Aureus.

A new lasso peptide, huascopeptin, was isolated following genome-mined discovery of a new biosynthetic gene cluster in extremotolerant Streptomyces huasconensis HST28T from Salar de Huasco, Atacama Desert, Chile. Compound 1 is a 13-residue class II lasso peptide containing a novel Gly1-Asp7 macrolactam ring, a three-residue loop, and a three-residue tail, making it the smallest lasso peptide isolated to date. The lasso structure was confirmed using NOE restraint-based molecular dynamics simulations.

Lasso peptides are a diverse class of ribosomally synthesized and post-translationally modified peptides (RiPPs). Their proteolytic and thermal stability alongside their growing potential as therapeutics has increased attention to these antimicrobial peptides. With the advent of genome mining, the discovery of RiPPs allows for the accurate prediction of putatively encoded structures, however, MSn experiments only provide partial sequence confirmation, therefore 2D NMR experiments are necessary for characterisation. Multiple MS/MS techniques were applied to two structurally characterized lasso peptides, huascopeptin and leepeptin, and one uncharacterized lasso peptide, citrulassin C, which was not isolable in sufficient quantity for NMR analysis. We have shown that MS2 can be used to elucidate the full amino acid sequences previously predicted with genome mining for this compound class. HCD was able to open the macrocycles and fragment the newly opened linear peptides, confirming the complete amino acid sequences of the characterised lasso peptides. In addition, to determine if this technique could be applied at the earliest stages of the isolation process, we targeted a lasso peptide found by genome mining, citrulassin C, and were able to fully elucidate the amino acid sequence using only MS2 from a semi-crude extract of Streptomyces huasconensis HST28T.

Analysis of the genome sequence of Streptomyces leeuwenhoekii C34T identified biosynthetic gene clusters (BGCs) for three different lasso peptides (Lp1, Lp2, and Lp3) which were not known to be made by the strain. Lasso peptides represent relatively new members of the RiPP (ribosomally synthesized and posttranslationally modified peptides) family of natural products and have not been extensively studied. Lp3, whose production could be detected in culture supernatants from S. leeuwenhoekii C34T and after heterologous expression of its BGC in Streptomyces coelicolor, is identical to the previously characterized chaxapeptin. Lp1, whose production could not be detected or achieved heterologously, appears to be identical to a recently identified member of the citrulassin family of lasso peptides. Since production of Lp2 by S. leeuwenhoekii C34T was not observed, its BGC was also expressed in S. coelicolor The lasso peptide was isolated and its structure confirmed by mass spectrometry and nuclear magnetic resonance analyses, revealing a novel structure that appears to represent a new family of lasso peptides.IMPORTANCE Recent developments in genome sequencing combined with bioinformatic analysis have revealed that actinomycetes contain a plethora of unexpected BGCs and thus have the potential to produce many more natural products than previously thought. This reflects the inability to detect the production of these compounds under laboratory conditions, perhaps through the use of inappropriate growth media or the absence of the environmental cues required to elicit expression of the corresponding BGCs. One approach to overcoming this problem is to circumvent the regulatory mechanisms that control expression of the BGC in its natural host by deploying heterologous expression. The generally compact nature of lasso peptide BGCs makes them particularly amenable to this approach, and, in the example given here, analysis revealed a new member of the lasso peptide family of RiPPs. This approach should be readily applicable to other cryptic lasso peptide gene clusters and would also facilitate the design and production of nonnatural variants by changing the sequence encoding the core peptide, as has been achieved with other classes of RiPPs.

A Micromonospora strain, designated 5R2A7(T), isolated from a high altitude Atacama Desert soil was examined by using a polyphasic approach. Strain 5R2A7(T) was found to have morphological, chemotaxonomic and cultural characteristics typical of members of the genus Micromonospora. The cell wall contains meso- and hydroxy-diaminopimelic acid, the major whole-cell sugars are glucose, ribose and xylose, the predominant menaquinones MK-10(H-4), MK-10(H-6), MK-10(H-8) and MK-9(H-6), the major polar lipids diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and an unknown glycolipid, and the predominant cellular fatty acids iso-C-16:0, iso(C15)(:0) and 10-methyl C-17:0 . The digital genomic DNA G+C content is 72.3 mol%. Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain 5R2A7(T) was closely related to Micromonospora coriariae DSM 44875(T) (99.8 %) and Micromonospora cremea CR30(T) (99.7 %), and was separated readily from the latter, its closest phylogenetic neighbour, based on gyrB and multilocus sequence data, by low average nucleotide identity (92.59 %) and in silico DNA-DNA relatedness (51.7%) values calculated from draft genome assemblies and by a range of chemotaxonomic and phenotypic properties. Consequently, strain 5R2A7(T) is considered to represent a novel species of Micromonospora for which the name Micromonospora acroterricola sp. nov. is proposed. The type strain is 5R2A7(T) (=LMG 30755(T) =CECT 9656(T)).

A set of oligonucleotide primers, Rubro223f and Rubro454r, were found to amplify a 267 nucleotide sequence of 16S rRNA genes of Rubrobacter type strains. The primers distinguished members of this genus from other deeply-rooted actinobacterial lineages corresponding to the genera Conexibacter, Gaiella, Parviterribacter, Patulibacter, Solirubrobacter and Thermoleophilum of the class Thermoleophilia. Amplification of DNA bands of about 267 nucleotides were generated from environmental DNA extracted from soil samples taken from two locations in the Atacama Desert. Sequencing of a DNA library prepared from the bands showed that all of the clones fell within the evolutionary radiation occupied by the genus Rubrobacter. Most of the clones were assigned to two lineages that were well separated from phyletic lines composed of Rubrobacter type strains. It can be concluded that primers Rubro223f and Rubro454r are specific for the genus Rubrobacter and can be used to detect the presence and abundance of members of this genus in the Atacama Desert and other biomes.

α-Amylase is an endo-acting enzyme which catalyzes random hydrolysis of starch. These enzymes are used in various biotechnological processes including the textile, paper, food, biofuels, detergents and pharmaceutical industries. The use of active enzymes at low temperatures has a high potential because these enzymes would avoid the demand for heating during the process thereby reducing costs. In this work, the gene of α-amylase from Pseudoalteromonas sp. 2–3 (Antarctic bacteria) has been sequenced and expressed in Escherichia coli BL21(DE3). The ORF of the α-amylase gene cloned into pET22b(+) is 1824 bp long and codes for a protein of 607 amino acid residues including a His6-tag. The mature protein has a calculated molecular mass of 68.8 kDa. Recombinant α-amylase was purified with Ni-NTA affinity chromatography. The purified enzyme is active on potato starch with a Km of 6.94 mg/ml and Vmax of 0.27 mg/ml*min. The pH optimum is 8.0 and the optimal temperature is 20 °C. This enzyme was strongly activated by Ca2+; results consistent with other α-amylases. To the best of our knowledge, this enzyme has the lowest temperature optimum so far reported for α-amylases.

BACKGROUND:There is little published regarding metabolism of Salinispora species. In continuation with efforts performed towards this goal, this study is focused on new insights into the metabolism of the three-identified species of Salinispora using constraints-based modeling. At present, only one manually curated genome-scale metabolic model (GSM) for Salinispora tropica strain CNB-440T has been built despite the role of Salinispora strains in drug discovery.RESULTS:Here, we updated, and expanded the scope of the model of Salinispora tropica CNB-440T, and GSMs were constructed for two sequenced type strains covering the three-identified species. We also constructed a Salinispora core model that contains the genes shared by 93 sequenced strains and a few non-conserved genes associated with essential reactions. The models predicted no auxotrophies for essential amino acids, which was corroborated experimentally using a defined minimal medium (DMM). Experimental observations suggest possible sulfur accumulation. The Core metabolic content shows that the biosynthesis of specialised metabolites is the less conserved subsystem. Sets of reactions were analyzed to explore the differences between the reconstructions. Unique reactions associated to each GSM were mainly due to genome sequence data except for the ST-CNB440 reconstruction. In this case, additional reactions were added from experimental evidence. This reveals that by reaction content the ST-CNB440 model is different from the other species models. The differences identified in reaction content between models gave rise to different functional predictions of essential nutrient usage by each species in DMM. Furthermore, models were used to evaluate in silico single gene knockouts under DMM and complex medium. Cluster analysis of these results shows that ST-CNB440, and SP-CNR114 models are more similar when considering predicted essential genes.CONCLUSIONS:Models were built for each of the three currently identified Salinispora species, and a core model representing the conserved metabolic capabilities of Salinispora was constructed. Models will allow in silico metabolism studies of Salinispora strains, and help researchers to guide and increase the production of specialised metabolites. Also, models can be used as templates to build GSMs models of closely related organisms with high biotechnology potential.

Streptomyces strain HST28T isolated from the Salar de Huasco, an athalassohaline and poly-extreme high altitude saline wetland located in northern Chile, was the subject of a polyphasic taxonomic study. Strain HST28T showed morphological and chemotaxonomic features in line with its classification in the genus Streptomyces . Optimal growth of strain HST28T was obtained at 28 °C, pH 8-9 and up to 10 % (w/v) NaCl. Single (16S rRNA) and multi-locus gene sequence analyses showed that strain HST28T had a distinct phylogenetic position from its closest relatives, the type strains of Steptomyces aureus and Streptomyces kanamyceticus. Digital DNA-DNA hybridization (23.3 and 31.0 %) and average nucleotide identity (79.3 and 85.6 %) values between strain HST28T and its corresponding relatives mentioned above were below the threshold of 70 and 96 %, respectively, defined for assigning a prokaryotic strains to the same species. Strain HST28T was characterised by the presence of ll-diaminopimelic acid in its peptidoglycan layer; galactose, glucose, ribose and traces of arabinose and mannose as whole-cell sugars; phosphatidylmethylethanolamine, phosphatidylinositol, aminolipid, glycophospholipid and an unidentified lipid as polar lipids; and the predominating menaquinones MK-9(H6), MK-9(H8) and MK-9(H4) (>20 %) as well as anteiso-C15 : 0 and anteiso-C17 : 0 as major fatty acids (>15 %). Based on the phenotypic and genetic results, strain HST28T (DSM 107268T=CECT 9648T) merits recognition as a new species named Streptomyces huasconensis sp. nov.

Salar de Huasco at the Chilean Altiplano of the Atacama Desert is considered a polyextreme environment, where solar radiation, salinity and aridity are extremely high and occur simultaneously. In this study, a total of 76 bacterial isolates were discovered from soil samples collected at two different sites in the east shoreline of Salar de Huasco, including H0 (base camp next to freshwater stream in the north part) and H6 (saline soils in the south part). All isolated bacteria were preliminarily identified using some of their phenotypic and genotypic data into the genera Streptomyces (86%), Nocardiopsis (9%), Micromonospora (3%), Bacillus (1%), and Pseudomonas (1%). Streptomyces was found dominantly in both sites (H0 = 19 isolates and H6 = 46 isolates), while the other genera were found only in site H0 (11 isolates). Based on the genotypic and phylogenetic analyses using the 16S rRNA gene sequences of all Streptomyces isolates, 18% (12 isolates) revealed <98.7% identity of the gene sequences compared to those in the publicly available databases and were determined as highly possibly novel species. Further studies suggested that many Streptomyces isolates possess the nonribosomal peptide synthetases-coding gene, and some of which could inhibit growth of at least two test microbes (i.e., Gram-positive and Gram-negative bacteria and fungi) and showed also the cytotoxicity against hepatocellular carcinoma and or mouse fibroblast cell lines. The antimicrobial activity and cytotoxicity of these Streptomyces isolates were highly dependent upon the nutrients used for their cultivation. Moreover, the HPLC-UV-MS profiles of metabolites produced by the selected Streptomyces isolates unveiled apparent differences when compared to the public database of existing natural products. With our findings, the polyextreme environments like Salar de Huasco are promising sources for exploring novel and valuable bacteria with pharmaceutical potentials.

The Atacama Desert is one of the oldest and driest places on Earth. In the last decade, microbial richness and diversity has been acknowledged as an important biological resource of this region. Owing to the value of the microbial diversity apparent in potential biotechnology applications and conservation purposes, it is necessary to catalogue these microbial communities to promote research activities and help to preserve the wide range of ecological niches of the Atacama region. A prototype Atacama Database has been designed and it provides a description of the rich microbial diversity of the Atacama Desert, and helps to visualise available literature resources. Data has been collected, curated, and organised into several categories to generate a single record for each organism in the database that covers classification, isolation metadata, morphology, physiology, genome and metabolism information. The current version of Atacama Database contains 2302 microorganisms and includes cultured and uncultured organisms retrieved from different environments within the desert between 1984 and 2016. These organisms are distributed in bacterial, archaeal or eukaryotic domains, along with those that are unclassified taxonomically. The initial prototype of the Atacama Database includes a basic search and taxonomic and advanced search tools to allow identification and comparison of microbial populations, and space distribution within this biome. A geolocation search was implemented to visualise the microbial diversity of the ecological niches defined by sectors and extract general information of the sampling sites. This effort will aid understanding of the microbial ecology of the desert, microbial population dynamics, seasonal behaviour, impact of climate change over time, and reveal further biotechnological applications of these microorganisms. The Atacama Database is freely available at: https://www.atacamadb.cl.

A polyphasic approach was used for evaluating the taxonomic status of strain HST21T isolated from Salar de Huasco in the Atacama Desert. The results of 16S rRNA gene and multilocus sequence phylogenetic analyses assigned strain HST21T to the genus Streptomyceswith Streptomyces albidochromogenes DSM 41800Tand Streptomyces flavidovirens DSM 40150T as its nearest neighbours. Digital DNA-DNA hydridization (dDDH) and average nucleotide identity (ANI) values between the genome sequences of strain HST21T and S. albidochromogenes DSM 41800T (35.6 and 88.2 %) and S. flavidovirens DSM 40105T (47.2 and 88.8 %) were below the thresholds of 70  and 95-96 % for prokaryotic conspecific assignation. Phenotypic, chemotaxonomic and genetic results distinguished strain HST21T from its closest neighbours. Strain HST21T is characterized by the presence of ll-diaminopimelic acid in its peptidoglycan layer; glucose and ribose as whole cell sugars; diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylethanolamine, phosphatidylinositol, glycophospholipids, unknown lipids and phospholipids as polar lipids; and anteiso-C15 : 0 (21.6 %) and anteiso-C17 : 0 (20.5 %) as major fatty acids (>15 %). Based on these results, strain HST21T merits recognition as a novel species, for which the name Streptomyces altiplanensis sp. nov. is proposed. The type strain is HST21T=DSM 107267T=CECT 9647T. While analysing the phylogenies of strain HST21T, Streptomyces chryseus DSM 40420T and Streptomyces helvaticus DSM 40431T were found to have 100 % 16S rRNA gene sequence similarity with digital DNA-DNA hydridization (dDDH) and average nucleotide identity (ANI) values of 95.3 and 99.4 %, respectively. Therefore, S. helvaticus is considered as a later heterotypic synonym of S. chryseus and, consequently, we emend the description of S. chryseus.

Streptomyces leeuwenhoekii strains C34T, C38, C58 and C79 were isolated from a soil sample collected from the Chaxa Lagoon, located in the Salar de Atacama in northern Chile. These streptomycetes produce a variety of new specialised metabolites with antibiotic, anti-cancer and anti-inflammatory activities. Moreover, genome mining performed on two of these strains has revealed the presence of biosynthetic gene clusters with the potential to produce new specialised metabolites. This review focusses on this new clade of Streptomyces strains, summarises the literature and presents new information on strain C34T.