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In this work, the antifungal activity of rhamnolipids produced by Pseudomonas aeruginosa #112 was evaluated against Aspergillus niger MUM 92.13 and Aspergillus carbonarius MUM 05.18. It was demonstrated that the di-rhamnolipid congeners were responsible for the antifungal activity exhibited by the crude rhamnolipid mixture, whereas mono-rhamnolipids showed a weak inhibitory activity. Furthermore, in the presence of NaCl (from 375 mM to 875 mM), the antifungal activity of the crude rhamnolipid mixture and the purified di-rhamnolipids was considerably increased. Dynamic Light Scattering studies showed that the size of the structures formed by the rhamnolipids increased as the NaCl concentration increased, being this effect more pronounced in the case of di-rhamnolipids. These results were confirmed by Confocal Scanning Laser Microscopy, which revealed the formation of giant vesicle-like structures (in the µm range) by self-assembling of the crude rhamnolipid mixture in the presence of 875 mM NaCl. In the case of the purified mono- and di-rhamnolipids, spherical structures (also in the µm range) were observed at the same conditions. The results herein obtained demonstrated a direct relationship between the rhamnolipids antifungal activity and their aggregation behaviour, opening the possibility to improve their biological activities for application in different fields.

The Escherichia coli heat shock response (HSR) is a complex mechanism triggered by heat shock and by a variety of other growth-impairing stresses. We explore here the potential use of the E. coli HSR mechanism in synthetic biology approaches. Several components of the regulatory mechanism (such as heat shock promoters, proteins, and RNA thermosensors) can be extremely valuable in the creation of a toolbox of well-characterized biological parts to construct biosensors or microbial cell factories with applications in the environment, industry, or healthcare. In the future, these systems can be used for instance to detect a pollutant in water, to regulate and optimize the production of a compound with industrial relevance, or to administer a therapeutic agent in vivo. Synthetic biology is an exciting field that aims to redesign biological systems for different applications. The development of a toolbox containing well-characterized biological parts is essential to create systems that function in predictable ways to develop innovative solutions for various challenges. Bacteria have been synthetically designed to carry out specific tasks. The components of E. coli HSR mechanism, such as heat shock promoters, proteins, and RNA thermometers, may be extremely valuable in the creation of organisms with novel functionalities. Biosensors/reporter bacteria are being developed to detect and measure the presence of harmful chemicals in food, soil, and water, or to detect a specific disease. Biological parts may also enable advances in the genetic control of metabolic pathways with relevant industrial applications.

Coumarins and furanocoumarins are plant secondary metabolites with known biological activities. As they are present in low amounts in plants, their heterologous production emerged as a more sustainable and efficient approach to plant extraction. Although coumarins biosynthesis has been positively established, furanocoumarin biosynthesis has been far more challenging. This study aims to evaluate if Escherichia coli could be a suitable host for furanocoumarin biosynthesis. The biosynthetic pathway for coumarins biosynthesis in E. coli was effectively constructed, leading to the production of umbelliferone, esculetin and scopoletin (128.7, 17.6, and 15.7 µM, respectively, from tyrosine). However, it was not possible to complete the pathway with the enzymes that ultimately lead to furanocoumarins production. Prenyltransferase, psoralen synthase, and marmesin synthase did not show any activity when expressed in E. coli. Several strategies were tested to improve the enzymes solubility and activity with no success, including removing potential N-terminal transit peptides and expression of cytochrome P450 reductases, chaperones and/or enzymes to increase dimethylallylpyrophosphate availability. Considering the results herein obtained, E. coli does not seem to be an appropriate host to express these enzymes. However, new alternative microbial enzymes may be a suitable option for reconstituting the furanocoumarins pathway in E. coli. Nevertheless, until further microbial enzymes are identified, Saccharomyces cerevisiae may be considered a preferred host as it has already been proven to successfully express some of these plant enzymes.

The renewable, abundant , and low-cost nature of lignocellulosic biomass can play an important role in the sustainable production of bioenergy and several added-value bioproducts, thus providing alternative solutions to counteract the global energetic and industrial demands. The efficient conversion of lignocellulosic biomass greatly relies on the catalytic activity of carbohydrate-active enzymes (CAZymes). Finding novel and robust biocatalysts, capable of being active under harsh industrial conditions, is thus imperative to achieve an economically feasible process. In this study, thermophilic compost samples from three Portuguese companies were collected, and their metagenomic DNA was extracted and sequenced through shotgun sequencing. A novel multi-step bioinformatic pipeline was developed to find CAZymes and characterize the taxonomic and functional profiles of the microbial communities, using both reads and metagenome-assembled genomes (MAGs) as input. The samples’ microbiome was dominated by bacteria, where the classes Gammaproteobacteria, Alphaproteobacteria, and Balneolia stood out for their higher abundance, indicating that the degradation of compost biomass is mainly driven by bacterial enzymatic activity. Furthermore, the functional studies revealed that our samples are a rich reservoir of glycoside hydrolases (GH), particularly of GH5 and GH9 cellulases, and GH3 oligosaccharide-degrading enzymes. We further constructed metagenomic fosmid libraries with the compost DNA and demonstrated that a great number of clones exhibited β-glucosidase activity. The comparison of our samples with others from the literature showed that, independently of the composition and process conditions, composting is an excellent source of lignocellulose-degrading enzymes. To the best of our knowledge, this is the first comparative study on the CAZyme abundance and taxonomic/functional profiles of Portuguese compost samples.Key points• Sequence- and function-based metagenomics were used to find CAZymes in compost samples.• Thermophilic composts proved to be rich in bacterial GH3, GH5, and GH9 enzymes.• Compost-derived fosmid libraries are enriched in clones with β-glucosidase activity.

Cancer is a major cause of mortality and morbidity worldwide. Detection and quantification of cancer biomarkers plays a critical role in cancer early diagnosis, screening, and treatment. Clinicians, particularly in developing countries, deal with high costs and limited resources for diagnostic systems. Using low-cost substrates to develop sensor devices could be very helpful. The interest in paper-based sensors with colorimetric detection increased exponentially in the last decade as they meet the criteria for point-of-care (PoC) devices. Cellulose and different nanomaterials have been used as substrate and colorimetric probes, respectively, for these types of devices in their different designs as spot tests, lateral-flow assays, dipsticks, and microfluidic paper-based devices (μPADs), offering low-cost and disposable devices. However, the main challenge with these devices is their low sensitivity and lack of efficiency in performing quantitative measurements. This review includes an overview of the use of paper for the development of sensing devices focusing on colorimetric detection and their application to cancer biomarkers. We highlight recent works reporting the use of paper in the development of colorimetric sensors for cancer biomarkers, such as proteins, nucleic acids, and others. Finally, we discuss the main advantages of these types of devices and highlight their major pitfalls.

Zymomonas mobilis ZM4 is an attractive host for the development of microbial cell factories to synthesize high-value compounds, including prebiotics. In this study, a straightforward process to produce fructooligosaccharides (FOS) from sucrose was established. To control the relative FOS composition, recombinant Z. mobilis strains secreting a native levansucrase (encoded by sacB) or a mutated β-fructofuranosidase (Ffase-Leu196) from Schwanniomyces occidentalis were constructed. Both strains were able to produce a FOS mixture with high concentration of 6-kestose. The best results were obtained with Z. mobilis ZM4 pB1- sacB that was able to produce 73.4 ± 1.6 g L −1 of FOS, with a productivity of 1.53 ± 0.03 g L −1  h −1 and a yield of 0.31 ± 0.03 g FOS g sucrose −1 . This is the first report on the FOS production using a mutant Z. mobilis ZM4 strain in a one-step process. Key points • Zymomonas mobilis was engineered to produce FOS in a one-step fermentation process. • Mutant strains produced FOS mixtures with high concentration of 6-kestose. • A new route to produce tailor-made FOS mixtures was presented. Graphical abstract

Infectious agents, especially bacteria and viruses, account for a vast number of hospitalisations and mortality worldwide. Providing effective and timely diagnostics for the multiplicity of infectious diseases is challenging. Conventional diagnostic solutions, although technologically advanced, are highly complex and often inaccessible in resource-limited settings. An alternative strategy involves convenient rapid diagnostics which can be easily administered at the point-of-care (POC) and at low cost without sacrificing reliability. Biosensors and other rapid POC diagnostic tools which require biorecognition elements to precisely identify the causative pathogen are being developed. The effectiveness of these devices is highly dependent on their biorecognition capabilities. Naturally occurring biorecognition elements include antibodies, bacteriophages and enzymes. Recently, modified molecules such as DNAzymes, peptide nucleic acids and molecules which suffer a selective screening like aptamers and peptides are gaining interest for their biorecognition capabilities and other advantages over purely natural ones, such as robustness and lower production costs. Antimicrobials with a broad-spectrum activity against pathogens, such as antibiotics, are also used in dual diagnostic and therapeutic strategies. Other successful pathogen identification strategies use chemical ligands, molecularly imprinted polymers and Clustered Regularly Interspaced Short Palindromic Repeats-associated nuclease. Herein, the latest developments regarding biorecognition elements and strategies to use them in the design of new biosensors for pathogens detection are reviewed.

Curcuminoids are well-known for their therapeutic properties. However, their extraction from natural sources is environmentally unfriendly, expensive and limited by seasonal variability, highlighting the need for alternative production processes. We propose an optimized artificial biosynthetic pathway to produce curcuminoids, including curcumin, in . This pathway involves six enzymes, tyrosine ammonia lyase (TAL), 4-coumarate 3-hydroxylase (C3H), caffeic acid O-methyltransferase (COMT), 4-coumarate-CoA ligase (4CL), diketide-CoA synthase (DCS), and curcumin synthase (CURS1). Curcuminoids pathway was divided in two modules, the first module included TAL, C3H and COMT and the second one 4CL, DCS and CURS1. Optimizing the first module of the pathway, from tyrosine to ferulic acid, enabled obtaining the highest ferulic acid titer reported so far (1325.1 μM). Afterward, ferulic acid was used as substrate to optimize the second module of the pathway. We achieved the highest concentration of curcumin ever reported (1529.5 μM), corresponding to a 59.4% increase. Subsequently, curcumin and other curcuminoids were produced from tyrosine (using the whole pathway) in mono-culture. The production increased comparing to a previously reported pathway that used a caffeoyl-CoA O-methyltransferase enzyme (to convert caffeoyl-CoA to feruloyl-CoA) instead of COMT (to convert caffeic to ferulic acid). Additionally, the potential of a co-culture approach was evaluated to further improve curcuminoids production by reducing cells metabolic burden. We used one strain able to convert tyrosine to ferulic acid and another able to convert the hydroxycinnamic acids produced by the first one to curcuminoids. The co-culture strategies tested led to 6.6 times increase of total curcuminoids (125.8 μM) when compared to the mono-culture system. The curcuminoids production achieved in this study corresponds to a 6817% improvement. In addition, by using an inoculation ratio of 2:1, although total curcuminoids production decreased, curcumin production was enhanced and reached 43.2 μM, corresponding to an improvement of 160% comparing to mono-culture system. To our knowledge, these values correspond to the highest titers of curcuminoids obtained to date. These results demonstrate the enormous potential of modular co-culture engineering to produce curcumin, and other curcuminoids, from tyrosine.

Therapeutic ultrasound (US) holds promise as a potential therapy to treat articular cartilage; however, optimized and targeted US protocols are lacking. This study aimed at determining an appropriate set of US protocols for stimulating human chondrocyte activity. Human chondrocytes were exposed to various US parameters, including different central frequencies, power densities, operation modes, pulse parameters, and durations, either daily or every other day over a three-day period. Temperature was monitored during stimulation. Protocols that showed the greatest potential for increasing chondrocyte metabolic activity and proliferation were further investigated and extended to a seven-day application. Cartilage matrix synthesis was analyzed by immunocytochemistry, qPCR, and glycosaminoglycans staining. Chondrocytes exposed to 250 mW/cm 2 at 1.25 MHz (continuous or pulsed, every other day), 0.45 MHz (pulsed, daily), or 2.00 MHz (pulsed, every other day) exhibited significantly higher proliferation and metabolic activity. Immunocytochemistry analyses revealed that those protocols markedly increased COL II expression by up to 2.7-fold and aggrecan up to 1.7-fold compared to non-stimulated chondrocytes. Collagen type II and aggrecan mRNA levels were elevated by up to 2.0-fold and 4.5-fold, respectively. No significant effect on glycosaminoglycan production was detected. US stimulation also played an important role in counteracting chondrocyte phenotype loss, reducing collagen type I protein and mRNA expression by up to 68% and 90%, respectively, compared to untreated chondrocytes. Temperature remained stable during US stimulation. The US protocol 250 mW/cm 2 at 2.00 MHz (pulsed, 1 Hz, 50% duty cycle) for 20 min, every other day, appears to be the most effective protocol for eliciting cartilage components synthesis and decelerating the dedifferentiation process in chondrocytes and, thus, it should be further explored to regenerate articular cartilage.

Fructooligosaccharides (FOS) have gained attention due to their prebiotic properties and potential health benefits. This study explores the production of FOS using Zymomonas mobilis ZM4, a promising candidate for biotechnological processes, utilizing corn steep liquor (CSL) and sugarcane molasses as alternative and sustainable carbon and nitrogen sources. Two distinct media formulations were investigated, namely one composed of CSL supplemented with yeast extract (YE), and another utilizing sugarcane molasses. CSL was evaluated at concentrations of 10 g L −1 and 12 g L −1 in combination with YE. The optimal combination, 12 g L −1 CSL and 8 g L −1 YE, yielded 60.00 ± 0.44 g L −1 FOS with a productivity of 1.250 ± 0.009 g L −1  h −1 , comparable to synthetic media. Molasses, another agro-industrial by-product, was tested at sucrose-equivalent concentrations of 150, 200, and 350 g L −1 . The highest FOS concentration, 58.67 ± 1.64 g L −1 , was achieved with 200 g L −1 of molasses. Combining CSL and molasses (CSLM media) resulted in 58.15 ± 0.21 g L −1 of FOS with a yield of 0.307 ± 0.003 g FOS g sucrose −1 . The FOS mixture included 1-kestose, 6-kestose, nystose, and neokestose. Although scaling up to a bioreactor led to a lower FOS concentration of 42.31 ± 0.16 g L −1 , the yield remained promising at 0.482 ± 0.008 g FOS g sucrose −1 . This study not only highlights the efficient production of FOS using Z. mobilis ZM4 but also demonstrates the potential of using CSL and molasses, byproducts of agro-industrial processes, as cost-effective and sustainable substrates for industrial-scale FOS production. The findings provide valuable insights for the development of bio-based processes for functional oligosaccharide production. Graphical abstract