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Abstract: An early diagnosis has the potential to greatly decrease cancer mortality. For that purpose, specific cancer biomarkers have been molecularly targeted by aptamer sequences to enable an accurate and rapid detection. Aptamer-based biosensors for cancer diagnostics are a promising alternative to those using antibodies, due to their high affinity and specificity to the target molecules and advantageous production. Synthetic nucleic acid aptamers are generated by in vitro Systematic Evolution of Ligands by Exponential enrichment (SELEX) methodologies that have been improved over the years to enhance the efficacy and shorten the selection process. Aptamers have been successfully applied in electrochemical, optical, photoelectrochemical and piezoelectrical-based detection strategies. These aptasensors comprise a sensitive, accurate and inexpensive option for cancer detection being used as point-of-care devices. This review highlights the recent advances in cancer biomarkers, achievements and optimizations made in aptamer selection, as well as the different aptasensors developed for the detection of several cancer biomarkers.

Enrichment and diagnosis tools for pathogens currently available are time consuming, thus the development of fast and highly sensitive alternatives is desirable. In this study, a novel approach was described that enables selective capture of bacteria expressing hydrolyzed collagen-binding adhesins with hydrolyzed collagen-coated magnetic nanoparticles (MNPs). This platform could be useful to shorten the time needed to confirm the presence of a bacterial infection. MNPs were synthesized by a simple two-step approach through a green co-precipitation method using water as solvent. These MNPs were specifically designed to interact with pathogenic bacteria by establishing a hydrolyzed collagen-adhesin linker. The bacterial capture efficacy of hydrolyzed collagen MNPs (H-Coll@MNPs) for bacteria expressing collagen binding adhesins was 1.3 times higher than that of arginine MNPs (Arg@MNPs), herein used as control. More importantly, after optimization of the MNP concentration and contact time, the H-Coll@MNPs were able to capture 95% of bacteria present in the samples. More importantly, the bacteria can be enriched within 30 min and the time for bacterial identification is effectively shortened in comparison to the "gold standard" in clinical diagnosis. These results suggest that H-Coll@MNPs can be used for the selective isolation of specific bacteria from mixed populations present, for example, in biological samples.

10 pags., 2 figs., 1 tab., 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 gFOS gsucrose-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., The study received financial support from Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and the project FoSynBio—Synthetic biology approaches to design and construct microbial cell factories for the production of fructooligosaccharides (POCI-01–0145-FEDER-029549).

For the first time, an aptamer that specifically binds to cathepsin B (CatB)-overexpressing prostate cancer cells was identified by combining SELEX and computational tools.

The world economy is currently moving towards more sustainable approaches. Lignocellulosic biomass has been widely used as a substitute for fossil sources since it is considered a low-cost bio-renewable resource due to its abundance and continuous production. Compost habitats presenting high content of lignocellulosic biomass are considered a promising source of robust lignocellulose-degrading enzymes. Recently, several novel biocatalysts from different environments have been identified using metagenomic techniques. A key point of the metagenomics studies is the extraction and purification of nucleic acids. Nevertheless, the isolation of high molecular weight DNA from soil-like samples, such as compost, with the required quality for metagenomic approaches remains technically challenging, mainly due to the complex composition of the samples and the presence of contaminants like humic substances. In this work, a rapid and cost-effective protocol for metagenomic DNA extraction from compost samples composed of lignocellulosic residues and containing high content of humic substances was developed. The metagenomic DNA was considered as representative of the global environment and presented high quality (>\thinspace99\\% of humic acids effectively removed) and sufficient quantity (10.5--13.8 \textmug g1 of compost) for downstream applications, namely functional metagenomic studies. The protocol takes about 4 h of bench work, and it can be performed using standard molecular biology equipment and reagents available in the laboratory., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and the Project LIG-NOZYMES—Metagenomics approach to unravel the potential of lignocellulosic residues towards the discovery of novel enzymes (POCI-01–0145-FEDER-029773)., info:eu-repo/semantics/publishedVersion

Microbial exopolysaccharides (EPS) are promising alternatives to synthetic polymers in a variety of applications. Their high production costs, however, limit their use despite their outstanding properties. The use of low-cost substrates such as agro-industrial wastes in their production, can help to boost their market competitiveness. In this work, an alternative low-cost culture medium (CSLM) was developed for EPS production by

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

Despite recent preclinical progress with oncolytic bacteria in cancer therapy, dose-limiting toxicity has been a long-standing challenge for clinical application. Genetic and chemical modifications for enhancing the bacterial tumor-targeting ability have been unable to establish a balance between increasing its specificity and effectiveness while decreasing side effects. Herein, we report a simple, highly efficient method for rapidly self-assembling a clinically used lipid on bacterium and for reducing its minimum effective dose and toxicity to normal organs. The resultant bacteria present the ability to reverse-charge between neutral and acidic solutions, thus enabling weak interactions with the negatively charged normal cells, hence increasing their biocompatibility with blood cells and with the immune system. Additionally, the lipid-coated bacteria exhibit a longer blood circulation lifetime and low tissue trapping compared with the wild-type strains. Thereby, the engineered bacteria show enhanced tumor specificity and effectiveness even at low doses. Multiple visualization techniques are used for vividly demonstrating the time course of bacterial circulation in the blood and normal organs after intravenous administration. We believe that these methods for biointerfacial lipid self-assembly and evaluation of bacterial systemic circulation possess vast potential in exquisitely fabricating engineered bacteria for cancer therapy in the future., This research was supported by the National Natural Science Foundation of China (grant no. 81971726), the Natural Science Foundation of Jiangsu Province (grant no. BK20191351), the China Postdoctoral Science Foundation (grant no. 2020M670091ZX), and the Nanjing Healthcare Science and Technology Development Special Funded Project (YKK20191)., info:eu-repo/semantics/publishedVersion

The milk-derived bovine lactoferrin (bLf) is an iron-binding glycoprotein with remarkable selective anticancer activity towards highly metastatic cancer cells displaying the proton pump V-ATPase at the plasma membrane. As studies aiming to dissect the bLf mechanisms of action are critical to improve its efficacy and boost its targeted clinical use, herein we sought to further uncover the molecular basis of bLf anticancer activity. We showed that bLf co-localizes with V-ATPase and cholesterol-rich lipid rafts at the plasma membrane of highly metastatic cancer cells. Our data also revealed that bLf perturbs cellular trafficking, induces intracellular accumulation of cholesterol and lipid rafts disruption, downregulates PI3K, and AKT or p-AKT and inhibits glycolysis of cancer cells harbouring V-ATPase at the plasma membrane lipid rafts. Altogether, our results can lay the foundation for future bLf-based targeted anticancer strategies as they unravel a novel cascade of molecular events that explains and further reinforces bLf selectivity for cancer cells displaying plasmalemmal V-ATPase.

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

Microbial exopolysaccharides (EPS) are promising alternatives to synthetic polymers in a variety of applications. Their high production costs, however, limit their use despite their outstanding properties. The use of low-cost substrates such as agro-industrial wastes in their production, can help to boost their market competitiveness. In this work, an alternative low-cost culture medium (CSLM) was developed for EPS production by Rhizobium viscosum CECT908, containing sugarcane molasses (60 g/L) and corn steep liquor (10 mL/L) as sole ingredients. This medium allowed the production of 6.1 ± 0.2 g EPS/L, twice the amount produced in the standard medium (Syn), whose main ingredients were glucose and yeast extract. This is the first report of EPS production by R. viscosum using agro-industrial residues as sole substrates. EPSCSLM and EPSSyn exhibited a similar carbohydrate composition, mainly 4-linked galactose, glucose and mannuronic acid. Although both EPS showed a good fit to the Herschel–Bulkley model, EPSCSLM displayed a higher yield stress and flow consistency index when compared with EPSSyn, due to its higher apparent viscosity. EPSCSLM demonstrated its potential use in Microbial Enhanced Oil Recovery by enabling the recovery of nearly 50% of the trapped oil in sand-pack column experiments using a heavy crude oil.

Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.seppur.2018.07.062., Esterases catalyze the cleavage and formation of ester bonds of a broad range of substrates presenting a widespread spectrum of industrial applications. This work aimed to partially purify and characterize an esterase from Aureobasidium pullulans LABIOTEC 01 produced in a culture medium containing olive mill wastewater. Esterase purification was evaluated using different strategies, namely the enzyme recovery by PEG-salt aqueous two-phase systems (ATPSs); and the enzyme precipitation with ammonium sulfate, acetone, and ethanol. The best purification factor (18±2) was obtained when the ATPS composed of 20% (w/w) polyethylene glycol (PEG) 6000 and 5.8% (w/w) potassium phosphate buffer (PPB) pH 8.0 was combined with acetone precipitation. The partially purified enzyme presented an optimum pH of 5.0, although it remained active in the pH range of 4.5 to 7.5 ( 50% relative activity). The optimum temperature was found to be 60°C. Furthermore, the addition of salts such as FeCl3, CuSO4 and MnCl2 promoted an increase in the enzymatic activity (above 100%). The enzyme was found to be stable and showed high activity when exposed to polar solvents such as dimethyl sulfoxide, dimethylformamide, and methanol. The use of an integrated strategy of purification combining simple purification methods such as ATPS and precipitation was herein reported for the first time for esterase. This strategy proved to be an interesting approach to partially purify the esterase produced under submerged fermentation by A. pullulans. Furthermore, the enzyme showed potential to be applied in industrial biocatalytic processes using high temperature and different salts or solvents. Also, the production of esterase using olive mill wastewater as substrate demonstrated to be a suitable alternative to reduce and valorize agro-industrial residues., ACL acknowledges his post-doctoral grant and research funds from CAPES and CNPq (Brazilian funding agencies). SCS and LRR acknowledge their grants SFRH/BPD/88584/2012 and SFRH/BSAB/142873/ 2018, respectively, from the Portuguese Foundation for Science and Technology (FCT). The study also received financial support from FCT under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684), the Project MultiBiorefinery - Multi‐purpose strategies for broadband agro‐forest and fisheries by‐products valorisation: a step forward for a truly integrated biorefinery (POCI-01-0145-FEDER-016403) and the Project Lignozymes - Metagenomics approach to unravel the potential of lignocellulosic residues towards the discovery of novel enzymes (POCI-010145-FEDER-029773). The authors also acknowledge financial support from the BioTecNorte operation (NORTE-01-0145-FEDER-000004) fundedbytheEuropeanRegionalDevelopment Fundunderthescopeof Norte2020 - Programa Operacional Regional do Norte., info:eu-repo/semantics/publishedVersion

Triple-negative breast cancer is the most aggressive subtype of invasive breast cancer with a poor prognosis and no approved targeted therapy. Hence, the identification of new and specific ligands is essential to develop novel targeted therapies. In this study, we aimed to identify new aptamers that bind to highly metastatic breast cancer MDA-MB-231 cells using the cell-SELEX technology aided by high throughput sequencing. After 8 cycles of selection, the aptamer pool was sequenced and the 25 most frequent sequences were aligned for homology within their variable core region, plotted according to their free energy and the key nucleotides possibly involved in the target binding site were analyzed. Two aptamer candidates, Apt1 and Apt2, binding specifically to the target cells with Kd values of 44.3 ± 13.3 nM and 17.7 ± 2.7 nM, respectively, were further validated. The binding analysis clearly showed their specificity to MDA-MB-231 cells and suggested the targeting of cell surface receptors. Additionally, Apt2 revealed no toxicity in vitro and showed potential translational application due to its affinity to breast cancer tissue sections. Overall, the results suggest that Apt2 is a promising candidate to be used in triple-negative breast cancer treatment and/or diagnosis. © 2021, The Author(s)., Tis study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte. Débora Ferreira (DF) is the recipient of a fellowship supported by a doctoral advanced training (call NORTE-69-2015-15) funded by the European Social Fund under the scope of Norte2020—Programa Operacional Regional do Norte. Joaquim Barbosa (JB) and Diana A. Sousa (DAS) acknowledge FCT for the Grants SFRH/BD/51109/2010 and PD/BD/139083/2018, respectively., info:eu-repo/semantics/publishedVersion

Consumers are conscientiously changing their eating preferences toward healthier options, such as functional foods enriched with pre- and probiotics. Prebiotics are attractive bioactive compounds with multidimensional beneficial action on both human and animal health, namely on the gastrointestinal tract, cardiometabolism, bones or mental health. Conventionally, prebiotics are non-digestible carbohydrates which generally present favorable organoleptic properties, temperature and acidic stability, and are considered interesting food ingredients. However, according to the current definition of prebiotics, application categories other than food are accepted, as well as non-carbohydrate substrates and bioactivity at extra-intestinal sites. Regulatory issues are considered a major concern for prebiotics since a clear understanding and application of these compounds among the consumers, regulators, scientists, suppliers or manufacturers, health-care providers and standards or recommendation-setting organizations are of utmost importance. Prebiotics can be divided in several categories according to their development and regulatory status. Inulin, galactooligosaccharides, fructooligosaccharides and lactulose are generally classified as well established prebiotics. Xylooligosaccharides, isomaltooligosaccharides, chitooligosaccharides and lactosucrose are classified as "emerging" prebiotics, while raffinose, neoagaro-oligosaccharides and epilactose are "under development." Other substances, such as human milk oligosaccharides, polyphenols, polyunsaturated fatty acids, proteins, protein hydrolysates and peptides are considered "new candidates." This chapter will encompass actual information about the non-established prebiotics, mainly their physicochemical properties, market, legislation, biological activity and possible applications. Generally, there is a lack of clear demonstrations about the effective health benefits associated with all the non-established prebiotics. Overcoming this limitation will undoubtedly increase the demand for these compounds and their market size will follow the consumer's trend.

Consumers are conscientiously changing their eating preferences toward healthier options, such as functional foods enriched with pre- and probiotics. Prebiotics are attractive bioactive compounds with multidimensional beneficial action on both human and animal health, namely on the gastrointestinal tract, cardiometabolism, bones or mental health. Conventionally, prebiotics are non-digestible carbohydrates which generally present favorable organoleptic properties, temperature and acidic stability, and are considered interesting food ingredients. However, according to the current definition of prebiotics, application categories other than food are accepted, as well as non-carbohydrate substrates and bioactivity at extra-intestinal sites. Regulatory issues are considered a major concern for prebiotics since a clear understanding and application of these compounds among the consumers, regulators, scientists, suppliers or manufacturers, health-care providers and standards or recommendation-setting organizations are of utmost importance. Prebiotics can be divided in several categories according to their development and regulatory status. Inulin, galactooligosaccharides, fructooligosaccharides and lactulose are generally classified as well established prebiotics. Xylooligosaccharides, isomaltooligosaccharides, chitooligosaccharides and lactosucrose are classified as "emerging" prebiotics, while raffinose, neoagaro-oligosaccharides and epilactose are "under development." Other substances, such as human milk oligosaccharides, polyphenols, polyunsaturated fatty acids, proteins, protein hydrolysates and peptides are considered "new candidates." This chapter will encompass actual information about the non-established prebiotics, mainly their physicochemical properties, market, legislation, biological activity and possible applications. Generally, there is a lack of clear demonstrations about the effective health benefits associated with all the non-established prebiotics. Overcoming this limitation will undoubtedly increase the demand for these compounds and their market size will follow the consumer's trend.

A levan-type fructooligosaccharide was produced by a Paenibacillus strain isolated from Brazilian crude oil, the purity of which was 98.5% after precipitation with ethanol and dialysis. Characterization by FTIR, NMR spectroscopy, GC-FID and ESI-MS revealed that it is a mixture of linear (26) fructosyl polymers with average degree of polymerization (DP) of 18 and branching ratio of 20. Morphological structure and physicochemical properties were investigated to assess levan microstructure, degradation temperature and thermomechanical features. Thermal Gravimetric Analysis highlighted degradation temperature of 218°C, Differential Scanning Calorimetry (DSC) glass transition at 81.47°C, and Dynamic Mechanical Analysis three frequency-dependent transition peaks. These peaks, corresponding to a first thermomechanical transition event at 86.60°C related to the DSC endothermic event, a second at 170.9°C and a third at 185.2°C, were attributed to different glass transition temperatures of oligo and polyfructans with different DP. Levan showed high morphological versatility and technological potential for the food, nutraceutical, and pharmaceutical industries., São Paulo Research Foundation (FAPESP) within Grants 2020/13271-6 and 2018/25511-1. Additionally, the authors acknowledge the financial support by National Council for Scientific and Technological Development – CNPq and by Coordination for the Improvement of Higher Education Personnel (CAPES), Finance Code 001. In addition, this study was supported by the Portuguese Science Foundation (FCT) under the scope of the strategic funding of UID/BIO/04469/2020 unit and the Project FOSYNBIO (POCI-01-0145-FEDER-029549)., info:eu-repo/semantics/publishedVersion

Saccharomyces cerevisiae has been for a long time a common model for fundamental biological studies and a popular biotechnological engineering platform to produce chemicals, fuels, and pharmaceuticals due to its peculiar characteristics. Both lines of research require an effective editing of the native genetic elements or the inclusion of heterologous pathways into the yeast genome. Although S. cerevisiae is a well-known host with several molecular biology tools available, a more precise tool is still needed. The clustered, regularly interspaced, short palindromic repeats–associated Cas9 (CRISPR-Cas9) system is a current, widespread genome editing tool. The implementation of a reprogrammable, precise, and specific method, such as CRISPR-Cas9, to edit the S. cerevisiae genome has revolutionized laboratory practices. Herein, we describe and discuss some applications of the CRISPR-Cas9 system in S. cerevisiae from simple gene knockouts to more complex processes such as artificial heterologous pathway integration, transcriptional regulation, or tolerance engineering.

Aflatoxins are hepatotoxic and carcinogenic fungal secondary metabolites that usually contaminate crops and represent a serious health hazard for humans and animals worldwide. In this work, the effect of rhamnolipids (RLs) produced by Pseudomonas aeruginosa #112 on the growth and aflatoxins production by Aspergillus flavus MUM 17.14 was studied in vitro. At concentrations between 45 and 1500 mg/L, RLs reduced the mycelial growth of A. flavus by 2340% and the production of aflatoxins by 93.999.5%. Purified mono-RLs and di-RLs exhibited a similar inhibitory activity on fungal growth. However, the RL mixture had a stronger inhibitory effect on aflatoxins production at concentrations up to 190 mg/L, probably due to a synergistic effect resulting from the combination of both congeners. Using transmission electron microscopy, it was demonstrated that RLs damaged the cell wall and the cytoplasmic membrane of the fungus, leading to the loss of intracellular content. This disruptive phenomenon explains the growth inhibition observed. Furthermore, RLs down-regulated the expression of genes aflC, aflE, aflP and aflQ involved in the aflatoxins biosynthetic pathway (6.4, 44.3, 38.1 and 2.0-fold, respectively), which is in agreement with the almost complete inhibition of aflatoxins production. Overall, the results herein gathered demonstrate for the first time that RLs could be used against aflatoxigenic fungi to attenuate the production of aflatoxins, and unraveled some of their mechanisms of action., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER 000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. The authors also acknowledge the support of the i3S Scientific Platform Histology and Electron Microscopy (HEMS), member of the PPBI (PPBI POCI-01-0145-FEDER-022122). A.I. Rodrigues and L. Abrunhosa acknowledge FCT for the grant SFRH/BD/111600/2015 and the Assistant Research contract CEECIND/00728/2017, respectively., info:eu-repo/semantics/publishedVersion

The potential use of alternative culture media towards the development of a sustainable bioprocess to produce lipases by Diutina rugosa is clearly demonstrated. First, a synthetic medium containing glucose, peptone, yeast extract, oleic acid, and ammonium sulfate was proposed, with lipase activity of 143 U/L. Then, alternative culture media formulated with agro-industrial residues, such as molasses, corn steep liquor (CSL), and olive mill waste (OMW), were investigated. An experimental design was conducted, and only CSL concentration was found to have a positive effect in lipase production. The highest lipase activity (561 U/L) was produced on a mixture of molasses (5 g/L), CSL (6 g/L), OMW (0.5% v/v), 0.5 g/L of ammonium sulfate, and 3 g/L of peptone at 24 h of cultivation. Lipase production was also carried out in a 1-L bioreactor leading to a slightly higher lipase activity at 24 h of cultivation. The semi-purified enzyme exhibits an optimum temperature and pH of 40 °C and 7.0, respectively. Finally, the media cost per unit of lipase produced (UPC) was influenced by the medium components, specially by the inducer used. The lowest UPC was obtained when the agro-industrial residues were combined and used at the improved concentrations.

Acrylic acid is a value-added chemical used in industry to produce diapers, coatings, paints, and adhesives, among many others. Due to its economic importance, there is currently a need for new and sustainable ways to synthesise it. Recently, the focus has been laid in the use ofEscherichia colito express the full bio-based pathway using 3-hydroxypropionate as an intermediary through three distinct pathways (glycerol, malonyl-CoA, andβ-alanine). Hence, the goals of this work were to use COPASI software to assess which of the three pathways has a higher potential for industrial-scale production, from either glucose or glycerol, and identify potential targets to improve the biosynthetic pathways yields.When compared to the available literature, the models developed during this work successfully predict the production of 3-hydroxypropionate, using glycerol as carbon source in the glycerol pathway, and using glucose as a carbon source in the malonyl-CoA andβ-alanine pathways. Finally, this work allowed to identify four potential over-expression targets (glycerol-3-phosphate dehydrogenase (G3pD), acetyl-CoA carboxylase (AccC), aspartate aminotransferase (AspAT), and aspartate carboxylase (AspC)) that should, theoretically, result in higher AA yields.Author summaryAcrylic acid is an economically important chemical compound due to its high market value. Nevertheless, the majority of acrylic acid consumed worldwide its produced from petroleum derivatives by a purely chemical process, which is not only expensive, but it also contributes towards environment deterioration. Hence, justifying the current need for sustainable novel production methods that allow higher profit margins. Ideally, to minimise production cust, the pathway should consist in the direct bio-based production from microbial feedstocks, such as Escherichia coli, but the current yields achieved are still to low to compete with conventional method. In this work, even though the glycerol pathway presented higher yields, we identified the malonyl-CoA route, when using glucose as carbon source, as having the most potential for industrial-scale production, since it is cheaper to implement. Furthermore, we also identified potential optimisation targets for all the tested pathways, that can help the bio-based method to compete with the conventional process.

Polyphenols are plant secondary metabolites with diverse biological and potential therapeutic activities such as antioxidant, anti-inflammatory and anticancer, among others. However, their extraction from the native plants is not enough to satisfy the increasing demand for this type of compounds. The development of microbial cell factories to effectively produce polyphenols may represent the most attractive solution to overcome this limitation and produce high amounts of these bioactive molecules. With the advances in the synthetic biology field, the development of efficient microbial cell factories has become easier, largely due to the development of the molecular biology techniques and by the identification of novel isoenzymes in plants or simpler organisms to construct the heterologous pathways. Furthermore, efforts have been made to make the process more profitable through improvements in the host chassis. In this review, advances in the production of polyphenols by genetically engineered Saccharomyces cerevisiae as well as by synthetic biology and metabolic engineering approaches to improve the production of these compounds at industrial settings are discussed.

The semi-synthesis of 11 isatin derivatives was achieved through bimolecular nucleophilic substitution and click chemistry. Seven new compounds were obtained. All chemical structures were determined by infrared spectroscopy (IR), nuclear magnetic resonance spectrometry (NMR) and high-resolution mass spectrometry (HRMS) data. These derivatives were evaluated for their anti-GSK-3 activity and all isatin derivatives (N-alkyl and 1,2,3-triazolic) exhibited strong inhibitory activity, with 2b and 4h exhibiting remarkable potency. In addition, docking studies were performed with 2b and 2e models to unravel the molecular mechanism underlying the polar interactions on the GSK-3 ATP-binding site., This study was funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brasil (CAPES), Finance Code 001, by the National Council of Scientific and Technological Development (CNPq) and Foundation of Support to Research and Innovation of Espírito Santo (FAPES PPE-Agro No. 76418880/16). We also would like to acknowledge INCTBioNat (CNPq 465637/2014-0) for additional support and NCQP-UFES, as well as the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020, Programa Operacional Regional do Norte. L. R. R. also acknowledges her sabbatical leave fellowship (SFRH/BSAB/142991/2018) funded by FCT. D. F. is recipient of a doctoral fellowship (call NORTE-69-2015-15) funded by the European Social Fund under the scope of Norte2020, Programa Operacional Regional do Norte., info:eu-repo/semantics/publishedVersion

Bovine lactoferrin (bLF) has been extensively described as a wide spectrum antimicrobial protein. bLF bactericidal activity has been mainly attributed to two different mechanisms: environmental iron depletion and cell membrane destabilization. Due to its antimicrobial properties, bLF has been included in the formulation nutraceutical food products and edible active packages. This work comprises the experimental evidence of the requirement of bLF unrestricted mobility (free bLF) to effectively perform its bactericidal action. To assess the unrestricted and restricted bLF activity, a nontoxic matrix of bacterial nanocellulose (BNC) was used as carrier, and as an anchoring scaffold, respectively. Therefore, BNC was functionalized with bLF through two different methodologies: (i) bLF was embedded within the three-dimensional structure of BNC and; (ii) bLF was covalently bounded to the nanofibrils of BNC. bLF efficiency was tested against two bacteria isolated from clinical specimens, Escherichia coli and Staphylococcus aureus. bLF concentration after covalent binding to BNC was two-fold higher in comparison to the embedding method. Nevertheless, only the embedded bLF exhibited a significant bactericidal activity, due to bLF ability to permeate the BNC matrix and execute its bactericidal action., Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/ 04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER- 000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte, Portuguese Foundation for Science and Technology (FCT) in the framework of the Strategic Funding of UID/FIS/04650/2020 and projects PTDC/BTM-MAT/28237/ 2017 and PTDC/EMDEMD/28159/2017, Portuguese Foundation for Science and Technology (FCT) SFRH/BD/64901/2009 and SFRH/BD/111478/2015, Spanish State Research Agency (AEI) and the European Regional Development Fund through the project PID2019-106099RBC43/ AEI/10.13039/501100011033 and from the Basque Government Industry and Education Department under the ELKARTEK, HAZITEK and PIBA (PIBA-2018-06) programs, respectively, info:eu-repo/semantics/publishedVersion

A sustainable and rational use of the existing resources with limited generation of residues and using environmentally friendly processes is a current concern that is being addressed under the concepts of green chemistry and circular economy. Biotechnological processes, namely, the use of microorganisms and/or enzymes to cost-efficiently produce and convert renewable biological resources and waste streams into valuable products across several economic sectors (such as food, feed, bio-based products, and bioenergy), comprise a powerful alternative. The so-called circular bioeconomy is currently taking advantage of the recent developments in omics techniques, gene editing, systems, and synthetic biology, as well as of the novel technological developments of bioreactors and downstream processes. In addition, the potential of many existing residues from different sources, which remain to be mapped and thoroughly characterized, is largely unexplored and represents a great opportunity to design new products and processes, hence a great opportunity for the economy, for the environment, and ultimately for the society.

Sugar fatty acid esters (SFAEs) are composed of one or more fatty acid chains linked to a carbohydrate molecule. They are commonly used as non-ionic and biodegradable surfactants and emulsifiers. However, SFAEs also present other interesting features such as tasteless, odorless, non-toxicity, and bioactivity which make them suitable for industrial applications in the food, pharmaceutical, and cosmetics industries. Traditionally, SFAEs are obtained from chemical synthesis despite the recognized disadvantages associated to this process. More recently, the synthesis of SFAEs using enzymes as biocatalysts proved to be a suitable and greener alternative. However, the use of organic solvents as reaction medium has been pointed out as one of the greatest limitations of the enzymatic process. To overcome this issue, ionic liquids (ILs) have been proposed as alternative solvents. ILs are frequently described as “tunable” solvents due to the possibility of combining different cations and anions to obtain the desired physicochemical properties. Based on their unique characteristics, ILs have been recognized as suitable solvents for biocatalytic processes involving different enzymes and reactions. Nevertheless, the selection of the suitable IL for the enzymatic synthesis of SFAEs is considered a key aspect in the bioprocess since compounds chemically different (sugars, fatty acids, and enzymes) are simultaneously present in the reaction medium. Ideally, the IL used as solvent should facilitate the solubilization of the substrates and not negatively affect the enzymatic activity and stability. The enzymatic synthesis of SFAEs can occur though two different approaches, namely esterification and transesterification. Several ILs and biocatalysts have been successfully reported for the synthesis of different types of SFAEs. Considering the enormous versatility of combining different sugars with fatty acids, it is expected that novel and promising SFAEs could be synthesized in the future using ILs as reaction medium.

Brewers spent grain (BSG) is an inexpensive and abundant brewery by-product that can be used to produce prebiotic arabino-xylooligosaccharides (AXOS). In this study, Bacillus subtilis 3610 was used, for the first time, to produce AXOS through direct fermentation of BSG. Additionally, the microorganism was genetically modified to improve the AXOS production. The xylanase gene xyn2 from Trichoderma reesei coupled with a secretion tag endogenous to B. subtilis was cloned in pDR111 and integrated into its chromosome. After optimization by experimental design, AXOS with a degree of polymerization ranging from 2 to 6 were obtained. The maximum production yield expressed in xylose equivalents per amount of BSG (54.2 ± 1.1 mg/g) represents an increase of 33% comparing to the wild type. When compared with the enzymatic hydrolysis process, single-step fermentation with B. subtilis proved to be a very promising low-cost strategy for the simultaneous production of AXOS and valorization of BSG., CA, SCS, EC and LRR acknowledge their grants (PD/BD/105870/ 2014, SFRH/BPD/88584/2012, SFRH/BPD/70589/2010 and SFRH/ BSAB/142873/2018) from Portuguese Foundation for Science and Technology(FCT).Thestudyreceived financialsupportfromFCTunder the scope of the strategic funding of UID/BIO/04469/2013 unit; COMPETE2020 (POCI-01-0145-FEDER-006684); QOPNAresearch Unit (FCT UID/QUI/00062/2013), through national founds and where applicable co-financed by the FEDER, within the PT2020 Partnership Agreement; the projects MultiBiorefinery (POCI-01-0145-FEDER016403); FoSynBio (POCI-01-0145-FEDER-029549) and Lignozymes (POCI-01-0145-FEDER-029773). The authors also acknowledge financial support from the BioTecNorte operation (NORTE-01-0145FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte., info:eu-repo/semantics/publishedVersion

"Available online 21 November 2017", The antimicrobial and anti-adhesive activities of the cell-bound biosurfactants, produced by Lactobacillus pentosus (PEB), characterized as glycolipopeptide macromolecules, were evaluated against several microorganisms present in the skin microflora, envisaging its potential use as a natural ingredient in cosmetic and personal care formulations. Their performance was compared with another cell-bound biosurfactants also characterized as glycolipopeptides produced by Lactobacillus paracasei (PAB). At concentrations of 50 mg/mL, the PEB showed an important antimicrobial activity against Pseudomonas aeruginosa (85% when extracted with phosphate buffer (PB) and 100% when extracted with phosphate buffer saline (PBS)), Streptococcus agalactiae (100% for both extracts), Staphylococcus aureus (67% when extracted with PBS and 100% when extracted with PB), Escherichia coli (72% when extracted with PB and 89% when extracted with PBS), Streptococcus pyogenes (about 85% for both extracts) and Candida albicans (around 70% for both extracts), comparable with that obtained for the PAB. However, at lower concentrations the PAB exhibited in general higher antimicrobial activities. Biosurfactants produced by both microorganisms also showed significant anti-adhesive properties against all the microorganisms under study, except for E. coli and C. albicans (less than 30%). Overall, these cell-bound biosurfactants could be used as potential antimicrobial and anti-adhesive agents in cosmetic and pharmaceutical formulations., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI01-0145-FEDER-006684), the Project MultiBiorefinery − Multipurposestrategies for broadband agro-forest and fisheries byproducts valorisation: a step forward for a truly integrated biorefinery (POCI-01-0145-FEDER-016403) and the project RECI/BBBEBI/0179/2012 (FCOMP-01-0124-FEDER-027462), as well as X. Vecino post-doctoral grant (SFRH/BPD/101476/2014). Also, L. Rodríguez-López acknowledges to the Spanish Ministry of Education, Culture and Sport for her pre-doctoral fellowship (FPU15/00205)., info:eu-repo/semantics/publishedVersion

Curcumin is a polyphenolic compound found in Curcuma longa. This bioactive molecule has several reported health-benefit effects, being the anticarcinogenic activity among the most promising ones. However, curcumin extraction from natural sources is hampered by impure products obtained from harsh chemicals and limited by plant seasonality and high prices. Therefore, curcumin heterologous production emerged as an interesting alternative. Escherichia coli has been explored as chassis but the implementation of the pathway in Saccharomyces cerevisiae can have several advantages, including its generally regarded as safe status. Hence, S. cerevisiae was engineered for the first time to produce curcumin from its precursor ferulic acid.The enzymes 4-coumarate-CoA ligase (4CL1) from Arabidopsis thaliana or feruloyl-CoA synthetase (FerA) from Pseudomonas paucimobilis and type III polyketide synthases (PKSs) from Oryza sativa or C. longa were expressed in BY4741 strain. To avoid ferulic acid deviation, the gene FDC1 coding a ferulic acid decarboxylase was deleted. The maximum curcumin titer was obtained with FerA combined with C. longa PKSs (2.7 mg LUp to our knowledge, this is the first work reporting the expression of a feruloyl-CoA synthase and also curcuminoid biosynthetic enzymes in S. cerevisiae, and consequently, curcumin production.

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., This research is affiliated with the VibrANT project that received funding from the EU Horizon 2020 Research and Innovation Programme under the Marie Sklowdowska-Curie Grant, agreement no 765042. In addition, the authors acknowledge the financial support from Fundação para a Ciência e Tecnologia (FCT) under the scope of the strategic funding of UID/BIO/04469/2020 unit. Débora Ferreira (DF) is the recipient of a fellowship supported by a doctoral advanced training (call NORTE-69-2015-15) funded by the European Social Fund under the scope of Norte2020., info:eu-repo/semantics/publishedVersion

Cosmetic and personal care products including toothpaste, shampoo, creams, makeup, among others, are usually formulated with petroleum-based surfactants, although in the last years the consume trend for green products is inducing the replacement of surface-active agents in these formulations by natural surfactants, so-called biosurfactants. In addition to their surfactant capacity, many biosurfactants can act as good emulsifiers, which is an extra advantage in the preparation of green cosmetic products. In this work, a biosurfactant obtained from Lactobacillus paracasei was used as a stabilizing agent in oil-in-water emulsions containing essential oils and natural antioxidant extract. In the presence of biosurfactant, maximum percentages of emulsion volumes (EV = 100%) were observed, with droplets sizes about 199 nm. These results were comparable with the ones obtained using sodium dodecyl sulfate (SDS), a synthetic well known surfactant with high emulsify capacity. Moreover, the biosurfactant and emulsions cytotoxicity was evaluated using a mouse fibroblast cell line. Solutions containing 5 g/L of biosurfactant presented cell proliferation values of 97%, whereas 0.5 g/L of SDS showed a strong inhibitory effect. Overall, the results herein gathered are very promising towards the development of new green cosmetic formulations., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01-0145-FEDER-006684) and the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462), as well as X. Vecino post-doctoral grant (SFRH/BPD/101476/2014). Additionally, the authors acknowledge the financial support from Spanish Ministry of Economy and Competitiveness (FEDER funds) under the project CTM2015-68904. Also, A. Ferreira acknowledges to the Region Aquitaine Limousin Poitou-Charentes for her Erasmus + internship., info:eu-repo/semantics/publishedVersion

Biosurfactants, surface active molecules synthesized by microorganisms, represent a promising alternative to the synthetic surfactants in many different applications. Among them, rhamnolipids have attracted considerable attention in the last years due to their extraordinary surface-active properties and biological activities. Rhamnolipids are usually synthesized by the gram-negative bacterium Pseudomonas aeruginosa as complex mixtures of different congeners. In this chapter, we describe the most common techniques that can be used for the production, recovery and purification of rhamnolipids, using two sequential chromatographic techniques to recover and separate the monorhamnolipid and dirhamnolipid congeners.

This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and under the scope of the Project RECI/BBB-EBI/0179/ 2012 (FCOMP-01-0124-FEDER-027462). The authors also acknowledge financial support from BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. D. Ferreira is recipient of a fellowship (UMINHO/BD/21/2016) supported by a doctoral advanced training (call NORTE-69-2015-15) funded by the European Social Fund under the scope of Norte2020. J. L. Rodrigues acknowledges the post-doctoral grant (UMINHO/BPD/37/2015) funded by FCT.

Biosurfactants, surface active molecules synthesized by microorganisms, represent a promising alternative to the synthetic surfactants in many different applications. Among them, rhamnolipids have attracted considerable attention in the last years due to their extraordinary surface-active properties and biological activities. Rhamnolipids are usually synthesized by the gram-negative bacterium Pseudomonas aeruginosa as complex mixtures of different congeners. In this chapter, we describe the most common techniques that can be used for the production, recovery and purification of rhamnolipids, using two sequential chromatographic techniques to recover and separate the monorhamnolipid and dirhamnolipid congeners.

Early breast cancer detection remains a challenge and comes at high costs for governments, health institutions, and the patients. Risk factors (age, genetics and geographical variations) and disease heterogeneity are complex and not always easy to understand using the conventional diagnosis methodologies (e.g. mammography, X-rays, ultrasounds and magnetic resonance imaging (MRI)). This chapter provides insight into the current developments on breast cancer and the role of biosensors for its rapid detection as a potential solution to the limitations of current approaches for the detection and differentiation of the different types of breast cancer.

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.bej.2021.107954., Aureobasidium pullulans URM 7059 produced esterase using residual glycerol from biodiesel as the sole carbon source. The culture medium containing residual glycerol (0.1% v/v), (NH4)2SO4 (4g/L), and yeast extract (8g/L) resulted in the highest esterase production using shake-flasks. The enzyme exhibited a molar mass of 50kDa and was stable at neutral pH and temperatures below 30°C. The cations Cu2+ and Al3+ did not affect the esterase activity, while Ca2+ promoted the highest activity loss. The enzyme kinetic parameters were determined using different substrates (p-nitrophenylcaprylate and p-nitrophenylbutyrate). Km and Vmax were 1.4mM and 218µmolmin-1 for p-NPC, and 1.55mM and 76.7µmolmin-1 for p-NPB. The esterase production was further evaluated using stirred tank and 2-L airlift bioreactors. The airlift reactor operating at the highest air flow rate (8L/min) increased the enzyme productivity 3-fold compared to the shake-flasks experiments. However, the crude enzymatic extract showed 3 active protein bands by zymography with molecular masses of 172kDa, 66kDa, and 40kDa approximately, suggesting that the pattern of enzyme production changed due to aeration. The crude enzyme degraded the MACO-Sty biopolymer in 14 days, being stable in a wide range of pH (7.0 9.0) and temperatures (40°C 80°C). The results suggest that this enzyme is a promising catalyst in remediation processes., This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004), projects MultiBiorefinery (POCI-01-0145-FEDER016403) and Lignozymes (POCI-01-0145-FEDER-029773), all funded by the European Regional Development Fund under the scope of Norte2020 – Programa Operacional Regional do Norte. In Brazil, this study was financed in part by the Coordenaçao ˜ de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. The authors also thank the CNPq and FUNCAP for financial support., info:eu-repo/semantics/publishedVersion

Bacterial cellulose (BC) films from two distinct sources (obtained by static culture with Gluconacetobacter xylinus ATCC 53582 (BC1) and from a commercial source (BC2)) were modified by bovine lactoferrin (bLF) adsorption. The functionalized films (BC+bLF) were assessed as edible antimicrobial packaging, for use in direct contact with highly perishable foods, specifically fresh sausage as a model of meat products. BC+bLF films and sausage casings were characterized regarding their water vapour permeability (WVP), mechanical properties, and bactericidal efficiency against two food pathogens, Escherichia coli and Staphylococcus aureus. Considering their edibility, an in vitro gastrointestinal tract model was used to study the changes occurring in the BC films during passage through the gastrointestinal tract. Moreover, the cytotoxicity of the BC films against 3T3 mouse embryo fibroblasts was evaluated. BC1 and BC2 showed equivalent density, WVP and maximum tensile strength. The percentage of bactericidal efficiency of BC1 and BC2 with adsorbed bLF (BC1+bLF and BC2+bLF, respectively) in the standalone films and in inoculated fresh sausages, was similar against E. coli (mean reduction 69 % in the films per se versus 94 % in the sausages) and S. aureus (mean reduction 97 % in the films per se versus 36 % in the case sausages). Moreover, the BC1+bLF and BC2+bLF films significantly hindered the specific growth rate of both bacteria. Finally, no relevant cytotoxicity against 3T3 fibroblasts was found for the films before and after the simulated digestion. BC films with adsorbed bLF may constitute an approach in the development of bio-based edible antimicrobial packaging systems., The authors would like to acknowledge Portuguese Foundation for Science and Technology (Fundação para a Ciência e Tecnologia) for the research grants: Jorge Padrão SFRH/BD/64901/2009, Sara Gonçalves SFRH/BD/63578/2009, João Pedro Silva SFRH/BPD/ 64958/2009, Ana Cristina Pinheiro SFRH/BPD/101181/2014. V. Sencadas thanks support from the COST Action MP1206: “Electrospun nano-fibres for bio inspired composite materials and innovative industrial applications” and MP1301: “New Generation Biomimetic and Customized Implants for Bone Engineering”. The authors would also like to thank the co-funded by the Programa Operacional Regional do Norte (ON.2 e O Novo Norte), QREN, FEDER Projects “BioHealth e Biotechnology and Bioengineering approaches to improve health quality”, Ref. NORTE-07-0124- FEDER-000027; “BioInd e Biotechnology and Bioengineering for improved Industrial and Agro-Food processes”, REF. NORTE-07- 0124-FEDER-000028; Strategic Project PEST-C/FIS/UI607/2014; Matepro eOptimizing Materials and Processes”, ref. NORTE-07- 0124-FEDER-000037; Strategic Project PEst-OE/EQB/LA0023/2013 and project ref. RECI/BBB-EBI/0179/2012 (project number FCOMP- 01-0124-FEDER-027462). Finally, the authors thank the Fundação para a Ciência e Tecnologia for the strategic funding from the UID/ BIO/04469/2013 unit.

Lactulose is a lactose-based carbohydrate with well-known prebiotic effect and recognized medical applications. Currently, the commercially available lactulose is chemically synthesized. Nevertheless, the process leads to low yields and high levels of by-products. Alternatively, lactulose can be produced by enzymatic synthesis, which provides a cleaner production under mild conditions. Two different enzymatic routes were reported for lactulose production. Lactulose can be obtained through hydrolysis and transfer reactions catalyzed by a glycosidase. Alternatively, lactulose can be produced by direct isomerization of lactose to lactulose catalyzed by cellobiose-2-epimerase. An interesting characteristic of lactulose is also its capacity to act as substrate in additional enzymatic synthesis which leads to the formation of attractive compounds, such as lactulose-based oligosaccharides and lactulose esters. Besides increasing the interest and potential of lactulose, these lactulose-based compounds can also offer new and promising functionalities and applications. Herein, we review the enzymes involved in the synthesis of lactulose, as well as the reaction conditions and yields. The potential of different enzymes is discussed and it is shown that reaction conditions and composition of products depend on the type of enzyme and its microbial source. The conversion of lactulose into lactulose-based compounds is also covered, describing in detail the biocatalysts involved, the reaction conditions used, and the potential of the final products obtained.