Genetic engineering of Saccharomyces boulardii: Tools, strategies and advances for enhanced probiotic and therapeutic applications.

Saccharomyces boulardii is a probiotic yeast that has been extensively studied in clinical trials, and it is used to treat several gut disorders. Its high survivability and the ability of secreting recombinant proteins, make S. boulardii an attractive delivery vessel for molecules of interest in the gut.

A first-in-class inhibitor of homologous recombination DNA repair counteracts tumour growth, metastasis and therapeutic resistance in pancreatic cancer.

Background: Pancreatic ductal adenocarcinoma (PDAC) is among the cancer types with poorest prognosis and survival rates primarily due to resistance to standard-of-care therapies, including gemcitabine (GEM) and olaparib. Particularly, wild-type (wt)BRCA tumours, the most prevalent in PDAC, are more resistant to DNA-targeting agents like olaparib, restraining their clinical application. Recently, we disclosed a monoterpene indole alkaloid derivative (BBIT20) as a new inhibitor of homologous recombination (HR) DNA repair with anticancer activity in breast and ovarian cancer.

Whole yeast cell synthesis of 5-hydroxymethylfurfural-derivatives from apple waste processed by green technologies.

The transition to renewable resources and bioprocesses is essential to address climate change and meet energy demands. Biorefineries offer a sustainable solution by valorising agro-industrial wastes, such as apple pomace, a by-product of the juice and cider industries. This study presents an efficient process to produce 5-hydroxymethylfurfural (HMF) derivatives using deep eutectic solvent (DES) and microwave-assisted heating.

CRISPR-Cas9 engineered Saccharomyces cerevisiae for endolysin delivery to combat Listeria monocytogenes.

Listeriosis is an infection caused by the consumption of food contaminated with Listeria monocytogenes. It leads to febrile gastroenteritis, central nervous system infections, and even death in risk populations. Bacteriophage endolysins selectively kill bacteria hydrolyzing their cell walls and have emerged as a potential tool for listeriosis control.

Production of HMF-derivatives from wine residues using Saccharomyces cerevisiae as whole-cell biocatalyst.

Background: There is an urgent need to develop bioprocesses independent of fossil resources to address resource depletion and mitigate environmental harm. Transitioning to a bio-based economy requires prioritizing chemical production processes that utilize renewable resources, ensuring sustainability and environmental responsibility. 5-Hydroxymethylfurfural (HMF) and its derivatives are promising building blocks, ranked among the top 12 bio-based molecules derived from biomass.

Overview of resveratrol properties, applications, and advances in microbial precision fermentation.

Resveratrol is an antioxidant abundant in plants like grapes and peanuts and has garnered significant attention for its potential therapeutic applications. This review explores its chemical attributes, stability, and solubility, influencing its diverse applications and bioavailability. Resveratrol's multifaceted therapeutic roles encompass: antioxidant, cardioprotective, anti-inflammatory, neuroprotective, anti-aging, and anticancer properties.

Metabolic Versatility of acetogens in syngas Fermentation: Responding to varying CO availability.

Syngas fermentation using acetogenic bacteria offers a promising route for sustainable chemical production. However, gas-liquid mass transfer limitations and efficient co-utilization of CO and H pose significant challenges. This study investigated the kinetics of syngas conversion to acetate by Acetobacterium wieringae and Clostridium species in batch conditions under varying initial CO partial pressures (19 - 110 kPa).

Engineering thermotolerant Kluyveromyces marxianus strains for resveratrol production by simultaneous saccharification and fermentation of whole slurry corn cob.

The microbial biosynthesis of the antioxidant compound resveratrol offers an eco-friendly and sustainable alternative to chemical synthesis or plant extraction. Here, we showed that Kluyveromyces marxianus strains produce p-coumaric acid, a key precursor of resveratrol, with higher titres achieved under increased agitation conditions. Through further strain engineering, resveratrol production was achieved using glucose, xylose, and/or ethanol as substrates.

Recombinant bovine serum albumin domain II as bioreceptor for ochratoxin A capture.

Established chromatographic techniques for mycotoxin control in foodstuffs require prior sample enrichment and clean-up, typically achieved using immunoaffinity columns (IACs). Bovine serum albumin (BSA) has recently emerged as a cost-effective alternative to antibodies used in IACs. This study aimed at exploring the BSA domain II (BDII), which houses the primary binding site for ochratoxin A (OTA), as a bioreceptor for OTA capture.

Pioneering precision in markerless strain development for Synechococcus sp. PCC 7002.

Marine cyanobacteria such as Picosynechococcus sp. (formerly called Synechococcus sp.) PCC 7002 are promising chassis for photosynthetic production of commodity chemicals with low environmental burdens.

Enhanced bacterial cellulose production in Komagataeibacter sucrofermentans: impact of different PQQ-dependent dehydrogenase knockouts and ethanol supplementation.

Background: Bacterial cellulose (BC) is a biocompatible material with unique mechanical properties, thus holding a significant industrial potential. Despite many acetic acid bacteria (AAB) being BC overproducers, cost-effective production remains a challenge. The role of pyrroloquinoline quinone (PQQ)-dependent membrane dehydrogenases (mDH) is crucial in the metabolism of AAB since it links substrate incomplete oxidation in the periplasm to energy generation.

Process modeling, environmental and economic sustainability of the valorization of whey and eucalyptus residues for resveratrol biosynthesis.

Biomass is one of the renewable resources with the greatest potential, not only because of the possibility of energy recovery but also because of its content in components of interest. In this context, the regions of Galicia and Portugal have large areas of land dedicated to forestry, agriculture and livestock, and the large amount of waste generated represents a cost for the producer. The importance of these facts has aroused great interest in society to focus its interest on improving the current situation while seeking a benefit, both environmental and economic, from existing resources.

Resveratrol-based biorefinery models for favoring its inclusion along the market value-added chains: A critical review.

Resveratrol, a natural organic polyhydroxyphenolic compound, has gained significant attention in the last years given its potential health benefits, including antioxidant, anti-cancer, and anti-inflammatory properties. It can be directly extracted from plants, vegetables, and related products and waste resources, but also chemically/enzymatically/microbially synthesized. However, certain process strategies have some limitations, such as high costs, reduced yield or high energy demand, thus implying significant environmental loads.

Megaprimer-Based PCR to Synthesize Fusion Genes for Cloning.

Megaprimer-based polymerase chain reaction (PCR) strategies allow the versatile and fast assembly and amplification of a myriad of tailor-made or random DNA sequences readily available for conventional or restriction-free (RF) cloning.In this chapter, we present a megaprimer-based PCR protocol that enables the expeditious construction of customized fusion genes ready for cloning into commercial expression plasmids. With the expanding use of protein tag technology in the most diverse application fields, this protocol remains a versatile and affordable solution for the synthesis and fusion of peptide tags/domains of interest.

Mitochondrial DNA D-Loop Amplification and Sequencing for Species Differentiation in Milk.

Adulteration of dairy products, mainly through the substitution of high-quality milk for lower-quality milk, results in the production of low-value products, raising health, social, and economic concerns. As such, the development of methods to ensure dairy products' safety and quality is of great concern for governments and consumers. Although several methods have been developed for species differentiation in dairy products, their application and the establishment of reliable molecular markers for authentication purposes still need to be improved.

Recombinant protein purification and immobilization strategies based on peptides with dual affinity to iron oxide and silica.

Iron oxide and silica-based materials have emerged as attractive protein purification and immobilization matrices. His6 has been reported as an effective affinity tag for both iron oxide and silica. Here, the silica-binding tags CotB1p and Car9 were shown to work as effectively as iron oxide-binding tags.

Towards T7 RNA polymerase (T7RNAP)-based expression system in yeast: challenges and opportunities.

During the last decades, we have witnessed unprecedented advances in biological engineering and synthetic biology. These disciplines aim to take advantage of gene pathway regulation and gene expression in different organisms, to enable cells to perform desired functions. Yeast has been widely utilized as a model for the study of eukaryotic protein expression while bacteriophage T7RNAP and its promoter constitute the preferred system for prokaryotic protein expression (such as pET-based expression systems).

Exploiting Non-Conventional Yeasts for Low-Alcohol Beer Production.

Non- yeasts represent a very appealing alternative to producing beers with zero or low ethanol content. The current study explores the potential of seven non- yeasts to produce low-alcohol or non-alcoholic beer, in addition to engineered/selected yeasts for low-alcohol production. The yeasts were first screened for their sugar consumption and ethanol production profiles, leading to the selection of strains with absent or inefficient maltose consumption and consequently with low-to-null ethanol production.

Developing a genetic engineering method for Acetobacterium wieringae to expand one-carbon valorization pathways.

Background: Developing new bioprocesses to produce chemicals and fuels with reduced production costs will greatly facilitate the replacement of fossil-based raw materials. In most fermentation bioprocesses, the feedstock usually represents the highest cost, which becomes the target for cost reduction. Additionally, the biorefinery concept advocates revenue growth from the production of several compounds using the same feedstock.

Potato peels waste as a sustainable source for biotechnological production of biofuels: Process optimization.

Potato peel waste (PPW) is a starchy by-product generated in great amounts during the industrial processing of potatoes. It can be used as a low cost alternative, and renewable feedstock for the production of second generation bioethanol. In order to intensify this process, Saccharomyces cerevisiae Ethanol Red®, a robust and thermotolerant yeast strain, was selected and two experimental designs and response surfaces assessment were conducted to enable very high gravity fermentations (VHGF) using PPW as feedstock.

Multi-feedstock biorefinery concept: Valorization of winery wastes by engineered yeast.

The wine industry produces significant amounts of by-products and residues that are not properly managed, posing an environmental problem. Grape must surplus, vine shoots, and wine lees have the potential to be used as renewable resources for the production of energy and chemicals. Metabolic engineering efforts have established Saccharomyces cerevisiae as an efficient microbial cell factory for biorefineries.

Expression of Yarrowia lipolytica acetyl-CoA carboxylase in Saccharomyces cerevisiae and its effect on in-vivo accumulation of Malonyl-CoA.

Malonyl-CoA is an energy-rich molecule formed by the ATP-dependent carboxylation of acetyl coenzyme A catalyzed by acetyl-CoA carboxylase. This molecule is an important precursor for many biotechnologically interesting compounds such as flavonoids, polyketides, and fatty acids. The yeast remains one of the preferred cell factories, but has a limited capacity to produce malonyl-CoA compared to oleaginous organisms.

Kluyveromyces marxianus as a microbial cell factory for lignocellulosic biomass valorisation.

The non-conventional yeast Kluyveromyces marxianus is widely used for several biotechnological applications, mainly due to its thermotolerance, high growth rate, and ability to metabolise a wide range of sugars. These cell traits are strategic for lignocellulosic biomass valorisation and strain diversity prompts the development of robust chassis, either with improved tolerance to lignocellulosic inhibitors or ethanol. This review summarises bioethanol and value-added chemicals production by K.

Resveratrol production for the valorisation of lactose-rich wastes by engineered industrial Saccharomyces cerevisiae.

Resveratrol is an antioxidant with applications in the food and cosmetic industries. Its biosynthesis can side the hindrances of its extraction from plants. The dairy industry generates tonnes of lactose-rich wastes, which can be a carbon source.

Genome-wide effect of non-optimal temperatures under anaerobic conditions on gene expression in Saccharomyces cerevisiae.

Understanding of thermal adaptation mechanisms in yeast is crucial to develop better-adapted strains to industrial processes, providing more economical and sustainable products. We have analyzed the transcriptomic responses of three Saccharomyces cerevisiae strains, a commercial wine strain, ADY5, a laboratory strain, CEN.PK113-7D and a commercial bioethanol strain, Ethanol Red, grown at non-optimal temperatures under anaerobic chemostat conditions.