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Food fraud has become an increasingly worldwide problem mainly driven by rapid innovation in the food sector and constantly changing consumer's choices. This led to an increased necessity to improve/establish reliable authentication processes, resulting in the replacement of protein-based techniques for dairy products authentication by higher sensitive and reproducible DNA-based methods. Most used molecular methods for dairy products authentication include PCR, Real-time PCR, multiplex PCR, and PCR-RFLP. Despite the several molecular methods available for species/breeds differentiation in dairy products, there is a need for the development of more efficient and reliable molecular tools. In this review, traditional and more recent DNA-based methods for dairy products authentication are discussed and analysed. Moreover, the increasingly important role of bioinformatic tools for analysis of large amount of data and for the development of DNA markers is also discussed. DNA quality is one of the major factors affecting molecular-based dairy products authentication, which can be influenced by the manufacturing process, extraction method employed, chemical composition of the food matrix, among others. PCR-based methods continue to be the most important and successfully used for dairy products authentication. The DNA markers chosen for species/breed detection are an important factor for PCR success. Although there are several molecular methods for the detection of adulterant species, there is still an unmet demand for methods to detect adulterant breeds. Available public databases and bioinformatics revolutionized the analysis of large amount of data and will be pivotal for the development of effective DNA markers. Image 1 • Improvement/establishment of authentication processes in dairy is mandatory. • Recent and revolutionary molecular methods for dairy products authentication are discussed. • Breed differentiation in dairy products remains a challenge. • Bioinformatics will be pivotal for the development of effective DNA markers. [ABSTRACT FROM AUTHOR]

Background: Consolidated bioprocessing, which combines saccharolytic and fermentative abilities in a single microorganism, is receiving increased attention to decrease environmental and economic costs in lignocellulosic biorefineries. Nevertheless, the economic viability of lignocellulosic ethanol is also dependent of an efficient utilization of the hemicellulosic fraction, which contains xylose as a major component in concentrations that can reach up to 40% of the total biomass in hardwoods and agricultural residues. This major bottleneck is mainly due to the necessity of chemical/enzymatic treatments to hydrolyze hemicellulose into fermentable sugars and to the fact that xylose is not readily consumed by Saccharomyces cerevisiae—the most used organism for large-scale ethanol production. In this work, industrial S. cerevisiae strains, presenting robust traits such as thermotolerance and improved resistance to inhibitors, were evaluated as hosts for the cell-surface display of hemicellulolytic enzymes and optimized xylose assimilation, aiming at the development of whole-cell biocatalysts for consolidated bioprocessing of corn cob-derived hemicellulose. Results: These modifications allowed the direct production of ethanol from non-detoxified hemicellulosic liquor obtained by hydrothermal pretreatment of corn cob, reaching an ethanol titer of 11.1 g/L corresponding to a yield of 0.328 g/g of potential xylose and glucose, without the need for external hydrolytic catalysts. Also, consolidated bioprocessing of pretreated corn cob was found to be more efficient for hemicellulosic ethanol production than simultaneous saccharification and fermentation with addition of commercial hemicellulases. Conclusions: These results show the potential of industrial S. cerevisiae strains for the design of whole-cell biocatalysts and paves the way for the development of more efficient consolidated bioprocesses for lignocellulosic biomass valorization, further decreasing environmental and economic costs. [ABSTRACT FROM AUTHOR]

Lignocellulose-based biorefineries have been gaining increasing attention to substitute current petroleum-based refineries. Biomass processing requires a pretreatment step to break lignocellulosic biomass recalcitrant structure, which results in the release of a broad range of microbial inhibitors, mainly weak acids, furans, and phenolic compounds. Saccharomyces cerevisiae is the most commonly used organism for ethanol production; however, it can be severely distressed by these lignocellulose-derived inhibitors, in addition to other challenging conditions, such as pentose sugar utilization and the high temperatures required for an efficient simultaneous saccharification and fermentation step. Therefore, a better understanding of the yeast response and adaptation towards the presence of these multiple stresses is of crucial importance to design strategies to improve yeast robustness and bioconversion capacity from lignocellulosic biomass. This review includes an overview of the main inhibitors derived from diverse raw material resultants from different biomass pretreatments, and describes the main mechanisms of yeast response to their presence, as well as to the presence of stresses imposed by xylose utilization and high-temperature conditions, with a special emphasis on the synergistic effect of multiple inhibitors/stressors. Furthermore, successful cases of tolerance improvement of S. cerevisiae are highlighted, in particular those associated with other process-related physiologically relevant conditions. Decoding the overall yeast response mechanisms will pave the way for the integrated development of sustainable yeast cell-based biorefineries. [ABSTRACT FROM AUTHOR]

In this work, the industrial Saccharomyces cerevisiae PE-2 strain, presenting innate capacity for xylitol accumulation, was engineered for xylitol production by overexpression of the endogenous GRE3 gene and expression of different xylose reductases from Pichia stipitis . The best-performing GRE3 -overexpressing strain was capable to produce 148.5 g/L of xylitol from high xylose-containing media, with a 0.95 g/g yield, and maintained close to maximum theoretical yields (0.89 g/g) when tested in non-detoxified corn cob hydrolysates. Furthermore, a successful integrated strategy was developed for the production of xylitol from whole slurry corn cob in a presaccharification and simultaneous saccharification and fermentation process (15% solid loading and 36 FPU) reaching xylitol yield of 0.93 g/g and a productivity of 0.54 g/L·h. This novel approach results in an intensified valorization of lignocellulosic biomass for xylitol production in a fully integrated process and represents an advance towards a circular economy. [ABSTRACT FROM AUTHOR]

Acetic acid tolerance and xylose consumption are desirable traits for yeast strains used in industrial biotechnological processes. In this work, overexpression of a weak acid stress transcriptional activator encoded by the gene HAA1 and a phosphoribosyl pyrophosphate synthetase encoded by PRS3 in a recombinant industrial Saccharomyces cerevisiae strain containing a xylose metabolic pathway was evaluated in the presence of acetic acid in xylose- or glucose-containing media. HAA1 or PRS3 overexpression resulted in superior yeast growth and higher sugar consumption capacities in the presence of 4 g/L acetic acid, and a positive synergistic effect resulted from the simultaneous overexpression of both genes. Overexpressing these genes also improved yeast adaptation to a non-detoxified hardwood hydrolysate with a high acetic acid content. Furthermore, the overexpression of HAA1 and/or PRS3 was found to increase the robustness of yeast cell wall when challenged with acetic acid stress, suggesting the involvement of the modulation of the cell wall integrity pathway. This study clearly shows HAA1 and/or, for the first time, PRS3 overexpression to play an important role in the improvement of industrial yeast tolerance towards acetic acid. The results expand the molecular toolbox and add to the current understanding of the mechanisms involved in higher acetic acid tolerance, paving the way for the further development of more efficient industrial processes. [ABSTRACT FROM AUTHOR]

Serra da Estrela Protected Designation of Origin (PDO) cheese is the most famous Portuguese cheese and has a high commercial value. However, the adulteration of production with cheaper/lower-quality milks from non-autochthones ovine breeds compromises the quality of the final product and undervalues the original PDO cheese. A Randomly Amplified Polymorphic DNA (RAPD) method was developed for efficient detection of adulterant breeds in milk mixtures used for fraudulent production of this cheese. Furthermore, Sequence Characterized Amplified Region (SCAR) markers were designed envisioning the detection of milk adulteration in processed dairy foods. The RAPD-SCAR technique is here described, for the first time, to be potentially useful for detection of milk origin in dairy products. In this sense, our findings will play an important role on the valorization of Serra da Estrela cheese, as well as on other high-quality dairy products prone to adulteration, contributing to the further development of the dairy industry. [ABSTRACT FROM AUTHOR]

PRS3 , RPB4 and ZWF1 were previously identified as key genes for yeast tolerance to lignocellulose-derived inhibitors. To better understand their contribution to yeast resistance to the multiple stresses occurring during lignocellulosic hydrolysate fermentations, we overexpressed these genes in two industrial Saccharomyces cerevisiae strains, CCUG53310 and PE-2, and evaluated their impact on the fermentation of Eucalyptus globulus wood and corn cob hydrolysates. PRS3 overexpression improved the fermentation rate (up to 32%) and productivity (up to 48%) in different hydrolysates. ZWF1 and RPB4 overexpression did not improve the fermentation performance, but their increased expression in the presence of acetic acid, furfural and hydroxymethylfurfural was found to contribute to yeast adaptation to these inhibitors. This study expands our understanding about the molecular mechanisms involved in industrial yeast tolerance to the stresses occurring during lignocellulosic bioethanol production and highlights the importance of selecting appropriate strain backgrounds/hydrolysates combinations when addressing further improvement of these processes. [ABSTRACT FROM AUTHOR]

The implementation of cost-effective and sustainable biorefineries to substitute the petroleum-based economy is dependent on coupling the production of bioenergy with high-value chemicals. For this purpose, the US Department of Energy identified a group of key target compounds to be produced from renewable biomass. Among them, 5-hydroxymethylfurfural (HMF) can be obtained by dehydration of the hexoses present in biomass and is an extremely versatile molecule that can be further converted into a wide range of higher value compounds. HMF derivatives include 2,5-bis(hydroxymethyl)furan (BHMF), 5-hydroxymethyl-furan-2-carboxylic acid (HMFCA), 2,5-diformylfuran (DFF), 5-formyl-2-furancarboxylic acid (FFCA) and 2,5-furandicarboxylic acid (FDCA), all presenting valuable applications, in polymers, bioplastics and pharmaceuticals. Biocatalysis conversion of HMF into its derivatives emerges as a green alternative, taking into account the high selectivity of enzymes and the mild reaction conditions used. Considering these factors, this work reviews the use of microorganisms as whole-cell biocatalysts for the production of HMF derivatives. In the last years, a large number of whole-cell biocatalysts have been discovered and developed for HMF conversion into BHMF, FDCA and HMFCA, however there are no reports on microbial production of DFF and FFCA. While the production of BHMF and HMFCA mainly relies on wild type microorganisms, FDCA production, which requires multiple bioconversion steps from HMF, is strongly dependent on genetic engineering strategies. Together, the information gathered supports the possibility for the development of cell factories to produce high-value compounds, envisioning economical viable biorefineries. [ABSTRACT FROM AUTHOR]

The establishment of lignocellulosic biorefineries is dependent on microorganisms being able to cope with the stressful conditions resulting from the release of inhibitory compounds during biomass processing. The yeast Kluyveromyces marxianus has been explored as an alternative microbial factory due to its thermotolerance and ability to natively metabolize xylose. The lignocellulose-derived inhibitors furfural and 5-hydroxymethylfurfural (HMF) are considered promising building-block platforms that can be converted into a wide variety of high-value derivatives. Here, several K. marxianus strains, isolated from cocoa fermentation, were evaluated for xylose consumption and tolerance towards acetic acid, furfural, and HMF. The potential of this yeast to reduce furfural and HMF at high inhibitory loads was disclosed and characterized. Our results associated HMF reduction with NADPH while furfural-reducing activity was higher with NADH. In addition, furans' inhibitory effect was higher when combined with xylose consumption. The furan derivatives produced by K. marxianus in different conditions were identified. Furthermore, one selected isolate was efficiently used as a whole-cell biocatalyst to convert furfural and HMF into their derivatives, furfuryl alcohol and 2,5-bis(hydroxymethyl)furan (BHMF), with high yields and productivities. These results validate K. marxianus as a promising microbial platform in lignocellulosic biorefineries. [ABSTRACT FROM AUTHOR]

[Display omitted] • Evaluation of industrial Saccharomyces cerevisiae for consolidated bioprocessing. • Development of a lactose-consuming cellulolytic Saccharomyces cerevisiae. • Cell-surface display of cellulases and β-galactosidase in a robust industrial strain. • High ethanol titers from a mixture of corn cob (high loads) and cheese whey. • Increased ethanol productivity and reduced costs in this multi-feedstock approach. The viability of 2nd generation bioethanol processes is dependent on achieving high ethanol titers, which requires the use of high solid loadings that will negatively affect the fermentative microorganism besides increasing enzyme-associated costs. To solve this, and also problems of feedstock availability, lignocellulosic biomass can be mixed with dairy by-products to increase carbon content. In this study, industrial strains of Saccharomyces cerevisiae , with improved thermotolerance and stress resistance, were engineered for the cell surface display of cellulolytic enzymes and were evaluated in consolidated bioprocessing of cellulose. Additionally, β-galactosidase was also displayed to enable lactose consumption, resulting in high ethanol titers (>50 g/L) from the simultaneous use of cheese whey and pretreated corn cob as substrate. The multi-feedstock valorization approach together with this lactose-consuming cellulolytic yeast allowed the reduction on materials costs by 60% with a 2.5-fold increase in the annual ethanol production, therefore contributing to the establishment of economic viable ethanol processes. [ABSTRACT FROM AUTHOR]

Background: Xylose isomerase (XI) and xylose reductase/xylitol dehydrogenase (XR/XDH) pathways have been extensively used to confer xylose assimilation capacity to Saccharomyces cerevisiae and tackle one of the major bottlenecks in the attainment of economically viable lignocellulosic ethanol production. Nevertheless, there is a lack of studies comparing the efficiency of those pathways both separately and combined. In this work, the XI and/or XR/XDH pathways were introduced into two robust industrial S. cerevisiae strains, evaluated in synthetic media and corn cob hemicellulosic hydrolysate and the results were correlated with the differential enzyme activities found in the xylose-pathway engineered strains. Results: The sole expression of XI was found to increase the fermentative capacity of both strains in synthetic media at 30 °C and 40 °C: decreasing xylitol accumulation and improving xylose consumption and ethanol production. Similar results were observed in fermentations of detoxified hydrolysate. However, in the presence of lignocellulosic-derived inhibitors, a positive synergistic effect resulted from the expression of both XI and XR/XDH, possibly caused by a cofactor equilibrium between the XDH and furan detoxifying enzymes, increasing the ethanol yield by more than 38%. Conclusions: This study clearly shows an advantage of using the XI from Clostridium phytofermentans to attain high ethanol productivities and yields from xylose. Furthermore, and for the first time, the simultaneous utilization of XR/XDH and XI pathways was compared to the single expression of XR/XDH or XI and was found to improve ethanol production from non-detoxified hemicellulosic hydrolysates. These results extend the knowledge regarding S. cerevisiae xylose assimilation metabolism and pave the way for the construction of more efficient strains for use in lignocellulosic industrial processes. [ABSTRACT FROM AUTHOR]

In this work, four robust yeast chassis isolated from industrial environments were engineered with the same xylose metabolic pathway. The recombinant strains were physiologically characterized in synthetic xylose and xylose-glucose medium, on non-detoxified hemicellulosic hydrolysates of fast-growing hardwoods ( Eucalyptus and Paulownia ) and agricultural residues (corn cob and wheat straw) and on Eucalyptus hydrolysate at different temperatures. Results show that the co-consumption of xylose-glucose was dependent on the yeast background. Moreover, heterogeneous results were obtained among different hydrolysates and temperatures for each individual strain pointing to the importance of designing from the very beginning a tailor-made yeast considering the specific raw material and process. [ABSTRACT FROM AUTHOR]

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. Current biorefineries designed for producing multiple products often rely on just one feedstock, but the bioeconomy would clearly benefit if these biorefineries could efficiently convert multiple feedstocks. Moreover, to reduce the environmental impact of fossil fuel consumption and maximize production economics, a biorefinery should be capable to supplement the manufacture of biofuel with the production of high-value products. This study proposes an integrated approach for the valorization of diverse wastes resulting from winemaking processes through the biosynthesis of xylitol and ethanol. Using genetically modified S. cerevisiae strains, the xylose-rich hemicellulosic fraction of hydrothermally pretreated vine shoots was converted into xylitol, and the cellulosic fraction was used to produce bioethanol. In addition, grape must, enriched in sugars, was efficiently used as a low-cost source for yeast propagation. The production of xylitol was optimized, in a Simultaneous Saccharification and Fermentation process configuration, by adjusting the inoculum size and enzyme loading. Furthermore, a yeast strain displaying cellulases in the cell surface was applied for the production of bioethanol from the glucan-rich cellulosic. With the addition of grape must and/or wine lees, high ethanol concentrations were reached, which are crucial for the economic feasibility of distillation. This integrated multi-feedstock valorization provides a synergistic alternative for converting a range of winery wastes and by-products into biofuel and an added-value chemical while decreasing waste released to the environment. • Design of a biorefinery capable of integrating multiple winery wastes. • Valorization of three winery wastes to produce biocatalyst, xylitol, and ethanol. • CRISPR-Cas9 system to construct a xylitol-producing Saccharomyces cerevisiae. • Xylitol production optimization from hemicellulosic vine shoots hydrolysate by SSF. [ABSTRACT FROM AUTHOR]

The implementation of biorefineries for a cost-effective and sustainable production of energy and chemicals from renewable carbon sources plays a fundamental role in the transition to a circular economy. The US Department of Energy identified a group of key target compounds that can be produced from biorefinery carbohydrates. In 2010, this list was revised and included organic acids (lactic, succinic, levulinic and 3-hydroxypropionic acids), sugar alcohols (xylitol and sorbitol), furans and derivatives (hydroxymethylfurfural, furfural and furandicarboxylic acid), biohydrocarbons (isoprene), and glycerol and its derivatives. The use of substrates like lignocellulosic biomass that impose harsh culture conditions drives the quest for the selection of suitable robust microorganisms. The yeast Saccharomyces cerevisiae , widely utilized in industrial processes, has been extensively engineered to produce high-value chemicals. For its robustness, ease of handling, genetic toolbox and fitness in an industrial context, S. cerevisiae is an ideal platform for the founding of sustainable bioprocesses. Taking these into account, this review focuses on metabolic engineering strategies that have been applied to S. cerevisiae for converting renewable resources into the previously identified chemical targets. The heterogeneity of each chemical and its manufacturing process leads to inevitable differences between the development stages of each process. Currently, 8 of 11 of these top value chemicals have been already reported to be produced by recombinant S. cerevisiae. While some of them are still in an early proof-of-concept stage, others, like xylitol or lactic acid, are already being produced from lignocellulosic biomass. Furthermore, the constant advances in genome-editing tools, e.g. CRISPR/Cas9, coupled with the application of innovative process concepts such as consolidated bioprocessing, will contribute for the establishment of S. cerevisiae -based biorefineries. • Sustainable biorefineries require bioethanol and high-value chemicals coproduction • Metabolic engineering strategies established S. cerevisiae as a cell factory for biorefineries • 8 of the 11 top-target biorefinery chemicals are produced by S. cerevisiae • Xylitol and lactic acid were already produced from lignocellulosic biomass • Industrial requirements may be fulfilled by advances on genetic tools and process configurations [ABSTRACT FROM AUTHOR]