Engineering Saccharomyces cerevisiae for the production of sugaralcohols

Excess sugar intake contributes to weight gain, obesity, and related diseases [1]. Considering the growing demand for healthier products, most food manufacturers are focused on the reformulation of foods and beverages to reduce added sugar, using natural sweeteners and combinations of these ingredients. Arabitol is a sugar alcohol presenting similar properties to its isomer xylitol, a well-established sugar substitute [2]. The microbiological production of these sugar alcohols has received growing interest as an alternative to the expensive chemical synthesis that involves negative environmental effects. The yeast Saccharomyces cerevisiae considered a platform cell factory for sustainable biorefineries [3], encodes in its genome an NADPH-dependent aldose reductase that converts aldoses into their corresponding alcohols [4]. Taking advantage of its broad substrate specificity, we demonstrate the feasibility of using an engineered industrial yeast strain for the simultaneous conversion of arabinose and xylose to arabitol and xylitol. In addition, the recombinant strain was further engineered to improve arabinose transport capacity, improving the arabinose to arabitol conversion yield. This strategy for the simultaneous production of sugar alcohols is a step forward in the development of a multi-chemical yeast production platform capable to convert bulk sugars present in agro-food residues, contributing to the establishment of a bioeconomy.This study was supported by the Portuguese Foundation for Science and Technology (FCT) - UID/BIO/04469/2020 unit; Ph.D grant SFRH/BD/132717/2017 to Sara L. Baptista and Ph.D. grant SFRH/BD/146367/2019 to Pedro O. Soares This study was also supported by BioVino project (0688_BIOVINO_6_E), funded by INTERREG España - Portugal and European Regionalinfo:eu-repo/semantics/publishedVersio

Arabitol production from lignocellulosic biomass through GRE3-overexpressing industrial Saccharomyces cerevisiae strains

Arabitol is a five carbon sugar alcohol that belongs to the pentitol family, the same of xylitol and ribitol, being one of the top 12 biomass-derivable building block chemicals. Due to its sweetness similar to glucose and low caloric content (0.2 kcal/g) it is used as an alternative sweetener in food industry [1]. The concerns about the depletion of fossil fuel reserves and the economic and environmental problems associated with their use have led to the search of renewable energy sources. Lignocellulosic biomass emerged as sustainable alternative for the production of value-added products, once lignocellulose is one the most abundantly renewable biomass available on earth. Thus, the development of a lignocellulose-based bioeconomy must compromise the valorisation of lignocellulosic biomass for the production of value-added products [2]. Currently, arabitol is industrially produced by chemical reduction of lactones [3]. However, bioconversion of sugars present in lignocellulosic biomass to arabitol could be a viable alternative to chemical production. An endogenous aldose reductase from Saccharomyces cerevisiae, with a broad substrate specificity, was previously reported to be able to convert xylose and arabinose to xylitol and arabitol, respectively [4,5]. In here, we demonstrate the feasibility of using an engineered yeast strain overexpressing an aldose reductase gene for the conversion of arabinose to arabitol. Due to the unspecificity of the enzyme, arabinose and xylose could be simultaneously converted to arabitol and xylitol, respectively, which can lead to the development of a multi-chemical yeast production platform, contributing to the establishment of a lignocellulose-based bioeconomy.Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit, BioTecNorte operation (NORTE-01-0145- FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte, COMPETE 2020 (POCI-01-0145-FEDER-006684) and MultiBiorefinery project (POCI-01-0145-FEDER-016403)info:eu-repo/semantics/publishedVersio

Yeast cell factories for sustainable whey-to-ethanol valorisation towards a circular economy

Cheese whey is the major by-product of the dairy industry, and its disposal constitutes an environmental concern. The production of cheese whey has been increasing, with 190 million tonnes per year being produced nowadays. Therefore, it is emergent to consider different routes for cheese whey utilization. The great nutritional value of cheese whey turns it into an attractive substrate for biotechnological applications. Currently, cheese whey processing includes a protein fractionating step that originates the permeate, a lactose-reach stream further used for valorisation.  In the last decades, yeast fermentation has brought several advances to the search for biorefinery alternatives. From the plethora of value-added products that can be obtained from cheese whey, ethanol is the most extensively explored since it is the alternative biofuel most used worldwide. Thus, this review focuses on the different strategies for ethanol production from cheese whey using yeasts as promising biological systems, including its integration in lignocellulosic biorefineries. These valorisation routes encompass the improvement of the fermentation process as well as metabolic engineering techniques for the introduction of heterologous pathways, resorting mainly to Kluyveromyces sp. and Saccharomyces cerevisiae strains. The solutions and challenges of the several strategies will be unveiled and explored in this review.This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020; the Ph.D. grants SFRH/BD/130739/2017 and SFRH/BD/132717/2017 to CEC and SLB, respectively.info:eu-repo/semantics/publishedVersio

Establishment of galactose utilization in Saccharomyces cerevisiae PE-2 through expression of the CEN.PK113-5D Gal2 galactose permease

Microbiotec'17 - Congress of Microbiology and Biotechnology 2017Background Saccharomyces cerevisiae PE - 2 is one of the most robust yeast chassis for use in second -generation bioprocesses [1 - 2]. Unfortunately, we noticed that it is incapable of utilizing galactose, which is abundant in diverse agro - industrial derived substrates. In this study we analysed the galactose utilization pathway (Leloir pathway) of this strain and identified the Gal2 galactose permease as the limiting step. Method The putative amino acid (aa) sequences of the S. cerevisiae JAY291 (haploid derivate of PE - 2) proteins involved in galactose utilization (Gal2, Gal1, Gal7, Gal10, Gal5, Gal4, Gal80 and Gal3) were aligned (Clustal Omega) with the corresponding proteins of other industrial and laboratorial strains, revealing several point mutations in the Gal2 permease. PE - 2 was then tr ansformed with a 2 micron plasmid containing the CEN.PK113 - 5D GAL2 under the regulation of the TDH3 promoter and PGI1 terminator, and the resulting transformants were physiologically characterized in liquid YP containing 2% galactose plus 150 μg/mL G418. Results & Conclusions Homology - based analysis of the S. cerevisiae PE - 2 Leloir pathway allowed the identification of 12 aa substitutions in the Gal2 sequence that are not conserved across other industrial and laboratorial strains. Three of these point mut ations were found in the transmembrane domain 7 (TM7), a region important for substrate recognition [3]. Among these, the most significant includes the substitution F336L, as the loss of aromatic aa in TM7 is reported to be critical for galactose transport activity [3]. These results suggested that the galactose permease of S. cerevisiae PE - 2 might lack galactose transport activity, which was further supported by the fact that expression of the CEN.PK113 - 5D GAL2 in PE - 2 established its galactose utilization capacity. In fact, with this modification PE - 2 was faster than CEN.PK113 - 5D in consuming 2% galactose (12h vs 14h, respectively). The high galactose utilization efficiency of this newly constructed PE - 2 strain opens new perspectives and opportunities for the valorisation of galactose - containing second - generation substrateStudy 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), Project MultiBiorefinery (POCI - 01 - 0145 - FEDER - 016403), RECI/BB B - EBI/0179/2012 (FCOMP - 01 - 0124 - FEDER - 027462) and BioTecNorte operation (NORTE - 01 - 0145 - FEDER - 000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norteinfo:eu-repo/semantics/publishedVersio

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. 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.info:eu-repo/semantics/publishedVersio

The L-arabinose isomerase from the food grade Bacillus subtilis for the production of tagatose: a natural sweetener

The increasing concern about adverse health impacts from excessive sugar consumption is the main driving force for the replacement of simple sugar by natural sweeteners (1). Tagatose is a hexose monosaccharide rarely found in nature, namely in some fruits and dairy products. This rare sugar represents a promising sweetener due its low calorie content (1.5 – 2.5 kcal/g), sweetness profile similar to sucrose and prebiotic and anticariogenic properties (2-3).Study supported by the Portuguese Foundation for Science and Technology (FCT, Portugal) under the scope of the strategic funding of UID/BIO/04469/2019 unit and COMPETE 2020 (POCI-01-0145- FEDER-006684), the BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte and Sara L. Baptista doctoral fellowship (SFRH/BD/132717)info:eu-repo/semantics/publishedVersio

Integrated approach for the valorisation of wine industry residues: production of xylitol and bioethanol

The wine industry is known to generate large volumes of by-products and residues that are not properly treated, representing an environmental problem (1). Vine pruning, wine lees, and grape must offer significant potential to serve as a sustainable source for the production of fuels and chemicals. Metabolic engineering strategies have been establishing Saccharomyces cerevisiae as a cell factory for biorefineries (2). For being sustainable, these biorefineries will require bioethanol and high-value chemical coproduction (2). In this work, an integrated approach was used for the valorisation of hydrothermally pre-treated vine pruning, using the xylose-rich hemicellulosic fraction for xylitol production (3) and the cellulosic fraction to produce bioethanol using genetically modified S. cerevisiae strains. Furthermore, wine lees were explored as nutritional supplements and grape must, rich in fermentable sugars, as both nutrient and carbon source. The xylitol production was optimized using different enzyme loading and inoculum size in a simultaneous saccharification and fermentation (SSF) process. Additionally, the production of bioethanol from the glucan enriched solid fraction was evaluated with the supplementation of wine lees and/or grape must, resulting in ethanol production higher than the critical threshold for distillation economic feasibility. This integrated multi-feedstock approach represents a possible solution for disposal problems of wine-producing industry, meeting the demands for the establishment of a circular economy.This study was supported by the Portuguese Foundation for Science and Technology (FCT, Portugal) under the scope of the strategic funding of UIDB/04469/2020, the PhD grant (SFRH/BD/132717/2017 to SLB) and BIOVINO project (0688_BIOVINO_6_E) funded by INTERREG España - Portugal and European Regionalinfo:eu-repo/semantics/publishedVersio

Yeast cell factory development for biorefineries

The development and establishment of biorefineries are key factors for the sustainable growth of a society based on a bioeconomy. However, to make these bioprocesses economically feasible, intensified and flexible processes, responsive to feedstock and market fluctuations, have to be considered. Fermentation is a core operation unit in this process, typically performed by the yeast Saccharomyces cerevisiae. In intensified lignocellulose conversion processes the yeast has to be able to work under very demanding conditions, that is, low pH, presence of fermentation inhibitors, high temperature and solid loadings, simultaneous consumption of C6 and C5 sugars[1,2]. Thus, the development of efficient yeast cell factories is mandatory for bringing lignocellulose-tobioethanol processes to a next level and also for upgrading these systems for the sustainable production of bioethanol and high-value biochemicals (such as xylitol and arabitol), a vital endeavor to successfully implement a bioeconomy. We will present examples of intensified and productive conditions attained by metabolic engineering strategies applied over robust industrial yeast chassis. The presented results attest the feasibility of intensifying biomass-to-ethanol processes[3] and show how this integrated strategy has the potential to be the driver for the emergence of economical and sustainable processes for biofuels and high-value chemicals production from lignocellulosic biomass.Study supported by FCT under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI01-0145-FEDER-006684), Project MultiBiorefinery (POCI-01-0145-FEDER-016403), and BioTecNorte operation (NORTE01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020.info:eu-repo/semantics/publishedVersio

Metabolic engineering of Saccharomyces cerevisiae for the production of top value chemicals from biorefinery carbohydrates

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.This work was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/ 04469/2020, the PhD grants (SFRH/BD/132717/2017 to SLB, SFRH/ BD/130739/2017 to CEC and SFRH/BD/146367/2019 to POS), the MIT-Portugal Program (Ph.D. Grant PD/BD/128247/2016 to JTC), BioTecNorte operation (NORTE-01-0145-FEDER-000004) and Biomass and Bioenergy Research Infrastructure (BBRI)- LISBOA-01-0145-FEDER- 022059 funded by the European Regional Development Fund (ERDF) under the scope of Norte2020 - Programa Operacional Regional do Norte.info:eu-repo/semantics/publishedVersio

Resíduos agroalimentares e plataformas de leveduras modificadas para produção de adoçantes naturais

Programa doutoral em Chemical and Biological EngineeringO açúcar é um componente prevalente nas dietas modernas, sendo valorizado pela sua capacidade singular de adoçar os alimentos. No entanto, o consumo excessivo de açúcar refinado tem sido associado a doenças, como obesidade e diabetes. Consequentemente, há um crescente interesse em substitutos naturais do açúcar. Além disso, a promoção de uma economia circular tem recebido destaque na agenda política de muitos países, com planos de ação que incluem a implementação de biorrefinarias para produzir energia e produtos de elevado valor. Nos últimos anos, a produção de biocombustíveis e outros produtos a partir de biomassas renováveis tem surgido como uma alternativa à economia baseada em petróleo. O desenvolvimento de processos inovadores que sejam rentáveis e ambientalmente responsáveis requer a utilização de fábricas celulares robustas. A levedura Saccharomyces cerevisiae tem sido amplamente estudada como uma plataforma para a produção de biocombustíveis e produtos naturais de alto valor a partir de biomassas renováveis. Este estudo visa explorar as propriedades atrativas das estirpes industriais de S. cerevisiae, como sua elevada capacidade fermentativa e habilidade de tolerar condições de processamento desafiadoras, para desenvolver estirpes recombinantes capazes de produzir adoçantes naturais. Face a este enquadramento, a presente tese resultou (1) na construção de uma estirpe de levedura capaz de alcançar títulos e rendimentos notáveis de xilitol através da expressão de uma aldose redutase endógena com atividade de xilose redutase, (2) num processo sustentável baseado em tecnologias verdes para a produção de xilitol a partir da fração sólida e liquida resultante do pré-tratamento do caroço do milho (3) numa abordagem integrada de valorização para converter múltiplos resíduos da indústria do vinho em xilitol, etanol e biomassa de levedura, (4) no estabelecimento da levedura S. cerevisiae como biocatalisador para a conversão de arabinose da polpa de beterraba em arabitol, aproveitando a atividade promíscua da aldose redutase anteriormente expressa, juntamente com a sobre expressão de um transportador de galactose, e (5) numa estratégia de produção sustentável de tagatose utilizando algas vermelhas, e os seus resíduos de processamento subvalorizados, como fonte de galactose numa isomerização mediada pela enzima L-arabinose isomerase. Estes resultados suportam o desenvolvimento de uma biorefinaria focada na produção de adoçantes naturais e evidenciam o potencial da S. cerevisiae como plataforma celular, contribuindo para o estabelecimento de uma economia circular e sustentável baseada em recursos biológicos.Sugar is a prevalent component of modern diets, valued for its special ability to sweeten food. However, consuming excessive amounts of refined sugar has been associated with non-communicable diseases and health issues such as obesity and diabetes. As a result, there has been growing interest in natural sugar substitutes. On the other hand, the promotion of a circular economy is ranked high on the political agenda of many countries and their action plan includes the implementation of cost-effective and sustainable biorefineries for energy with high-value chemicals production. In recent years, the production of biofuels and other chemicals from renewable biomasses using biotechnology has emerged as a viable alternative to the traditional petroleum-based economy. The development of sustainable and commercially viable processes relies heavily on the use of robust cell factories. The yeast Saccharomyces cerevisiae has gained significant attention as a cell factory to produce biofuels and high-value natural products from renewable biomasses. This study aims to utilize the attractive properties of industrial S. cerevisiae strains, including their high fermentative capacity and ability to tolerate harsh process conditions, to develop recombinant strains for natural sweeteners production. With this in mind, the present thesis resulted in: (1) a xylitol producing strain capable of attaining remarkable xylitol titers and yield, resulting from the expression of an endogenous aldose reductase with xylose reductase activity, (2) a sustainable bioprocess based on green technologies for xylitol production from corn cob whole slurry using the above mentioned strain (3) an integrated multi-feedstock valorization approach for converting a range of winery wastes into xylitol, ethanol and yeast biomass, (4) the establishment of S. cerevisiae as a biocatalyst for the conversion of arabinose from sugar beet pulp into arabitol, by harnessing the promiscuous activity of the endogenous aldose reductase previously expresses, together with the overexpression of a galactose transporter, and (5) sustainable tagatose production strategy by using red seaweed and its undervalued processing residues as source of galactose in a L-arabinose isomerase mediated-isomerization. Collectively, these findings support the development of a cost-effective biorefinery that focuses on natural sweetener production by tailoring S. cerevisiae as a cell factory platform, contributing to the establishment of a bio-based economy.The author would like to acknowledge the Portuguese Foundation for Science and Technology (FCT) for the financial support through the Ph.D. grant SFRH/BD/132717/2017. To the BIOVINO project (0688_BIOVINO_6_E), funded by the European Regional Development Fund through the Interreg VA España-Portugal (POCTEP). To CEB/UMinho, CfB/DTU, UVigo and UChalmers