• Energy Research

La biomasse lignocellulosique est une ressource commune à travers le monde, et sa fermentation offre une option prometteuse pour générer des carburants liquides de transport renouvelables. La déconstruction de la biomasse lignocellulosique libère des sucres qui peuvent être fermentés par des microbes, mais ces processus produisent également des inhibiteurs de fermentation, tels que des acides aromatiques et des aldéhydes. Plusieurs projets de recherche ont étudié la fermentation lignocellulosique de la biomasse par la levure de boulanger Saccharomyces cerevisiae. La plupart des projets ont adopté des approches biologiques synthétiques ou ont exploré la diversité naturelle de S. cerevisiae pour améliorer la tolérance au stress, la consommation de xylose ou la production d'éthanol. Malgré ces efforts, des souches améliorées avec de nouvelles propriétés sont nécessaires. Dans d'autres processus industriels, tels que la fermentation du vin et de la bière, les hybrides interspécifiques ont combiné des traits importants de plusieurs espèces, ce qui suggère que l'hybridation interspécifique peut également offrir un potentiel pour la recherche sur les biocarburants. Pour étudier l'efficacité de cette approche pour les traits pertinents pour la production de biocarburants lignocellulosiques, nous avons généré des hybrides synthétiques en croisant des souches de S. cerevisiae fermentant le xylose avec des souches sauvages de diverses espèces de Saccharomyces. Ces hybrides interspécifiques ont conservé des traits parentaux importants, tels que la consommation de xylose et la tolérance au stress, tout en présentant des paramètres cinétiques intermédiaires et, dans certains cas, une hétérosis (vigueur hybride). Ensuite, nous les avons exposés à une évolution adaptative dans l'hydrolysat de fourrage de maïs prétraité par expansion des fibres d'ammoniac et avons récupéré des souches présentant des traits fermentatifs améliorés. Le séquençage du génome a montré que les génomes de ces hybrides synthétiques évolués subissaient des réarrangements, des duplications et des délétions. Pour déterminer si le genre Saccharomyces contient un potentiel supplémentaire inexploité, nous avons examiné une collection génétiquement diversifiée de plus de 500 isolats de Saccharomyces sauvages non modifiés et découvert un large éventail de capacités pour les traits pertinents pour la production de biocarburants cellulosiques. Notamment, les souches de Saccharomyces mikatae ont une tolérance innée élevée aux toxines hydrolysées, tandis que certaines espèces de Saccharomyces ont une forte capacité native à consommer du xylose. Cette recherche démontre que l'hybridation est une méthode viable pour combiner des traits pertinents sur le plan industriel provenant de diverses espèces de levures et que les membres du genre Saccharomyces au-delà de S. cerevisiae peuvent offrir des gènes et des traits intéressants pour l'industrie des biocarburants lignocellulosiques. La biomasa lignocelulósica es un recurso común en todo el mundo, y su fermentación ofrece una opción prometedora para generar combustibles líquidos renovables para el transporte. La deconstrucción de la biomasa lignocelulósica libera azúcares que pueden ser fermentados por los microbios, pero estos procesos también producen inhibidores de la fermentación, como ácidos aromáticos y aldehídos. Varios proyectos de investigación han investigado la fermentación de biomasa lignocelulósica por la levadura de panadería Saccharomyces cerevisiae. La mayoría de los proyectos han adoptado enfoques biológicos sintéticos o han explorado la diversidad natural en S. cerevisiae para mejorar la tolerancia al estrés, el consumo de xilosa o la producción de etanol. A pesar de estos esfuerzos, se necesitan cepas mejoradas con nuevas propiedades. En otros procesos industriales, como la fermentación del vino y la cerveza, los híbridos entre especies han combinado rasgos importantes de múltiples especies, lo que sugiere que la hibridación entre especies también puede ofrecer potencial para la investigación de biocombustibles. Para investigar la eficacia de este enfoque para los rasgos relevantes para la producción de biocombustibles lignocelulósicos, generamos híbridos sintéticos cruzando cepas de fermentación de xilosa de S. cerevisiae con cepas silvestres de varias especies de Saccharomyces. Estos híbridos entre especies conservaron rasgos parentales importantes, como el consumo de xilosa y la tolerancia al estrés, al tiempo que mostraron parámetros cinéticos intermedios y, en algunos casos, heterosis (vigor híbrido). A continuación, los expusimos a la evolución adaptativa en el hidrolizado de rastrojo de maíz pretratado con expansión de fibra de amoníaco y cepas recuperadas con rasgos fermentativos mejorados. La secuenciación del genoma mostró que los genomas de estos híbridos sintéticos evolucionados sufrieron reordenamientos, duplicaciones y deleciones. Para determinar si el género Saccharomyces contiene un potencial adicional sin explotar, examinamos una colección genéticamente diversa de más de 500 aislados de Saccharomyces silvestres no modificados y descubrimos una amplia gama de capacidades para rasgos relevantes para la producción de biocombustibles celulósicos. En particular, las cepas de Saccharomyces mikatae tienen una alta tolerancia innata a las toxinas hidrolizadas, mientras que algunas especies de Saccharomyces tienen una sólida capacidad nativa para consumir xilosa. Esta investigación demuestra que la hibridación es un método viable para combinar rasgos industrialmente relevantes de diversas especies de levaduras y que los miembros del género Saccharomyces más allá de S. cerevisiae pueden ofrecer genes y rasgos ventajosos de interés para la industria de los biocombustibles lignocelulósicos. Lignocellulosic biomass is a common resource across the globe, and its fermentation offers a promising option for generating renewable liquid transportation fuels. The deconstruction of lignocellulosic biomass releases sugars that can be fermented by microbes, but these processes also produce fermentation inhibitors, such as aromatic acids and aldehydes. Several research projects have investigated lignocellulosic biomass fermentation by the baker's yeast Saccharomyces cerevisiae. Most projects have taken synthetic biological approaches or have explored naturally occurring diversity in S. cerevisiae to enhance stress tolerance, xylose consumption, or ethanol production. Despite these efforts, improved strains with new properties are needed. In other industrial processes, such as wine and beer fermentation, interspecies hybrids have combined important traits from multiple species, suggesting that interspecies hybridization may also offer potential for biofuel research.To investigate the efficacy of this approach for traits relevant to lignocellulosic biofuel production, we generated synthetic hybrids by crossing engineered xylose-fermenting strains of S. cerevisiae with wild strains from various Saccharomyces species. These interspecies hybrids retained important parental traits, such as xylose consumption and stress tolerance, while displaying intermediate kinetic parameters and, in some cases, heterosis (hybrid vigor). Next, we exposed them to adaptive evolution in ammonia fiber expansion-pretreated corn stover hydrolysate and recovered strains with improved fermentative traits. Genome sequencing showed that the genomes of these evolved synthetic hybrids underwent rearrangements, duplications, and deletions. To determine whether the genus Saccharomyces contains additional untapped potential, we screened a genetically diverse collection of more than 500 wild, non-engineered Saccharomyces isolates and uncovered a wide range of capabilities for traits relevant to cellulosic biofuel production. Notably, Saccharomyces mikatae strains have high innate tolerance to hydrolysate toxins, while some Saccharomyces species have a robust native capacity to consume xylose.This research demonstrates that hybridization is a viable method to combine industrially relevant traits from diverse yeast species and that members of the genus Saccharomyces beyond S. cerevisiae may offer advantageous genes and traits of interest to the lignocellulosic biofuel industry. تعد الكتلة الحيوية Lignocellulosic موردًا شائعًا في جميع أنحاء العالم، ويوفر تخميرها خيارًا واعدًا لتوليد وقود النقل السائل المتجدد. يؤدي تفكيك الكتلة الحيوية السليلوزية إلى إطلاق السكريات التي يمكن تخميرها بواسطة الميكروبات، ولكن هذه العمليات تنتج أيضًا مثبطات التخمير، مثل الأحماض العطرية والألدهيدات. قامت العديد من المشاريع البحثية بالتحقيق في تخمير الكتلة الحيوية اللجنوسليلوزية بواسطة خميرة الخباز Saccharomyces cerevisiae. اتخذت معظم المشاريع مناهج بيولوجية اصطناعية أو استكشفت التنوع الذي يحدث بشكل طبيعي في S. cerevisiae لتعزيز تحمل الإجهاد أو استهلاك الزيلوز أو إنتاج الإيثانول. على الرغم من هذه الجهود، هناك حاجة إلى سلالات محسنة مع خصائص جديدة. في العمليات الصناعية الأخرى، مثل تخمير النبيذ والبيرة، جمعت الهجينة بين الأنواع سمات مهمة من أنواع متعددة، مما يشير إلى أن التهجين بين الأنواع قد يوفر أيضًا إمكانات لأبحاث الوقود الحيوي. للتحقيق في فعالية هذا النهج للسمات ذات الصلة بإنتاج الوقود الحيوي السليولوزي، أنشأنا هجائن اصطناعية من خلال عبور سلالات تخمير الزيلوز المهندسة من S. cerevisiae مع سلالات برية من أنواع السكريات المختلفة. احتفظت هذه الهجينة بين الأنواع بسمات أبوية مهمة، مثل استهلاك الزيلوز وتحمل الإجهاد، مع عرض معلمات حركية وسيطة، وفي بعض الحالات، تغاير (قوة هجينة). بعد ذلك، عرّضناهم للتطور التكيفي في التحلل المائي لمخزن الذرة المعزز بألياف الأمونيا واستعدنا السلالات ذات الصفات التخمير المحسنة. أظهر تسلسل الجينوم أن جينومات هذه الهجائن الاصطناعية المتطورة خضعت لإعادة الترتيب والازدواجية والحذف. لتحديد ما إذا كان جنس السكريات يحتوي على إمكانات إضافية غير مستغلة، قمنا بفحص مجموعة متنوعة وراثيًا تضم أكثر من 500 من السكريات البرية غير المهندسة التي تعزل وتكشف عن مجموعة واسعة من القدرات للسمات ذات الصلة بإنتاج الوقود الحيوي السليلوزي. والجدير بالذكر أن سلالات السكريات الميكاتية لها قدرة فطرية عالية على تحمل السموم المتحللة، في حين أن بعض أنواع السكريات لديها قدرة أصلية قوية على استهلاك الزيلوز. يوضح هذا البحث أن التهجين هو طريقة قابلة للتطبيق للجمع بين السمات ذات الصلة صناعياً من أنواع الخميرة المتنوعة وأن أعضاء جنس السكريات خارج S. cerevisiae قد يقدمون جينات وسمات مفيدة تهم صناعة الوقود الحيوي الليجنيوسيليلوزي.

Abstract The fungal kingdom represents an extraordinary diversity of organisms with profound impacts across animal, plant, and ecosystem health. Fungi simultaneously support life, by forming beneficial symbioses with plants and producing life-saving medicines, and bring death, by causing devastating diseases in humans, plants, and animals. With climate change, increased antimicrobial resistance, global trade, environmental degradation, and novel viruses altering the impact of fungi on health and disease, developing new approaches is now more crucial than ever to combat the threats posed by fungi and to harness their extraordinary potential for applications in human health, food supply, and environmental remediation. To address this aim, the Canadian Institute for Advanced Research (CIFAR) and the Burroughs Wellcome Fund convened a workshop to unite leading experts on fungal biology from academia and industry to strategize innovative solutions to global challenges and fungal threats. This report provides recommendations to accelerate fungal research and highlights the major research advances and ideas discussed at the meeting pertaining to 5 major topics: (1) Connections between fungi and climate change and ways to avert climate catastrophe; (2) Fungal threats to humans and ways to mitigate them; (3) Fungal threats to agriculture and food security and approaches to ensure a robust global food supply; (4) Fungal threats to animals and approaches to avoid species collapse and extinction; and (5) Opportunities presented by the fungal kingdom, including novel medicines and enzymes.

Hybrid yeast strain co-produces isobutanol and ethanol at high yields. Reducing hydrolysis enzyme loading and enhancing xylose conversion greatly impact the economic potential of the biorefinery.

Fructophily is a rare trait that consists of the preference for fructose over other carbon sources. Here, we show that in a yeast lineage (the Wickerhamiella/Starmerella, W/S clade) comprised of fructophilic species thriving in the high-sugar floral niche, the acquisition of fructophily is concurrent with a wider remodeling of central carbon metabolism. Coupling comparative genomics with biochemical and genetic approaches, we gathered ample evidence for the loss of alcoholic fermentation in an ancestor of the W/S clade and subsequent reinstatement through either horizontal acquisition of homologous bacterial genes or modification of a pre-existing yeast gene. An enzyme required for sucrose assimilation was also acquired from bacteria, suggesting that the genetic novelties identified in the W/S clade may be related to adaptation to the high-sugar environment. This work shows how even central carbon metabolism can be remodeled by a surge of HGT events.

AbstractThe budding yeast Saccharomyces cerevisiae has been used extensively in fermentative industrial processes, including biofuel production from sustainable plant-based hydrolysates. Myriad toxins and stressors found in hydrolysates inhibit microbial metabolism and product formation. Overcoming these stresses requires mitigation strategies that include strain engineering. To identify shared and divergent mechanisms of toxicity and to implicate gene targets for genetic engineering, we used a chemical genomic approach to study fitness effects across a library of S. cerevisiae deletion mutants cultured anaerobically in dozens of individual compounds found in different types of hydrolysates. Relationships in chemical genomic profiles identified classes of toxins that provoked similar cellular responses, spanning inhibitor relationships that were not expected from chemical classification. Our results also revealed widespread antagonistic effects across inhibitors, such that the same gene deletions were beneficial for surviving some toxins but detrimental for others. This work presents a rich dataset relating gene function to chemical compounds, which both expands our understanding of plant-based hydrolysates and provides a useful resource to identify engineering targets.

Abstract Background Cost-effective production of biofuels from lignocellulose requires the fermentation of d-xylose. Many yeast species within and closely related to the genera Spathaspora and Scheffersomyces (both of the order Serinales) natively assimilate and ferment xylose. Other species consume xylose inefficiently, leading to extracellular accumulation of xylitol. Xylitol excretion is thought to be due to the different cofactor requirements of the first two steps of xylose metabolism. Xylose reductase (XR) generally uses NADPH to reduce xylose to xylitol, while xylitol dehydrogenase (XDH) generally uses NAD+ to oxidize xylitol to xylulose, creating an imbalanced redox pathway. This imbalance is thought to be particularly consequential in hypoxic or anoxic environments. Results We screened the growth of xylose-fermenting yeast species in high and moderate aeration and identified both ethanol producers and xylitol producers. Selected species were further characterized for their XR and XDH cofactor preferences by enzyme assays and gene expression patterns by RNA-Seq. Our data revealed that xylose metabolism is more redox balanced in some species, but it is strongly affected by oxygen levels. Under high aeration, most species switched from ethanol production to xylitol accumulation, despite the availability of ample oxygen to accept electrons from NADH. This switch was followed by decreases in enzyme activity and the expression of genes related to xylose metabolism, suggesting that bottlenecks in xylose fermentation are not always due to cofactor preferences. Finally, we expressed XYL genes from multiple Scheffersomyces species in a strain of Saccharomyces cerevisiae. Recombinant S. cerevisiae expressing XYL1 from Scheffersomyces xylosifermentans, which encodes an XR without a cofactor preference, showed improved anaerobic growth on xylose as the primary carbon source compared to S. cerevisiae strain expressing XYL genes from Scheffersomyces stipitis. Conclusion Collectively, our data do not support the hypothesis that xylitol accumulation occurs primarily due to differences in cofactor preferences between xylose reductase and xylitol dehydrogenase; instead, gene expression plays a major role in response to oxygen levels. We have also identified the yeast Sc. xylosifermentans as a potential source for genes that can be engineered into S. cerevisiae to improve xylose fermentation and biofuel production.