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At least 55 records · Page 3

Economic and environmental bottlenecks in the industrial‐scale production of lipid‐derived biofuels from oleaginous yeasts: A review of the current trends and future prospects

Abstract Concerns about climate change and the reliance on fossil fuel reserves have motivated researchers to identify new renewable sources of energy. Biomass holds the potential to replace fossil‐derived products with biofuels and bio‐based chemicals. Plant‐derived lipids are promising sources of biofuels; however, the production of plant oil often leads to the release of massive amounts of carbon dioxide due to deforestation and land‐use change. The production of biofuels via plant oils (such as soybean) also competes with food production and in turn, impacts biodiversity. To mitigate these issues, the production of lipids from oleaginous yeasts could be an excellent alternative by incorporating these microbes into biorefineries utilizing agricultural or forest residues. Eventually, these microbial lipids could be potential sources for producing lipid‐derived biofuels. However, the current conventional methods for the production and recovery of lipids from oleaginous microbes suffer from economic and ecological challenges that affect its industrial‐scale expansion. This review highlights the major economic and environmental bottlenecks for the production of lipid‐derived biofuels from oleaginous yeasts. It also provides perspectives on the strategies that could be adapted on economic and ecological fronts to assist the expansion of the production of microbial lipid‐derived biofuels at an industrial scale.

Agriculture↗

Machine learning identifies novel signatures of antifungal drug resistance in Saccharomycotina yeasts

Antifungal drug resistance is a major challenge in fungal infection management. Numerous genomic changes are known to contribute to acquired drug resistance in clinical isolates of specific pathogens, but whether they broadly explain natural resistance across entire lineages is unknown. We leveraged genomic, ecological, and phenotypic trait data from naturally sampled strains from nearly all known species in subphylum Saccharomycotina to examine the evolution of resistance to eight antifungal drugs. The phylogenetic distribution of drug resistance varied by drug; fluconazole resistance was widespread, while 5-fluorocytosine resistance was rare, except in Lipomycetales. A random forest algorithm trained on genomic data predicted drug-resistant yeasts with 54–75% accuracy. Fluconazole resistance was consistently predicted with the highest accuracy (75.2%). Furthermore, fluconazole resistance prediction accuracy was similar between models trained on genome-wide variation in the presence and number of InterPro protein annotations across Saccharomycotina (75.2%) and those trained on amino acid sequence alignment data of Erg11, a protein known to be involved in fluconazole resistance (74.3-74.9%). Interestingly, the top Erg11 residues for predicting fluconazole resistance across Saccharomycotina do not overlap with, are not spatially close to, and are less conserved than those previously linked to resistance in clinical isolates of Candida albicans. In silico deep mutational scanning of the C. albicans Erg11 protein reveals that amino acid variants implicated in clinical cases of resistance are almost universally destabilizing while variants in our most informative residues are energetically more neutral, explaining why the latter are much more common than the former in natural populations. Importantly, previous experimental analyses of C. albicans Erg11 have shown that amino acid variation in our most informative residues, despite having never been directly implicated in clinical cases, can directly contribute to resistance. Our results suggest that studies of natural resistance in yeast species never encountered in the clinic will yield a fuller understanding of antifungal drug resistance.

Harrison, Marie-Claire [Vanderbilt Univ., Nashvill↗

Genome-scale Design and Engineering of Non-model Yeast Organisms for Production of Biofuels and Bioproducts

The overall goal of this project was to develop genome-scale design and engineering tools for two non-model yeast organisms including Rhodotorula toruloides and Issatchenkia orientalis to produce high-levels of fatty acids-derived products and organic acids, respectively. The project was performed between 9/15/2017 and 9/14/2024 (the last two-years were no-cost extensions). The team consisted of Huimin Zhao (Lead PI) and Christopher Rao (Co-PI) from the University of Illinois at Urbana-Champaign (UIUC), Costas Maranas (Co-PI) from the Pennsylvania State University, Joshua Rabinowitz (Co-PI) and Martin Wuhr (Co-PI) from Princeton University, and Yasuo Yoshikuni (Co-PI) from the DOE Joint Genome Institute. The team has made great progress in both tool development and fundamental understanding of these two non-model yeasts. In total, there were 40 research publications (one of them is still under review) and one patent application as well as numerous oral presentations.

60 APPLIED LIFE SCIENCES↗

All You Can Eat Yeast: Substituting Hexose Transporters With AtSWEET7 Alleviates Glucose Repression, Enabling Simultaneous Utilization of Sugars in Renewable Feedstocks

Yeast sugar transporters have highly evolved for preferential glucose transport, a significant roadblock for utilizing non-glucose sugars in renewable feedstocks such as lignocellulosic biomass. To enable simultaneous transport of multiple sugars, native hexose transporters were replaced by SWEET7p from Arabidopsis thaliana in engineered Saccharomyces cerevisiae capable of fermenting xylose. Engineered S. cerevisiae exhibited reduced glucose preference, simultaneously co-fermenting glucose, mannose, fructose, and xylose both in synthetic and industrial media. Continuous culture experiments demonstrated the co-consuming phenotype and alleviation of glucose repression by engineered S. cerevisiae. In addition to hexose and pentose, the NKSW7-1 strain consumed xylitol as a carbon source. Through transcriptomic and metabolomic analysis of the NKSW7-1 strain, we show that the replacement of HXT1-7 with AtSWEET7 led to systemwide reprogramming of the central carbon metabolism. This broad transport capacity of AtSWEET7p holds promise for achieving co-consumption of all sugars in underutilized renewable feedstocks by microbial cell factory.

59 BASIC BIOLOGICAL SCIENCES↗

Resourceful and economical designing of fermentation medium for lab and commercial strains of yeast from alternative feedstock: ‘transgenic oilcane’

Sugarcane plant engineered to accumulate lipids in its vegetative tissue is being developed as a new bioenergy crop. The new crop would be a source of juice, oil, and cellulosic sugars. However, limited tolerance of industrially recognized yeasts towards inhibitors generated during the processing of lignocellulosic biomass to produce fermentable sugars is a major challenge in developing scalable processes for second-generation drop-in fuel production. To this end, hydrolysates generated from engineered sugarcane—‘oilcane’ bagasse contain added phenolics and fatty acids that further restrict the growth of fermenting microorganisms and necessitate nutrient supplementation and/or detoxification of hydrolysate which makes the fermentation process expensive. Herein, we propose a resourceful and economical approach for growing lab and commercial strains of S. cerevisiae on unrefined cellulosic sugars aerobically and fermentatively.

09 BIOMASS FUELS↗

Taxogenomic Analysis of a Novel Yeast Species, Lachancea rosae Sp. Nov. F.A., Isolated From the Wild Rose Rosa californica

A novel Saccharomycotina yeast strain, yHQL494, was isolated from the rose hip of the wild rose Rosa californica from Castle Crags State Park, California, USA. Phylogenetic analyses of both whole genome data and the sequences from the D1/D2 region of the large ribosomal subunit (LSU) rRNA gene placed strain yHQL494 within the genus Lachancea and grouped it into a clade with Lachancea lanzarotensis and Lachancea meyersii. Taxogenomic analyses were conducted on publicly available genome sequences to gain a deeper insight into the carbon and nitrogen gene-trait associations across the Lachancea clade. The results of these analyses were found to be consistent across Lachancea species. Growth assays and microscopic analyses were conducted to determine the physiological characteristics of strain yHQL494, including the presence of hyphae or pseudohyphae, ascospore formation, fermentation abilities, and assimilation of carbon and nitrogen compounds. Based on the phenotypic and genomic characteristics of the strain yHQL494 T (=NRRL Y-64858 T , =CBS 18,574 T ), we propose a new species, Lachancea rosae sp. nov. f.a.

Bajgain, Yakendra [Villanova Univ., PA (United Sta↗

Acetate-based biological platforms: Bridging carbon dioxide utilization and high-value bioproduct production in oleaginous yeasts

Acetate is emerging as a promising two-carbon substrate in the circular bioeconomy, bridging the gap between single-carbon sources and high-value biofuels and bioproducts. This review examines the key pathways for acetate production, including the electrochemical reduction of carbon dioxide, syngas fermentation, and biological acetogenesis. It focuses on acetate metabolism in oleaginous yeasts, such as Yarrowia lipolytica and Rhodotorula toruloides, which efficiently convert acetate-derived acetyl-CoA units into diverse bioproducts such as lipids, fatty alcohols, triacetic acid lactone, and carotenoids. Recent advances in metabolic engineering, transcriptomics, and metabolic flux analysis have improved the understanding of acetate assimilation in these organisms, thereby increasing their potential for industrial applications. In addition, the feasibility of a biological gas-to-liquid platform that utilizes acetate as a central intermediate for scalable biomanufacturing is discussed. Integrating acetate utilization with sustainable production strategies offers a promising path to advance the bio-based economy. Using acetate as a versatile metabolic intermediate enables the conversion of industrial emissions into biofuels and bioproducts while avoiding the energetic and toxicity constraints associated with direct fermentation of gaseous substrates.

59 BASIC BIOLOGICAL SCIENCES↗

The oleaginous yeast Rhodosporidium toruloides engineered for biomass hydrolysate-derived (E)-α-bisabolene production

The oleaginous yeast Rhodosporidium toruloides has been exploited for many bioproducts, including several terpenes, owing to its oleaginous nature and biomass inhibitor tolerance. Here, we built upon previous (E)-a-bisabolene work by iteratively stacking the complete mevalonate pathway from Saccharomyces cerevisiae onto a multicopy bisabolene synthase parent strain. Metabolomics and proteomics verified heterologous pathway expression and identified metabolic bottlenecks at three intermediate steps, with candidate feedback-resistant mevalonate kinases screening improving titers 15%. Subtle differences in codon optimization, and preliminary attenuation of competing flux toward lipids resulted in 6-fold, 7-fold higher titers relative to controls, respectively. Media optimization led to modest improvements, with zinc identified as the most promising at 10% titer improvement. Ultimately, high-performance strains were cultivated with corn-stover biomass hydrolysate in microtiter plates at 300g/L total sugar, achieving 20.8g/L bisabolene, the highest reported titer in the literature. A 2L glucose minimal medium bioreactor achieved 19.3 g/L bisabolene and a literature-high productivity of 0.11 g/L/h.

60 APPLIED LIFE SCIENCES↗

Anti-Pdc1p Nanobody as a Genetically Encoded Inhibitor of Ethanol Production Enables Dual Transcriptional and Post-translational Controls of Yeast Fermentations

Microbial fermentation provides a sustainable method of producing valuable chemicals. Adding dynamic control to fermentations can significantly improve titers, but most systems rely on transcriptional controls of metabolic enzymes, leaving existing intracellular enzymes unregulated. This limits the ability of transcriptional controls to switch off metabolic pathways, especially when metabolic enzymes have long half-lives. We developed a two-layer transcriptional/post-translational control system for yeast fermentations. Specifically, the system uses blue light to transcriptionally activate the major pyruvate decarboxylase PDC1, required for cell growth and concomitant ethanol production. Switching to darkness transcriptionally inactivates PDC1 and instead activates the anti-Pdc1p nanobody, NbJRI, to act as a genetically encoded inhibitor of Pdc1p accumulated during the growth phase. This dual transcriptional/post-translational control improves the production of 2,3-BDO and citramalate by up to 100 and 92% compared to using transcriptional controls alone in dynamic two-phase fermentations. Furthermore, this study establishes the NbJRI nanobody as an effective genetically encoded inhibitor of Pdc1p that can enhance the production of pyruvate-derived chemicals.

59 BASIC BIOLOGICAL SCIENCES↗

Decompartmentalization of the yeast mitochondrial metabolism to improve chemical production in Issatchenkia orientalis

Microbial production of chemicals may suffer from inadequate cofactor provision, a challenge further exacerbated in yeasts due to compartmentalized cofactor metabolism. Here, we perform cofactor engineering through the decompartmentalization of mitochondrial metabolism to improve succinic acid (SA) production in Issatchenkia orientalis. We localize the reducing equivalents of mitochondrial NADH to the cytosol through cytosolic expression of its pyruvate dehydrogenase (PDH) complex and couple a reductive tricarboxylic acid pathway with a glyoxylate shunt, partially bypassing an NADH-dependent malate dehydrogenase to conserve NADH. Cytosolic SA production reaches a titer of 104 g/L and a yield of 0.85 g/g glucose, surpassing the yield of 0.66 g/g glucose constrained by cytosolic NADH availability. Additionally, expressing cytosolic PDH, we expand our I. orientalis platform to enhance acetyl-CoA-derived citramalic acid and triacetic acid lactone production by 1.22- and 4.35-fold, respectively. Our work establishes I. orientalis as a versatile platform to produce markedly reduced and acetyl-CoA-derived chemicals.

59 BASIC BIOLOGICAL SCIENCES↗

Ancient co-option of LTR retrotransposons as yeast centromeres

Centromeres ensure accurate chromosome segregation, yet their DNA evolves rapidly across eukaryotes leaving the origins of new centromere architectures unclear. The brewer’s yeast Saccharomyces cerevisiae exemplifies this long-standing puzzle. Its centromeres shifted ancestrally from large, repeat-rich, epigenetically specified forms to the compact, genetically defined ‘point’ centromeres. How this transition occurred has remained unresolved6. Here we identify evolutionarily related ‘proto-point’ centromeres that provide a resolution to the evolutionary origins of point centromeres. Proto-point centromeres contain a single centromeric nucleosome positioned over an AT-rich core, accompanied by relaxed organization and sequence variability of flanking cis-elements. In two species, these proto-point centromeres lie within retrotransposon-derived repeat clusters, linking ancestral repeat-rich centromeres to genetically encoded ones. Comparative and phylogenetic analyses indicate that proto-point and point centromeres evolved in an ancestor with retrotransposon-rich centromeres. These results identify long-terminal-repeat retrotransposons, specifically Ty5 sequences, as the genetic substrate for point-centromere evolution and provide a mechanistic route by which an epigenetic centromere can become genetically specified. More broadly, they show how selfish elements can be co-opted to perform essential chromosomal functions.

Haase, Max A. B. [Max Planck Institute of Molecula↗

The genome of the polyextremophilic yeast, Naganishia friedmannii, reveals adaptations involved in stress response pathways, carbohydrate metabolism expansion, and a limited DNA repair repertoire

Here we report the draft genome sequence of Naganishia friedmannii (formerly Cryptococcus friedmannii) isolate, a Basidiomycota yeast commonly found in some of the most extreme environments of the Earth's cryosphere. We isolated N. friedmannii strain Llullensis from soils at 6000 m above sea level on Volcán Llullaillaco, Argentina. The genome was 22.2 Mb with 6251 identified protein coding genes. Proteins known to be associated with thermal, osmotic, and radiation stress were identified in the genome. Comparative analysis with seven other Naganishia genomes revealed unique features underlying its polyextremophilic lifestyle. Naganishia friedmannii showed an expansion of genes involved in breaking down plant-derived carbohydrates, supporting the hypothesis that it survives at high elevations by metabolizing wind-deposited organic matter. Surprisingly, many genes involved in cell-cycle checkpoints and DNA repair were missing, as in several other Naganishia species. This extensive loss may be adaptive in extreme environments prone to abiotic stress, where a high mutation rate could generate advantageous traits, and reduced cell-cycle control may allow for faster reproduction that would be advantageous for rapid growth during brief periods of soil wetting following rare snow events.

Vimercati, Lara↗

Targeted genetic manipulation and yeast-like evolutionary genomics in the green alga Auxenochlorella

Auxenochlorella spp. are diploid oleaginous green algae whose streamlined genomes can be readily manipulated by homologous recombination, making them highly amenable to discovery research and bioengineering. Vegetatively diploid organisms experience specific evolutionary phenomena, including allodiploid hybridization, mitotic recombination, loss-of-heterozygosity, and aneuploidy; however, studies of these forces have largely focused on yeasts. Here, we present a telomere-to-telomere phased diploid genome assembly of Auxenochlorella UTEX 250-A (haploid length 22 Mb) and introduce a genetic toolkit for site-specific manipulation of the nuclear genome in multiple strains, featuring several selectable markers, inducible promoters, and fluorescent reporters for protein localization. UTEX 250-A is an allodiploid hybrid of Auxenochlorella protothecoides and Auxenochlorella symbiontica, two species differentiated by extensive chromosomal rearrangements. UTEX 250-A haplotypes are a mosaic of each parental species following mitotic recombination, and two chromosomes are trisomic. Loss-of-heterozygosity events are pervasive across Auxenochlorella and can evolve rapidly in the laboratory. High-quality structural annotation yielded ∼7,500 genes per haplotype. Auxenochlorella have experienced gene family loss and reduction, including core photosynthesis genes, and exhibit periodic adenine and cytosine methylation at promoters and gene bodies, respectively. Approximately 10% of genes, especially those involved in DNA repair and sex, overlap antisense long noncoding RNAs, which may participate in a regulatory mechanism. We demonstrate the utility of Auxenochlorella for fundamental research by knockout of a chlorophyll biosynthesis enzyme, and confirm one trisomy by allele-specific transformation. These results demonstrate the generality of several evolutionary forces associated with vegetative diploidy and provide a foundation for the use of Auxenochlorella as a reference organism.

CHL27↗

Vanderwaltozyma urihicola sp. nov., a yeast species isolated from rotting wood and beetles in a Brazilian Amazonian rainforest biome

Five yeast isolates belonging to a candidate for novel species were obtained from rotting wood and the gut of a passalid beetle larva in a site of Amazonian rainforest biome in Brazil. Sequence analysis of the Internal Transcribed Spacer (ITS)-5.8S region and the D1/D2 domains of the large subunit rRNA gene showed that the isolates represent a novel species of the genus Vanderwaltozyma. The closest relative of the novel species is Vanderwaltozyma huisunica. These species differs due to 44 nt substitutions and 21 indels in the sequences of the ITS region, as well as by 15 substitutions and four indels in the sequences of the D1/D2 domains. A phylogenomic analysis of the Vanderwaltozyma species with genomes sequenced showed that this novel species is an outgroup to the other species of this genus. We propose the name Vanderwaltozyma urihicola sp. nov. (CBS 18107T, MycoBank MB 856975) to accommodate these isolates. Furthermore, the species is homothallic, producing one to two ascospores per ascus. The habitat of V. urihicola is rotting wood in the Brazilian Amazonian rainforest biome.

Amazonian Forest↗

Yeast Transformation on Hamilton Vantage (YT Vantage) v1

Our software program is designed for the Hamilton Vantage liquid handling robot, automating the Build step in the Design-Build-Test-Learn (DBTL) cycle for Saccharomyces cerevisiae. This program minimizes human intervention, enabling rapid identification of pathway bottlenecks and genes that enhance verazine production. The program takes competent yeast and plasmid DNA as input and generates an output library of engineered strains compatible with automated colony picking, high-throughput culturing, and chemical extraction for downstream LC-MS analysis. A user-friendly interface, developed using the Hamilton Method Editor software, allows for on-demand parameter customization. By automating this process, our program streamlines the construction of Saccharomyces cerevisiae, reducing manual labor and increasing efficiency. While the manual process is well-documented, integration with robotic automation is less common, making our program a valuable tool for researchers. With this software, we achieved 2-5 fold increases in verazine production, demonstrating its potential to accelerate research in this field.

Louie, Randy [Lawrence Berkeley National Laborator↗

Data for "Anti-Pdc1p Nanobody as a Genetically Encoded Inhibitor of Ethanol Production Enables Dual Transcriptional and Post-translational Controls of Yeast Fermentations"

Microbial fermentation provides a sustainable method of producing valuable chemicals. Adding dynamic control to fermentations can significantly improve titers, but most systems rely on transcriptional controls of metabolic enzymes, leaving existing intracellular enzymes unregulated. This limits the ability of transcriptional controls to switch off metabolic pathways, especially when metabolic enzymes have long half-lives. We developed a two-layer transcriptional/post-translational control system for yeast fermentations. Specifically, the system uses blue light to transcriptionally activate the major pyruvate decarboxylase PDC1 , required for cell growth and concomitant ethanol production. Switching to darkness transcriptionally inactivates PDC1 and instead activates the anti-Pdc1p nanobody, NbJRI, to act as a genetically encoded inhibitor of Pdc1p accumulated during the growth phase. This dual transcriptional/post-translational control improves the production of 2,3-BDO and citramalate by up to 100 and 92% compared to using transcriptional controls alone in dynamic two-phase fermentations. This study establishes the NbJRI nanobody as an effective genetically encoded inhibitor of Pdc1p that can enhance the production of pyruvate-derived chemicals.

metabolic engineering↗

Data for "Decompartmentalization of the yeast mitochondrial metabolism to improve chemical production in Issatchenkia orientalis "

Microbial production of chemicals may suffer from inadequate cofactor provision, a challenge further exacerbated in yeasts due to compartmentalized cofactor metabolism. Here, we perform cofactor engineering through the decompartmentalization of mitochondrial metabolism to improve succinic acid (SA) production in Issatchenkia orientalis . We localize the reducing equivalents of mitochondrial NADH to the cytosol through cytosolic expression of its pyruvate dehydrogenase (PDH) complex and couple a reductive tricarboxylic acid pathway with a glyoxylate shunt, partially bypassing an NADH-dependent malate dehydrogenase to conserve NADH. Cytosolic SA production reaches a titer of 104 g/L and a yield of 0.85 g/g glucose, surpassing the yield of 0.66 g/g glucose constrained by cytosolic NADH availability. Additionally, expressing cytosolic PDH, we expand our I. orientalis platform to enhance acetyl-CoA-derived citramalic acid and triacetic acid lactone production by 1.22- and 4.35-fold, respectively. Our work establishes I. orientalis as a versatile platform to produce markedly reduced and acetyl-CoA-derived chemicals.

bioproducts↗

Improving cellulose attribution by selectively removing yeast glucans from grain fermentation intermediates

Abstract This article presents an industry-relevant method for quantifying cellulose in mixed substrate samples. We built upon the cellulosic glucan measurement proposed by Sluiter et al. (2021) to investigate significant cellulose loss under cold caustic conditions, which has hindered the establishment of an industrially relevant method. To overcome this issue, we used dimethyl sulfoxide (DMSO) as an alternative solvent, which avoids mercerization reactions (Budtova and Navard 2015) but may leave some resistant starch in the sample. Treatment via an enzyme mixture removed starch and conformed to US Environmental Protection Agency (EPA) guidance on cellulose measurement by establishing quantitative de-starching via nuclear magnetic resonance (EPA 2022). The selective removal of yeast beta-glucan was accomplished using a commercially available Zymolyase. Our results demonstrated excellent reproducibility, with coefficients of variance of 7.14% or less, when measuring cellulose in low cellulose content samples. The method was tested on relevant lab and plant samples, showing an average 0.9% ethanol derived from the conversion of cellulose when cellulase was added to the process and zero response when no cellulase was added. These findings indicate that a cellulose method using DMSO and pullulanase provides a complete and accurate view of cellulose content, composition, and conversion in industrial fermentation processes.

Sluiter, Justin [National Renewable Energy Laborat↗