Flash hydrolysis of yeast (Saccharomyces cerevisiae) for protein recovery
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Thakkar, Anuj↗
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The Model System Saccharomyces cerevisiae Versus Emerging Non-Model Yeasts for the Production of Biofuels
Microorganisms are effective platforms for the production of a variety of chemicals including biofuels, commodity chemicals, polymers and other natural products. However, deep cellular understanding is required for improvement of current biofuel cell factories to truly transform the Bioeconomy. Modifications in microbial metabolic pathways and increased resistance to various types of stress caused by the production of these chemicals are crucial in the generation of robust and efficient production hosts. Recent advances in systems and synthetic biology provide new tools for metabolic engineering to design strategies and construct optimal biocatalysts for the sustainable production of desired chemicals, especially in the case of ethanol and fatty acid production. Yeast is an efficient producer of bioethanol and most of the available synthetic biology tools have been developed for the industrial yeast Saccharomyces cerevisiae. Non-conventional yeast systems have several advantageous characteristics that are not easily engineered such as ethanol tolerance, low pH tolerance, thermotolerance, inhibitor tolerance, genetic diversity and so forth. Currently, synthetic biology is still in its initial steps for studies in non-conventional yeasts such as Yarrowia lipolytica, Kluyveromyces marxianus, Issatchenkia orientalis and Pichia pastoris. Therefore, the development and application of advanced synthetic engineering tools must also focus on these underexploited, non-conventional yeast species. Herein, we review the basic synthetic biology tools that can be applied to the standard S. cerevisiae model strain, as well as those that have been developed for non-conventional yeasts. In addition, we will discuss the recent advances employed to develop non-conventional yeast strains that are efficient for the production of a variety of chemicals through the use of metabolic engineering and synthetic biology.
09 BIOMASS FUELS↗
A FAIR-compliant parts catalogue for genome engineering and expression control in Saccharomyces cerevisiae
The synthetic biology toolkit for baker's yeast, Saccharomyces cerevisiae, includes extensive genome engineering toolkits and parts repositories. However, with the increasing complexity of engineering tasks and versatile applications of this model eukaryote, there is a continued interest to expand and diversify the rational engineering capabilities in this chassis by FAIR (findable, accessible, interoperable, and reproducible) compliance. In this study, we designed and characterised 41 synthetic guide RNA sequences to expand the CRISPR-based genome engineering capabilities for easy and efficient replacement of genomically encoded elements. Moreover, we characterize in high temporal resolution 20 native promoters and 18 terminators using fluorescein and LUDOX CL-X as references for GFP expression and OD600 measurements, respectively. Additionally, all data and reported analysis is provided in a publicly accessible jupyter notebook providing a tool for researchers with low-coding skills to further explore the generated data as well as a template for researchers to write their own scripts. We expect the data, parts, and databases associated with this study to support a FAIR-compliant resource for further advancing the engineering of yeasts.
59 BASIC BIOLOGICAL SCIENCES↗
PKA regulatory subunit Bcy1 couples growth, lipid metabolism, and fermentation during anaerobic xylose growth in Saccharomyces cerevisiae
Past work from our lab, focused on engineering the budding yeast Saccharomyces cerevisiae for fermentation of the non-native pentose sugar xylose, discovered that hyperactivation of the RAS/Protein Kinase A (PKA) pathway was needed for rapid anaerobic xylose fermentation. Interestingly, the mechanism of PKA hyperactivation has a dramatic impact on growth and metabolism on xylose; deletion of the RAS inhibitor IRA2 permits rapid growth and fermentation, while deletion of the PKA regulatory subunit BCY1 allows for fermentation without growth on xylose. To understand how a single deletion in the PKA pathway can decouple growth and metabolism, we performed transcriptomic analysis of these strains, predicting that altered PKA activity would impact global gene expression and identify pathways important for growth and metabolism coordination.
Gasch, Audrey↗
A deep learning-guided automated workflow in LipidOz for detailed characterization of fungal fatty acid unsaturation by ozonolysis
Understanding fungal lipid biology and metabolism is critical for antifungal target discovery as lipids play central roles in cellular processes. Nuances in lipid structural differences can significantly impact their functions, making it necessary to characterize lipids in detail to enable and understanding of their roles in these complex systems. In particular, lipid double bond (DB) locations are an important component of lipid structure that can only be determined using a few specialized analytical techniques. Ozone-induced dissociation mass spectrometry (OzID-MS) is one such technique that uses ozone to break lipid DBs, producing pairs of characteristic fragments that allow the determination of DB positions. In this work we apply OzID-MS and LipidOz software to analyze the complex lipids of Saccharomyces cerevisiae yeast strains transfected with different fatty acid desaturases from Histoplasma capsulatum to determine the specific unsaturated lipids produce. The automated data analysis in LipidOz made the determination of DB positions from this large dataset more practical, but manual verification for all targets was still time-consuming. The DL model reduces manual involvement in data analysis, but since it was trained using mammalian lipid extracts, the prediction accuracy on yeast-derived data was reduced. We addressed both shortcomings by retraining the DL model to act as a pre-filter to prioritize targets for automated analysis, providing confident manually verified results but requiring less computational time and manual effort. Our workflow resulted in the determination of novel DB positions and enzymatic specificity.
mass spectrometry, deep learning, Lipidomics, doub↗
Coupling High-Throughput and Targeted Screening for Identification of Nonobvious Metabolic Engineering Targets
Identification of metabolic engineering targets is a fundamental challenge in strain development programs. While high-throughput (HTP) genetic engineering methodologies capable of generating vast diversity are being developed at a rapid rate, a majority of industrially interesting molecules cannot be screened at sufficient throughput to leverage these techniques. We propose a workflow that couples HTP screening of common precursors (e.g., amino acids) that can be screened either directly or by artificial biosensors, with low-throughput targeted validation of the molecule of interest to uncover nonintuitive beneficial metabolic engineering targets and combinations hereof. Using this workflow, we identified several nonobvious novel targets for improving p-coumaric acid (p-CA) and L -DOPA production from two large 4k gRNA libraries each deregulating 1000 metabolic genes in the yeast Saccharomyces cerevisiae. We initially screened yeast cells transformed with gRNA library plasmids for individual regulatory targets improving the production of l-tyrosine-derived betaxanthins, identifying 30 targets that increased intracellular betaxanthin content 3.5–5.7 fold. Hereafter, we screened the targets individually in a high-producing p-CA strain, narrowing down the targets to six that increased the secreted titer by up to 15%. To investigate whether any of the six targets could be additively combined to improve p-CA production further, we created a gRNA multiplexing library and subjected it to our proposed coupled workflow. The combination of regulating PYC1 and NTH2 simultaneously resulted in the highest (threefold) improvement of the betaxanthin content, and an additive trend was also observed in the p-CA strain. Lastly, we tested the initial 30 targets in a L -DOPA producing strain, identifying 10 targets that increased the secreted titer by up to 89%, further validating our screening by proxy workflow. This coupled approach is useful for strain development in the absence of direct HTP screening assays for products of interest.
59 BASIC BIOLOGICAL SCIENCES↗
Novel viruses of the family Partitiviridae discovered in Saccharomyces cerevisiae
It has been 49 years since the last discovery of a new virus family in the model yeast Saccharomyces cerevisiae . A large-scale screen to determine the diversity of double-stranded RNA (dsRNA) viruses in S . cerevisiae has identified multiple novel viruses from the family Partitiviridae that have been previously shown to infect plants, fungi, protozoans, and insects. Most S . cerevisiae partitiviruses (ScPVs) are associated with strains of yeasts isolated from coffee and cacao beans. The presence of partitiviruses was confirmed by sequencing the viral dsRNAs and purifying and visualizing isometric, non-enveloped viral particles. ScPVs have a typical bipartite genome encoding an RNA-dependent RNA polymerase (RdRP) and a coat protein (CP). Phylogenetic analysis of ScPVs identified three species of ScPV, which are most closely related to viruses of the genus Cryspovirus from the mammalian pathogenic protozoan Cryptosporidium parvum . Molecular modeling of the ScPV RdRP revealed a conserved tertiary structure and catalytic site organization when compared to the RdRPs of the Picornaviridae . The ScPV CP is the smallest so far identified in the Partitiviridae and has structural homology with the CP of other partitiviruses but likely lacks a protrusion domain that is a conspicuous feature of other partitivirus particles. ScPVs were stably maintained during laboratory growth and were successfully transferred to haploid progeny after sporulation, which provides future opportunities to study partitivirus-host interactions using the powerful genetic tools available for the model organism S . cerevisiae .
59 BASIC BIOLOGICAL SCIENCES↗
Phosphoproteome Response to Dithiothreitol Reveals Unique Versus Shared Features of Saccharomyces cerevisiae Stress Responses
To cope with sudden changes in the external environment, the budding yeast Saccharomyces cerevisiae orchestrates a multifaceted response that spans many levels of physiology. Several studies have interrogated the transcriptome response to endoplasmic reticulum (ER) stress and the role of regulators such as the Ire1 kinase and Hac1 transcription factors. However, less is known about responses to ER stress at other levels of physiology. Here, we used quantitative phosphoproteomics and computational network inference to uncover the yeast phosphoproteome response to the reducing agent dithiothreitol (DTT) and the upstream signaling network that controls it. We profiled wild-type cells and mutants lacking IRE1 or MAPK kinases MKK1 and MKK2, before and at various times after DTT treatment. In addition to revealing downstream targets of these kinases, our inference approach predicted new regulators in the DTT response, including cell-cycle regulator Cdc28 and osmotic-response kinase Rck2, which we validated computationally. Our results also revealed similarities and surprising differences in responses to different stress conditions, especially in the response of protein kinase A targets. Furthermore, these results have implications for the breadth of signaling programs that can give rise to common stress response signatures.
59 BASIC BIOLOGICAL SCIENCES↗
Crabtree/Warburg-like aerobic xylose fermentation by engineered Saccharomyces cerevisiae
Bottlenecks in the efficient conversion of xylose into cost-effective biofuels have limited the widespread use of plant lignocellulose as a renewable feedstock. The yeast Saccharomyces cerevisiae ferments glucose into ethanol with such high metabolic flux that it ferments high concentrations of glucose aerobically, a trait called the Crabtree/Warburg Effect. In contrast to glucose, most engineered S. cerevisiae strains do not ferment xylose at economically viable rates and yields, and they require respiration to achieve sufficient xylose metabolic flux and energy return for growth aerobically. Here, we evolved respiration-deficient S. cerevisiae strains that can grow on and ferment xylose to ethanol aerobically, a trait analogous to the Crabtree/Warburg Effect for glucose. Through genome sequence comparisons and directed engineering, we determined that duplications of genes encoding engineered xylose metabolism enzymes, as well as TKL1, a gene encoding a transketolase in the pentose phosphate pathway, were the causative genetic changes for the evolved phenotype. Reengineered duplications of these enzymes, in combination with deletion mutations in HOG1, ISU1, GRE3, and IRA2, increased the rates of aerobic and anaerobic xylose fermentation. Importantly, we found that these genetic modifications function in another genetic background and increase the rate and yield of xylose-to-ethanol conversion in industrially relevant switchgrass hydrolysate, indicating that these specific genetic modifications may enable the sustainable production of industrial biofuels from yeast. We propose a model for how key regulatory mutations prime yeast for aerobic xylose fermentation by lowering the threshold for overflow metabolism, allowing mutations to increase xylose flux and to redirect it into fermentation products.
59 BASIC BIOLOGICAL SCIENCES↗
Xylose Assimilation for the Efficient Production of Biofuels and Chemicals by Engineered Saccharomyces cerevisiae
Microbial conversion of plant biomass into fuels and chemicals offers a practical solution to global concerns over limited natural resources, environmental pollution, and climate change. Pursuant to these goals, researchers have put tremendous efforts and resources towards engineering the yeast Saccharomyces cerevisiae to efficiently convert xylose, the second most abundant sugar in lignocellulosic biomass, into various fuels and chemicals. Here, we summarize recent advances in metabolic engineering of yeast to address bottlenecks on xylose assimilation, and to enable simultaneous co-utilization of xylose and other carbon sources in lignocellulosic hydrolysates. We also highlight distinct characteristics of xylose metabolism that can be harnessed to produce advanced biofuels and chemicals. Although many challenges remain, recent research investments have facilitated efficient fermentation of xylose and simultaneous co-consumption of xylose and glucose. In particular, understanding of xylose-induced metabolic rewiring in engineered yeast has encouraged use of xylose as a carbon source for producing various non-ethanol bioproducts. Furthermore, to boost the lignocellulosic biomass-based bioeconomy, much attention is expected to promote xylose-utilizing efficiency via reprogramming cellular regulatory networks, to attain robust co-fermentation of xylose and other cellulosic carbon sources under industrial conditions, and to exploit the advantageous traits of yeast xylose metabolism for producing diverse fuels and chemicals.
59 BASIC BIOLOGICAL SCIENCES↗
Data for High-Level β-Carotene Production from Xylose by Engineered Saccharomyces cerevisiae without Overexpression of a Truncated HMG1 (t HMG1 )
β‐Carotene is a natural pigment and health‐promoting metabolite, and has been widely used in the nutraceutical, feed, and cosmetic industries. Here, we engineered a GRAS yeast Saccharomyces cerevisiae to produce β‐carotene from xylose, the second most abundant and inedible sugar component of lignocellulose biomass. Specifically, a β‐carotene biosynthetic pathway containing crtYB, crtI, and crtE from Xanthophyllomyces dendrorhous was introduced into a xylose‐fermenting S. cerevisiae . The resulting strain produced β‐carotene from xylose at a titer threefold higher than from glucose. Interestingly, overexpression of t HMG1 , which has been reported as a critical genetic perturbation to enhance metabolic fluxes in the mevalonate pathway and β‐carotene production in yeast when glucose is used, did not further improve the production of β‐carotene from xylose. Through fermentation profiling, metabolites analysis, and transcriptional studies, we found the advantages of using xylose as a carbon source, instead of glucose, for β‐carotene production to be a more respiratory feature of xylose consumption, a larger cytosolic acetyl‐CoA pool, and an upregulated expression level of rate‐limiting genes in the β‐carotene‐producing pathway, including ACS1 and HMG1 . As a result, 772.8 mg/L of β‐carotene was obtained in a fed‐batch bioreactor culture with xylose feeding. Considering the inevitable large scale production of xylose when cellulosic biomass‐based bioeconomy is implemented, our results suggest xylose utilization is a promising strategy for overproduction of carotenoids and other isoprenoids in engineered S. cerevisiae .
Conversion↗
High–level β–carotene production from xylose by engineered Saccharomyces cerevisiae without overexpression of a truncated HMG1 (t HMG1 )
Beta-carotene is a natural pigment and health-promoting metabolite, and has been widely used in the nutraceutical, feed, and cosmetic industries. Here, we engineered a GRAS yeast Saccharomyces cerevisiae to produce beta-carotene from xylose, the sec- ond most abundant and inedible sugar component of lignocellulose biomass. Specifically, a beta-carotene biosynthetic pathway containing crtYB, crtI, and crtE from Xanthophyllomyces dendrorhous was introduced into a xylose-fermenting S. cerevisiae. Here, the resulting strain produced beta-carotene from xylose at a titer threefold higher than from glucose. Interestingly, overexpression of tHMG1, which has been reported as a critical genetic perturbation to enhance metabolic fluxes in the mevalonate pathway and beta-carotene production in yeast when glucose is used, did not further improve the production of beta-carotene from xylose. Through fermentation profiling, metabolites analysis, and transcriptional studies, we found the advantages of using xylose as a carbon source, instead of glucose, for beta-carotene production to be a more respiratory feature of xylose consumption, a larger cytosolic acetyl!CoA pool, and an upregulated expression level of rate-limiting genes in the beta-carotene!producing pathway, including ACS1 and HMG1. As a result, 772.8 mg/L of beta-carotene was ob- tained in a fed-batch bioreactor culture with xylose feeding. Considering the in- evitable large scale production of xylose when cellulosic biomass!based bioeconomy is implemented, our results suggest xylose utilization is a promising strategy for overproduction of carotenoids and other isoprenoids in engineered S. cerevisiae.
09 BIOMASS FUELS↗
Functional Divergence in a Multi-gene Family Is a Key Evolutionary Innovation for Anaerobic Growth in Saccharomyces cerevisiae
The amplification and diversification of genes into large multi-gene families often mark key evolutionary innovations, but this process often creates genetic redundancy that hinders functional investigations. When the model budding yeast Saccharomyces cerevisiae transitions to anaerobic growth conditions, the cell massively induces the expression of seven serine/threonine-rich anaerobically-induced cell wall mannoproteins (anCWMPs): TIP1, TIR1, TIR2, TIR3, TIR4, DAN1, and DAN4. Here, we show that these genes likely derive evolutionarily from a single ancestral anCWMP locus, which was duplicated and translocated to new genomic contexts several times both prior to and following the budding yeast whole genome duplication (WGD) event. Based on synteny and their phylogeny, we separate the anCWMPs into four gene subfamilies. To resolve prior inconclusive genetic investigations of these genes, we constructed a set of combinatorial deletion mutants to determine their contributions toward anaerobic growth in S. cerevisiae. We found that two genes, TIR1 and TIR3, were together necessary and sufficient for the anCWMP contribution to anaerobic growth. Overexpressing either gene alone was insufficient for anaerobic growth, implying that they encode non-overlapping functional roles in the cell during anaerobic growth. We infer from the phylogeny of the anCWMP genes that these two important genes derive from an ancient duplication that predates the WGD event, whereas the TIR1 subfamily experienced gene family amplification after the WGD event. Taken together, the genetic and molecular evidence suggests that one key anCWMP gene duplication event, several auxiliary gene duplication events, and functional divergence underpin the evolution of anaerobic growth in budding yeasts.
59 BASIC BIOLOGICAL SCIENCES↗
Discovery of additional ancient genome duplications in yeasts
Whole-genome duplication (WGD) has had profound macroevolutionary impacts on diverse lineages, preceding adaptive radiations in vertebrates, teleost fish, and angiosperms. In contrast to the many known ancient WGDs in animals, and especially plants, we are aware of evidence for only four WGDs in fungi. The oldest of these occurred ∼100 million years ago (mya) and is shared by ∼60 extant Saccharomycetales species, including the baker’s yeast Saccharomyces cerevisiae. Notably, this is the only known ancient WGD event in the yeast subphylum Saccharomycotina. The dearth of ancient WGD events in fungi remains a mystery. Some studies have suggested that fungal lineages that experience chromosome and genome duplication quickly go extinct, leaving no trace in the genomic record, while others contend that the lack of known WGDs is due to an absence of data. Under the second hypothesis, additional sampling and deeper sequencing of fungal genomes should lead to the discovery of more WGD events. Coupling hundreds of recently published genomes from nearly every described Saccharomycotina species, with three additional long-read assemblies, we discovered three novel WGD events. Although the functions of retained duplicate genes originating from these events are broad, they bear similarities to the well-known WGD that occurred in the Saccharomycetales. In conclusion, our results suggest that WGD may be a more common evolutionary force in fungi than previously believed.
convergent evolution↗
Switchgrass Steroidal Saponins Reduce Fungal Disease but Decrease Yeast Fermentation Yield
Increasing the production of bioproducts from lignocellulosic feedstocks requires improvement in both field production and biorefinery efficiency. When plant traits arise that improve field production but decrease biofuel yield, these trade-offs can represent challenges in the entire production process. To examine trade-offs between field and production traits, we examined factors underlying switchgrass resistance to fungal rust pathogens in field conditions and factors that impede yeast fermentation in the lab using repeated measurements on a switchgrass genetic diversity panel. We found that the same switchgrass genotypes that showed high fungal pathogen resistance also showed recalcitrance to yeast fermentation. These switchgrass genotypes were mostly from the Atlantic genetic group, which had high levels of specialized metabolites of the saponin class. Among 1589 metabolites identified through metabolomics, we found that saponins were among the most likely to explain variation in both rust infection and fermentation yield using random forest feature selection, and that only four of these were sufficient to explain 57.9% of the variation in rust susceptibility. Through follow-up testing in recalcitrant biomass, we found that the bacterium Zymomonas mobilis does not suffer the same inhibition as the yeast Saccharomyces cerevisiae, and that the addition of ergosterol (thought to be the fungal cellular target of saponin inhibition) rescues yeast fermentation. Several lines of evidence point to a central role for saponins as key metabolites protecting switchgrass from fungal pathogens and interfering with yeast fermentation, underscoring an ongoing need for collaboration between plant breeders and biofuel production scientists.
VanWallendael, Acer [North Carolina State Universi↗
Advances in S. cerevisiae Engineering for Xylose Fermentation and Biofuel Production: Balancing Growth, Metabolism, and Defense
Genetically engineering microorganisms to produce chemicals has changed the industrialized world. The budding yeast Saccharomyces cerevisiae is frequently used in industry due to its genetic tractability and unique metabolic capabilities. S. cerevisiae has been engineered to produce novel compounds from diverse sugars found in lignocellulosic biomass, including pentose sugars, like xylose, not recognized by the organism. Engineering high flux toward novel compounds has proved to be more challenging than anticipated since simply introducing pathway components is often not enough. Several studies show that the rewiring of upstream signaling is required to direct products toward pathways of interest, but doing so can diminish stress tolerance, which is important in industrial conditions. As an example of these challenges, we reviewed S. cerevisiae engineering efforts, enabling anaerobic xylose fermentation as a model system and showcasing the regulatory interplay’s controlling growth, metabolism, and stress defense. Enabling xylose fermentation in S. cerevisiae requires the introduction of several key metabolic enzymes but also regulatory rewiring of three signaling pathways at the intersection of the growth and stress defense responses: the RAS/PKA, Snf1, and high osmolarity glycerol (HOG) pathways. The current studies reviewed here suggest the modulation of global signaling pathways should be adopted into biorefinery microbial engineering pipelines to increase efficient product yields.
59 BASIC BIOLOGICAL SCIENCES↗
Convergent evolution of aerobic fermentation through divergent mechanisms acting on the same key glycolytic genes
Expression profiling by high throughput sequencing The model yeast Saccharomyces cerevisiae has evolved aerobic fermentation in part through a whole genome duplication and subsequent retention of glycolytic genes. To identify independent yeast lineages with a high glycolytic rate phenotype we developed an assay measuring extracellular acidification rates (ECAR) to evaluate the glycolytic rates across diverse yeast species. This assay identified a novel group of yeasts in the genus Saturnispora with rapid glycolytic rates. Through comparative approaches, we found that several glycolytic genes encoding hexokinases and enzymes in lower glycolysis had higher expression and modifications in promoter sequences in rapid ECAR species. Intriguingly, many of the upregulated genes are the same genes that are duplicated in S. cerevisiae. When the transcription factor required for their activation was deleted, the mutants had a slow glycolytic rate and more respiratory phenotype. This work shows how the aerobic fermentation phenotype has convergently evolved in Saturnispora through transcriptional rewiring. This divergent mechanism nevertheless impacts the same glycolytic genes, which suggests that there are evolutionary constraints on how aerobic fermentation can arise.
Horianopoulos, Linda C. [Department of Food Scienc↗
Convergent evolution of aerobic fermentation through divergent mechanisms acting on key shared glycolytic genes
As the tree of life becomes increasingly accessible to molecular investigations, describing mechanisms underlying evolutionary convergence and constraint will be crucial to understanding diversification. The lineage including the model yeast Saccharomyces cerevisiae evolved aerobic fermentation in part through an ancient whole genome duplication and retention of glycolytic genes. To evaluate glycolytic rates across diverse yeasts, we developed and deployed an extracellular acidification rates (ECAR) assay on 299 species that span more than 400 million years of evolution and identified a clade in the genus Saturnispora that convergently evolved aerobic fermentation. Through comparative genomics and transcriptomics, we found that several glycolytic genes had higher expression and novel cis-regulatory elements in aerobically fermenting Saturnispora species. When the transcription factor required for their activation was deleted in Saturnispora dispora, the mutants had reduced glycolytic rates and increased respiration. Intriguingly, many of the upregulated genes are orthologous to duplicated glycolytic genes in S. cerevisiae. These divergent genetic mechanisms affecting the same set of genes suggest that there are strong evolutionary constraints on how aerobic fermentation can arise.
Horianopoulos, Linda C. [Great Lakes Bioenergy Res↗