Biology subjects

Skerker, J. M.

Publications and source records attributed to Skerker, J. M..

8 recordsLinked to original sources

bioRxiv2025

Construction of a randomly barcoded insertional mutant library in the filamentous fungus Trichoderma atroviride

Filamentous fungi play key roles in ecosystems, agriculture, biotechnology, symbiosis, and disease, yet the large-scale characterization of gene function in these organisms remains limited by low transformation efficiencies and their multinucleate, syncytial cells, which complicate high-throughput screening strategies. To address the challenge of high-throughput screening in filamentous fungi, we developed methods to construct a genome-wide barcoded insertional mutant library in Trichoderma atroviride, a filamentous fungus widely used as a biocontrol agent against bacterial and fungal plant pathogens. Our strategy leveraged randomly barcoded transfer DNA insertions from plasmid libraries containing hundreds of millions of unique DNA barcodes and a broad host-range drug resistance marker delivered via Agrobacterium tumefaciens into T. atroviride. By optimizing transformation conditions, we achieved up to 600 independent transformants per infection event, resulting in a library of over 31,000 mapped insertions disrupting 7,104 of the 11,863 predicted genes in the T. atroviride genome. This resource establishes a scalable platform for high-throughput functional genomics in filamentous fungi, enabling both fundamental investigations of fungal biology and engineering approaches toward improved medical applications, biotechnology, and sustainable agriculture.

systems biology↗

bioRxiv2022

Acetaminophen production in the edible, filamentous cyanobacterium Arthrospira platensis

Spirulina is the common name for the edible, non-heterocystous, filamentous cyanobacterium Arthrospira platensis that is grown industrially as a food supplement, animal feedstock, and pigment source. Although there are many applications for engineering this organism1-3, until recently no genetic tools or reproducible transformation methods have been published. While recent work showed the production of a diversity of proteins in A. platensis, including single domain antibodies for oral delivery, there remains a need for a modular, characterized genetic toolkit4. Here, we establish and characterize a genetic toolkit and reproducible method for the transformation of A. platensis and engineer this bacterium to produce acetaminophen as proof-of-concept for small molecule production in an edible host from CO2, H2O, and light. This work opens A. platensis to the wider scientific community for future engineering as a functional food for nutritional enhancement, modification of organoleptic traits, and production of pharmaceuticals for oral delivery.

bioengineering↗

bioRxiv2021

Barcoded RH-seq illuminates the complex genetic basis of yeast thermotolerance

Decades of successes in statistical genetics have revealed the molecular underpinnings of traits as they vary across individuals of a given species. But standard methods in the field cant be applied to divergences between reproductively isolated taxa. Genome-wide reciprocal hemizygosity mapping (RH-seq), a mutagenesis screen in an inter-species hybrid background, holds promise as a method to accelerate the progress of interspecies genetics research. Here we describe an improvement to RH-seq in which mutants harbor barcodes for cheap and straightforward sequencing after selection in a condition of interest. As a proof of concept for the new tool, we carried out genetic dissection of the difference in thermotolerance between two reproductively isolated budding yeast species. Experimental screening identified dozens of candidate loci at which variation between the species contributed to the thermotolerance trait. Hits were enriched for mitosis genes and other housekeeping factors, and among them were multiple loci with robust sequence signatures of positive selection. Together, these results shed new light on the mechanisms by which evolution solved the problems of cell survival and division at high temperature in the yeast clade, and they illustrate the power of the barcoded RH-seq approach.

evolutionary biology↗

bioRxiv2019

Genome-wide and enzymatic analysis reveals efficient D-galacturonic acid metabolism in the basidiomycete yeast Rhodosporidium toruloides

Biorefining of renewable feedstocks is one of the most promising routes to replace fossil-based products. Since many common fermentation hosts, such as Saccharomyces cerevisiae, are naturally unable to convert many component plant cell wall polysaccharides, the identification of organisms with broad catabolism capabilities represents an opportunity to expand the range of substrates used in fermentation biorefinery approaches. The red basidiomycete yeast Rhodosporidium toruloides is a promising and robust host for lipid and terpene derived chemicals. Previous studies demonstrated assimilation of a range of substrates, from C5/C6-sugars to aromatic molecules similar to lignin monomers. In the current study, we analyzed R. toruloides potential to assimilate D-galacturonic acid, a major sugar in many pectin-rich agricultural waste streams, including sugar beet pulp and citrus peels. D-galacturonic acid is not a preferred substrate for many fungi, but its metabolism was found to be on par with D-glucose and D-xylose in R. toruloides. A genome-wide analysis by combined RNAseq/RB-TDNAseq revealed those genes with high relevance for fitness on D-galacturonic acid. While R. toruloides was found to utilize the same non-phosphorylative catabolic pathway known from ascomycetes, the maximal velocities of several enzymes exceeded those previously reported. In addition, an efficient downstream glycerol catabolism and a novel transcription factor were found to be important for D-galacturonic acid utilization. These results set the basis for use of R. toruloides as a potential host for pectin-rich waste conversions and demonstrate its suitability as a model for metabolic studies in basidiomycetes.\n\nImportanceThe switch from the traditional fossil-based industry to a green and sustainable bio-economy demands the complete utilization of renewable feedstocks. Many currently used bio-conversion hosts are unable to utilize major components of plant biomass, warranting the identification of microorganisms with broader catabolic capacity and characterization of their unique biochemical pathways. D-galacturonic acid is a plant component of bio-conversion interest and is the major backbone sugar of pectin, a plant cell wall polysaccharide abundant in soft and young plant tissues. The red basidiomycete and oleaginous yeast Rhodosporidium toruloides has been previously shown to utilize a range of sugars and aromatic molecules. Using state-of-the-art functional genomic methods, physiological and biochemical assays, we elucidated the molecular basis underlying the efficient metabolism of D-galacturonic acid. This study identifies an efficient pathway for uronic acid conversion to guide future engineering efforts, and represents the first detailed metabolic analysis of pectin metabolism in a basidiomycete fungus.

systems biology↗

bioRxiv2019

A toolset of constitutive promoters for metabolic engineering of Rhodosporidium toruloides

BackgroundRhodosporidium toruloides is a promising host for the production of bioproducts from lignocellulosic biomass. A key prerequisite for efficient pathway engineering is the availability of robust genetic tools and resources. However, there is a lack of characterized promoters to drive expression of heterologous genes for strain engineering in R. toruloides.\n\nResultsOur data describes a set of native R. toruloides promoters, characterized over time in four different media commonly used for cultivation of this yeast. The promoter sequences were selected using transcriptional analysis and several of them were found to drive expression bidirectionally. We measured promoter expression strength by flow cytometry using a dual fluorescent reporter system. From these analyses, we found a total of 20 constitutive promoters (12 monodirectional and 8 bidirectional) of potential value for genetic engineering of R. toruloides.\n\nConclusionsWe present a list of robust and constitutive, native promoters to facilitate genetic engineering of R. toruloides. This set of thoroughly characterized promoters significantly expands the range of engineering tools available for this yeast and can be applied in future metabolic engineering studies.

synthetic biology↗

bioRxiv2019

Multiplexed CRISPR-Cas9 based genome editing of Rhodosporidium toruloides

Microbial production of biofuels and bioproducts offers a sustainable and economic alternative to petroleum-based fuels and chemicals. The basidiomycete yeast Rhodosporidium toruloides is a promising platform organism for generating bioproducts due to its ability to consume a broad spectrum of carbon sources (including those derived from lignocellulosic biomass) and to naturally accumulate high levels of lipids and carotenoids, two biosynthetic pathways that can be leveraged to produce a wide range of bioproducts. While R. toruloides has great potential, it has a more limited set of tools for genetic engineering relative to more advanced yeast platform organisms such as Yarrowia lipolytica and Saccharomyces cerevisiae. Significant advancements in the past few years have bolstered R. toruloides engineering capacity. Here we expand this capacity by demonstrating the first use of CRISPR-Cas9 based gene disruption in R. toruloides. Stably integrating a Cas9 expression cassette into the genome brought about successful targeted disruption of the native URA3 gene. While editing efficiencies were initially low (0.002%), optimization of the cassette increased efficiencies 364-fold (to 0.6%). Applying these optimized design conditions enabled disruption of another native gene involved in carotenoid biosynthesis, CAR2, with much greater success; editing efficiencies of CAR2 deletion reached roughly 50%. Finally, we demonstrated efficient multiplexed genome editing by disrupting both CAR2 and URA3 in a single transformation. Together, our results provide a framework for applying CRISPR-Cas9 to R. toruloides that will facilitate rapid and high throughput genome engineering in this industrially relevant organism. IMPORTANCEMicrobial biofuel and bioproduct platforms provide access to clean and renewable carbon sources that are more sustainable and environmentally friendly than petroleum-based carbon sources. Furthermore, they can serve as useful conduits for the synthesis of advanced molecules that are difficult to produce through strictly chemical means. R. toruloides has emerged as a promising potential host for converting renewable lignocellulosic material into valuable fuels and chemicals. However, engineering efforts to improve the yeasts production capabilities have been impeded by a lack of advanced tools for genome engineering. While this is rapidly changing, one key tool remains unexplored in R. toruloides; CRISPR-Cas9. The results outlined here demonstrate for the first time how effective multiplexed CRISPR-Cas9 gene disruption provides a framework for other researchers to utilize this revolutionary genome-editing tool effectively in R. toruloides.

synthetic biology↗

bioRxiv2017

The polygenic basis of an ancient divergence in yeast thermotolerance

Some of the most unique and compelling survival strategies in the natural world are fixed in isolated species. To date, molecular insight into these ancient adaptations has been limited, as classic experimental genetics has focused on interfertile individuals in populations. Here we use a new mapping approach, which screens mutants in a sterile interspecific hybrid, to identify eight housekeeping genes that underlie the growth advantage of Saccharomyces cerevisiae over its distant relative S. paradoxus at high temperature. Pro-thermotolerance alleles at these mapped loci were required for the adaptive trait in S. cerevisiae and sufficient for its partial reconstruction in S. paradoxus. The emerging picture is one in which S. cerevisiae improved the heat resistance of multiple components of the fundamental growth machinery in response to selective pressure. This study lays the groundwork for the mapping of genotype to phenotype in clades of sister species across Eukarya.

evolutionary biology↗

bioRxiv2017

Transposon insertional mutagenesis in Saccharomyces uvarum reveals trans-acting effects influencing species dependent essential genes

To understand how complex genetic networks perform and regulate diverse cellular processes, the function of each individual component must be defined. Comprehensive phenotypic studies of mutant alleles have been successful in model organisms in determining what processes depend on the normal function of a gene. These results are often ported to newly sequenced genomes by using sequence homology. However, sequence similarity does not always mean identical function or phenotype, suggesting that new methods are required to functionally annotate newly sequenced species. We have implemented comparative analysis by high-throughput experimental testing of gene dispensability in Saccharomyces uvarum, a sister species of S. cerevisiae. We created haploid and heterozygous diploid Tn7 insertional mutagenesis libraries in S. uvarum to identify species dependent essential genes, with the goal of detecting genes with divergent functions and/or different genetic interactions. Comprehensive gene dispensability comparisons with S. cerevisiae predicted diverged dispensability at 12% of conserved orthologs, and validation experiments confirmed 22 differentially essential genes. Surprisingly, despite their differences in essentiality, these genes were capable of cross-species complementation, demonstrating that trans-acting factors that are background-dependent contribute to differential gene essentiality. This study demonstrates that direct experimental testing of gene disruption phenotypes across species can inform comparative genomic analyses and improve gene annotation. Our method can be widely applied in microorganisms to further our understanding of genome evolution.

genomics↗