Biology subjects

Morrissey, J. P.

Publications and source records attributed to Morrissey, J. P..

4 recordsLinked to original sources

bioRxiv2022

Integrated data-driven reannotation of the Kluyveromyces marxianus genome reveals an expanded protein coding repertoire

The coding potential of the eukaryotic genome can be greatly expanded by the regulated use of mechanisms that generate more than one protein product from a gene. We combined techniques for mapping 5 and 3 ends of RNA transcripts with ribosome profiling to study the organisation of protein coding gene expression in the yeast Kluyveromyces marxianus. We uncovered over 1000 cases of novel proteoforms due to use of alternative transcription or translation start sites, identified 800 translated upstream open reading frames, observed surprising translation of antisense RNAs, and discovered a novel case of programmed ribosomal frameshifting. In some cases, features are conserved across yeast species, whereas others are species-specific. This offers new possibilities to explore the evolution of genomes and gene regulation in budding yeasts. Our analysis also enabled us to improve the genome annotation of K. marxianus by adding or correcting annotations of over 300 protein coding genes. The processed data has been made available on the GWIPS-viz and Trips-Viz browsers, thus providing an accurate data-driven annotation of transcripts and their protein coding regions along with quantitative information on their transcription and translation.

genomics↗

bioRxiv2022

Development of a Ribosome Profiling Protocol to Study Translation in the yeast Kluyveromyces marxianus

Kluyveromyces marxianus is an interesting and important yeast because of particular traits like thermotolerance and rapid growth, and applications in food and industrial biotechnology. Knowing how K. marxianus responds and adapts to changing environments is important to achieve a full understanding of the its biology and to develop bioprocesses. For this, a full suite of omics tools to measure and compare global patterns of gene expression and protein synthesis is needed. Whereas transcriptome analysis by RNA-Seq quantifies mRNA abundance, ribosome profiling allows codon-resolution of translation on a genome-wide scale by deep sequencing of ribosome locations on mRNAs and is emerging as a valuable tool to study translation control of gene expression. We report here the development of a ribosome profiling method for K. marxianus and we make the procedure available as a step by step protocol. To aid in the analysis and sharing of ribosome profiling data, we also added the K. marxianus genome as well as transcriptome and ribosome profiling data to the publicly accessible GWIPS-viz and Trips-Viz browsers. Users are able to upload custom ribosome profiling and RNA-Seq data to both browsers, therefore allowing easy analysis and sharing of data. As many studies only focus on the use of RNA-Seq to study K. marxianus in different environments, the availability of ribosome profiling is a powerful addition to the K. marxianus toolbox. Graphical abstractDevelopment of a Ribosome Profiling protocol to study gene expression in the thermotolerant yeast Kluyveromyces marxianus. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=150 SRC="FIGDIR/small/478964v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1268da7org.highwire.dtl.DTLVardef@1ddff1org.highwire.dtl.DTLVardef@1597da4org.highwire.dtl.DTLVardef@47c2ae_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2020

Rational engineering of Kluyveromyces marxianus to create a chassis for the production of aromatic products

The yeast Kluyveromyces marxianus offers unique potential for industrial biotechnology because of useful features like rapid growth, thermotolerance and a wide substrate range. As an emerging alternative platform, K. marxianus requires the development and validation of metabolic engineering strategies to best utilize its metabolism as a basis for bio-based production. To illustrate the synthetic biology strategies to be followed and showcase its potential, we describe a comprehensive approach to rationally engineer a metabolic pathway in K. marxianus. We use the phenylalanine biosynthetic pathway both as a prototype and because phenylalanine is a precursor for commercially valuable secondary metabolites. First, we modify and overexpress the pathway to be resistant to feedback inhibition so as to overproduce phenylalanine de novo from synthetic minimal medium. Second, we assess native and heterologous means to increase precursor supply to the biosynthetic pathway. Finally, we eliminate branch points and competing reactions in the pathway and rebalance precursors to redirect metabolic flux to a specific product, 2-phenylethanol (2-PE). As a result, we are able to construct robust strains capable of producing over 800 mg L-1 2-PE from minimal medium. The strains we constructed are a promising platform for the production of aromatic amino acid-based biochemicals, and our results illustrate challenges with attempting to combine individually beneficial modifications in an integrated platform.

synthetic biology↗

bioRxiv2019

Young Genes are More Responsive to Environmental Stress than Ancient Genes in Budding Yeasts

The Saccharomycotina subphylum (budding yeasts) spans more than 400 million years of evolution and includes species that thrive in many of Earths harsh environments. Characterizing species that grow in harsh conditions could enable the design of more robust yeast strains for biotechnology. However, tolerance to stressful conditions is a multifactorial response, which is difficult to understand since many of the genes involved are as yet uncharacterized. In this work, three divergent yeast species were grown under multiple stressful conditions to identify stress-induced genes. For each condition, duplicated and non-conserved genes were significantly enriched for stress responsiveness compared to single-copy conserved genes. To understand this further, we developed a sorting method that considers evolutionary origin and duplication timing to assign an evolutionary age to each gene. Subsequent analysis of the sets of genes that changed expression revealed a relationship between stress-induced genes and the youngest gene set, regardless of the species or stress in question. These young genes are rarely essential for growth and evolve rapidly, which may facilitate their functionalization for stress tolerance and may explain their stress-induced expression. These findings show that systems-level analyses that consider gene age can expedite the identification of stress tolerance genes.

systems biology↗