A systems biology understanding of protein constraints in the metabolism of budding yeasts

Fermentation technologies, such as bread making and production of alcoholic beverages, have been crucial for development of humanity throughout history. Saccharomyces cerevisiae provides a natural platform for this, due to its capability to transform sugars into ethanol. This, and other yeasts, are now used for production of pharmaceuticals, including insulin and artemisinic acid, flavors, fragrances, nutraceuticals, and fuel precursors. In this thesis, different systems biology methods were developed to study interactions between metabolism, enzymatic capabilities, and regulation of gene expression in budding yeasts. In paper I, a study of three different yeast species (S. cerevisiae, Yarrowia lipolytica and Kluyveromyces marxianus), exposed to multiple conditions, was carried out to understand their adaptation to environmental stress. Paper II revises the use of genome-scale metabolic models (GEMs) for the study and directed engineering of diverse yeast species. Additionally, 45 GEMs for different yeasts were collected, analyzed, and tested. In paper III, GECKO 2.0, a toolbox for integration of enzymatic constraints and proteomics data into GEMs, was developed and used for reconstruction of enzyme-constrained models (ecGEMs) for three yeast species and model organisms. Proteomics data and ecGEMs were used to further characterize the impact of environmental stress over metabolism of budding yeasts. On paper IV, gene engineering targets for increased accumulation of heme in S. cerevisiae cells were predicted with an ecGEM. Predictions were experimentally validated, yielding a 70-fold increase in intracellular heme. The prediction method was systematized and applied to the production of 102 chemicals in S. cerevisiae (Paper V). Results highlighted general principles for systems metabolic engineering and enabled understanding of the role of protein limitations in bio-based chemical production. Paper VI presents a hybrid model integrating an enzyme-constrained metabolic network, coupled to a gene regulatory model of nutrient-sensing mechanisms in S. cerevisiae. This model improves prediction of protein expression patterns while providing a rational connection between metabolism and the use of nutrients from the environment.This thesis demonstrates that integration of multiple systems biology approaches is valuable for understanding the connection of cell physiology at different levels, and provides tools for directed engineering of cells for the benefit of society

Insights into genomics and gene expression of oleaginous yeasts of the genus Rhodoturula

The Rhodotorula glutinis cluster includes oleaginous yeast species with high biotechnological potential as cell factories for lipid, carotenoid, and oleochemical production. They can grow on various low-cost residues, such as lignocellulose and crude glycerol. The accumulated microbial lipids have a fatty acid composition similar to vegetable oils, representing a sustainable alternative in food, feed, and biofuel industries. Strain manipulation could further increase its economic attractiveness. A basic prerequisite for this is to understand the genome organization of Rhodotorula spp. and the lipid and carotenoid metabolism on residual products in detail. This doctoral thesis made a substantial contribution to this:Hybrid genome assemblies from R. toruloides, R. glutinis, and R. babjevae were built de novo using a combination of short- and long-read sequencing. By doing this, high-quality genomes could be achieved, showing high completeness and contiguity (near-chromosome level). Among other things, this enabled the prediction of ploidy and the discovery of extrachromosomal structures. Phylogenetic analyses revealed high intraspecific divergence in R. babjevae. Using RNA-seq data generated from different growth conditions, gene annotation for R. toruloides could be significantly increased, and comprehensive gene expression data could be obtained. These allowed conclusions to be drawn about the activation and mechanism of lipid accumulation in R. toruloides grown on residual products such as crude glycerol and hemicellulosic hydrolysate

Genome-guided bioprospecting for novel antibiotic lead compounds.

Antimicrobial resistance continues to pose a threat to health and wellbeing. Unmitigated, it is predicted to be the leading cause of death by 2050. Hence, the sustained development of novel antibiotics is crucial. As over 60% of licensed antibiotics are based on scaffolds derived from less than 1% of all known bacterial species, bacterial secondary metabolites constitute an untapped source of novel antibiotics. The aim of this project therefore was to expand the chemical space of bacteria-derived antibiotic lead compounds, using genomics approach. To that end, a topsoil sample was collected from the rhizosphere in which antibiosis occurs naturally. Using starvation stress, sixty-five isolates were recovered from the sample, out of which four were selected based on morphology and designated A13BB, A23BA, A13AA and A23AA. A13BB was identified by 16S rRNA gene sequence comparison as a Pseudomonas spp. and the other three isolates as Hafnia/Obesumbacterium spp. A database search showed that species belonging to these genera have genomes larger than the 3 Mb size above which an increasing proportion of a bacterial genome is dedicated to secondary metabolism. Given their ecological origin, expected genome size and ability to withstand starvation stress, these four isolates were presumed to harbour antibiotic-encoding gene clusters. Isolates A13BB and A23BA were therefore selected for genome mining in the first instance. Illumina and GridION/MinION sequencing data were obtained for both isolates and assembled into high-quality genomes. Isolates' identities were confirmed by FastANI analysis as strains of P. fragi and H. alvei, with 4.94 and 4.77 Mb genomes, respectively. Assembled genomes were mined with antiSMASH. Amongst other secondary metabolite biosynthetic gene clusters (smBGCs) detected, the β-lactone smBGCs in both genomes were selected for activation as their end products bear the hallmarks of an 'ideal antibiotic' that can inhibit several bacteria-specific enzymes simultaneously. Analysis of these smBGCs revealed genes encoding two core enzymes: 2-isopropylmalate synthase (2-IPMS) and acyl CoA ligase homologues. In the biosynthetic pathway, 2-IPMS catalyses the condensation of acetyl CoA with the degradation product of valine or isoleucine to form 2-IPM. 2-IPM is isomerised to 3-IPM which then forms the β-lactone warhead through reactions catalysed by acyl CoA ligase. It was speculated that the β-lactone compound is biosynthesised to efficiently rid the organism of potentially harmful metabolic intermediates as it grows on poor carbon and nitrogen sources. Strain fermentation was therefore performed with 10.8 mM acetate as the main carbon source, and 5 mM L-valine or L-isoleucine as the nitrogen source. Fermentation extracts were analysed by LC-MS with at least thirty-seven metabolite ions detected. Many of these ions have masses in the range m/z 230-750, which is an ideal mass range for antibiotic molecules. As β-lactone compounds are difficult to identify in crude extracts, especially when utilising single-stage mass spectrometry, reactivity-guided screening of extracts with cysteine thiol probe was performed as the probe forms UV- and MS-visible adducts with β-lactone compounds. However, complete dimerization of probe at a faster-than-expected rate in extract matrices hindered successful screening. This meant that it was not possible to determine if any crude extract components were β-lactone compounds without further analysis. Measures to limit or eliminate probe dimerization are proposed, together with molecular networking strategies that can afford global visualisation and rapid dereplication of extract components, using tandem mass spectrometry fragmentation patterns of parent ions. This project provides an original and robust workflow that serves as a strong starting point in the isolation of novel β-lactone compounds from crude extracts, followed by structural optimisation and bioactivity profiling. The hitherto unrecognised potential of β-lactone natural compounds as 'ideal antibiotics' is highlighted, and several structural optimisation strategies required to harness this potential are proposed. The genomes assembled here, and associated data have been deposited in the repositories of the International Nucleotide Sequence Database Collaboration for repurposing by other researchers. Likewise, the hidden metabolic and biosynthetic potentials of P. fragi and H. alvei species uncovered by RASTtk and antiSMASH analyses have been catalogued and placed in the public domain, with many of these attributes reported for the first time

Sequence of the hyperplastic genome of the naturally competent Thermus scotoductus SA-01

<p>Abstract</p> <p>Background</p> <p>Many strains of <it>Thermus </it>have been isolated from hot environments around the world. <it>Thermus scotoductus </it>SA-01 was isolated from fissure water collected 3.2 km below surface in a South African gold mine. The isolate is capable of dissimilatory iron reduction, growth with oxygen and nitrate as terminal electron acceptors and the ability to reduce a variety of metal ions, including gold, chromate and uranium, was demonstrated. The genomes from two different <it>Thermus thermophilus </it>strains have been completed. This paper represents the completed genome from a second <it>Thermus </it>species - <it>T. scotoductus</it>.</p> <p>Results</p> <p>The genome of <it>Thermus scotoductus </it>SA-01 consists of a chromosome of 2,346,803 bp and a small plasmid which, together are about 11% larger than the <it>Thermus thermophilus </it>genomes. The <it>T. thermophilus </it>megaplasmid genes are part of the <it>T. scotoductus </it>chromosome and extensive rearrangement, deletion of nonessential genes and acquisition of gene islands have occurred, leading to a loss of synteny between the chromosomes of <it>T. scotoductus and T. thermophilus</it>. At least nine large inserts of which seven were identified as alien, were found, the most remarkable being a denitrification cluster and two operons relating to the metabolism of phenolics which appear to have been acquired from <it>Meiothermus ruber</it>. The majority of acquired genes are from closely related species of the Deinococcus-Thermus group, and many of the remaining genes are from microorganisms with a thermophilic or hyperthermophilic lifestyle. The natural competence of <it>Thermus scotoductus </it>was confirmed experimentally as expected as most of the proteins of the natural transformation system of <it>Thermus thermophilus </it>are present. Analysis of the metabolic capabilities revealed an extensive energy metabolism with many aerobic and anaerobic respiratory options. An abundance of sensor histidine kinases, response regulators and transporters for a wide variety of compounds are indicative of an oligotrophic lifestyle.</p> <p>Conclusions</p> <p>The genome of <it>Thermus scotoductus </it>SA-01 shows remarkable plasticity with the loss, acquisition and rearrangement of large portions of its genome compared to <it>Thermus thermophilus</it>. Its ability to naturally take up foreign DNA has helped it adapt rapidly to a subsurface lifestyle in the presence of a dense and diverse population which acted as source of nutrients. The genome of <it>Thermus scotoductus </it>illustrates how rapid adaptation can be achieved by a highly dynamic and plastic genome.</p

The salmon louse genome: Copepod features and parasitic adaptations

Copepods encompass numerous ecological roles including parasites, detrivores and phytoplankton grazers. Nonetheless, copepod genome assemblies remain scarce. Lepeophtheirus salmonis is an economically and ecologically important ectoparasitic copepod found on salmonid fish. We present the 695.4 Mbp L. salmonis genome assembly containing ≈60% repetitive regions and 13,081 annotated protein-coding genes. The genome comprises 14 autosomes and a ZZ-ZW sex chromosome system. Assembly assessment identified 92.4% of the expected arthropod genes. Transcriptomics supported annotation and indicated a marked shift in gene expression after host attachment, including apparent downregulation of genes related to circadian rhythm coinciding with abandoning diurnal migration. The genome shows evolutionary signatures including loss of genes needed for peroxisome biogenesis, presence of numerous FNII domains, and an incomplete heme homeostasis pathway suggesting heme proteins to be obtained from the host. Despite repeated development of resistance against chemical treatments L. salmonis exhibits low numbers of many genes involved in detoxification.publishedVersio

De novo Transcriptome Assembly and Differential Expression Analysis of Catharanthus roseus in Response to Salicylic acid

Publication history: Accepted - 12 September 2022; Published online - 24 October 2022The anti-cancer vinblastine and vincristine alkaloids can only be naturally found in periwinkle (Catharanthus roseus). Both of these alkaloids' accumulations are known to be influenced by salicylic acid (SA). The transcriptome data to reveal the induction effect (s) of SA, however, seem restricted at this time. In this study, the de novo approach of transcriptome assembly was performed on the RNA-Sequencing (RNA-Seq) data in C. roseus. The outcome demonstrated that SA treatment boosted the expression of all the genes in the Terpenoid Indole Alkaloids (TIAs) pathway that produces the vinblastine and vincristine alkaloids. These outcomes supported the time-course measurements of vincristine alkaloid, the end product of the TIAs pathway, and demonstrated that SA spray had a positive impact on transcription and alkaloid synthesis. Additionally, the abundance of transcription factor families including bHLH, C3H, C2H2, MYB, MYB-related, AP2/ ERF, NAC, bZIP, and WRKY suggests a role for a variety of transcription families in response to the SA stimuli. Di-nucleotide and tri-nucleotide SSRs were the most prevalent SSR markers in microsatellite analyses, making up 39% and 34% of all SSR markers, respectively, out of the 77,192 total SSRs discovered

Automatically exploiting genomic and metabolic contexts to aid the functional annotation of prokaryote genomes

Cette thèse porte sur le développement d'approches bioinformatiques exploitant de l'information de contextes génomiques et métaboliques afin de générer des annotations fonctionnelles de gènes prokaryotes, et comporte deux projets principaux. Le premier projet focalise sur les activités enzymatiques orphelines de séquence. Environ 27% des activités définies par le International Union of Biochemistry and Molecular Biology sont encore aujourd'hui orphelines. Pour celles-ci, les méthodes bioinformatiques traditionnelles ne peuvent proposer de gènes candidats; il est donc impératif d'utiliser des méthodes exploitant des informations contextuelles dans ces cas. La stratégie CanOE (fishingCandidate genes for Orphan Enzymes) a été développée et rajoutée à la plateforme MicroScope dans ce but, intégrant des informations génomiques et métaboliques sur des milliers d'organismes prokaryotes afin de localiser des gènes probants pour des activités orphelines. Le projet miroir au précédent est celui des protéines de fonction inconnue. Un projet collaboratif a été initié au Genoscope afin de formaliser les stratégies d'exploration des fonctions de familles protéiques prokaryotes. Une version pilote du projet a été mise en place sur la famille DUF849 dont une fonction enzymatique avait été récemment découverte. Des stratégies de proposition d'activités enzymatiques alternatives et d'établissement de sous familles isofonctionnelles ont été mises en place dans le cadre de cette thèse, afin de guider les expérimentations de paillasse et d'analyser leurs résultats.The subject of this thesis concerns the development of bioinformatic strategies exploiting genomic and metabolic contextual information in order to generate functional annotations for prokaryote genes. Two main projects were involved during this work: the first focuses on sequence-orphan enzymatic activities. Today, roughly 27% of activities defined by International Union of Biochemistry and Molecular Biology are sequence-orphans. For these, traditional bioinformatic approaches cannot propose candidate genes. It is thus imperative to use alternative, context-based approaches in such cases. The CanOE strategy fishing Candidate genes for Orphan Enzymes) was developed and added to the MicroScope bioinformatics platform in this aim. It integrates genomic and metabolic information across thousands of prokaryote genomes in order to locate promising gene candidates for orphan activities. The mirror project focuses on protein families of unknown function. A collaborative project has been set up at the Genoscope in hope of formalising functional exploration strategies for prokaryote protein families. A pilot version was created on the DUF849 Pfam family, for which a single activity had recently been elucidated. Strategies for proposing novel functions and activities and creating isofunctional sub-families were researched, so as to guide biochemical experimentations and to analyse their results.EVRY-Bib. électronique (912289901) / SudocSudocFranceF

Computational Genome and Pathway Analysis of Halophilic Archaea

Halophilic archaea inhabit hypersaline environments and share common physiological features such as acidic protein machineries in order to adapt to high internal salt concentrations as well as electron transport chains for oxidative respiration. Surprisingly, nutritional demands were found to differ considerably amongst haloarchaeal species, though, and in this project several complete genomes of halophilic archaea were analysed to predict their metabolic capabilities. Comparative analysis of gene equipments showed that haloarchaea adopted several strategies to utilize abundant cell material available in brines such as the acquisition of catabolic enzymes, secretion of hydrolytic enzymes, and elimination of biosynthesis gene clusters. For example, metabolic genes of the well-studied Halobacterium salinarum were found to be consistent with the known degradation of glycerol and amino acids. Further, the complex requirement of H. salinarum for various amino acids and vitamins in comparison with other halophiles was explained by the lack of several genes and gene clusters, e.g. for the biosynthesis of methionine, lysine, and thiamine. Nitrogen metabolism varied also among halophilic archaea, and the haloalkaliphile Natronomonas pharaonis was predicted to apply several modes of N-assimilation to cope with severe ammonium deficiencies in its highly alkaline habitat. This species was experimentally shown to possess a functional respiratory chain, but comparative analysis with several archaea suggests a yet unknown complex III analogue in N. pharaonis. Respiratory chains of halophilic and other respiratory archaea were found to share similar genes for pre-quinone electron transfer steps but show great diversity in post-quinone electron transfer steps indicating adaptation to changing environmental conditions in extreme habitats. Finally, secretomes of halophilic and non-halophilic archaea were predicted proposing that haloarchaea secretion proteins are predominantly exported via the twin-arginine pathway and commonly exhibit a lipobox motif for N-terminal lipid anchoring. In N. pharaonis, lipoboxcontaining proteins were most frequent suggesting that lipid anchoring might prevent protein extraction under alkaline conditions. By contrast, non-halophilic archaea seem to prefer the general secretion pathway for protein translocation and to retain only few secretion proteins by N-terminal lipid anchors. Membrane attachment was preferentially observed for interacting components of ABC transporters and respiratory chains and might further occur via postulated C-terminal anchors in archaea. Within this project, the complete genome of the newly sequenced N. pharaonis was analysed with focus on curation of automatically generated data in order to retrieve reliable gene prediction and protein function assignment results as a basis for additional studies. Through the development of a post-processing routine and expert validation as well as by integration of proteomics data, a highly reliable gene set was created for N. pharaonis which was subsequently used to assess various microbial gene finders. This showed that all automatic gene tools predicted a rather correct gene set for the GC-rich N. pharaonis genome but produced insufficient results in respect to their start codon assignments. Available proteomics results for N. pharaonis and H. salinarum were further analysed for posttranslational modifications, and N-terminal peptides of haloarchaeal proteins were found to be commonly processed by N-terminal methionine cleavage and to some extent further modified by N-acetylation. For general function assignment of predicted N. pharaonis proteins and for enzyme assignment in H. salinarum, similarity-based searches, genecontext methods such as neighbourhood analysis but also manual curation were applied in order to reduce the number of hypothetical proteins and to avoid cross-species transfer of misassigned functions. This permitted to reliably reconstruct the metabolism of H. salinarum and N. pharaonis. Generated metabolic data were stored in a newly developed metabolic database that also integrates experimental data retrieved from the literature. The pathway data can be assessed as coloured KEGG maps and were combined with data resulting from transcriptomics and proteomics techniques. In future, expert-curated reaction entries of the created metabolic database will be a valuable source for the design of metabolic experiments and will deliver a reliable input for metabolic models of halophilic archaea