Biology subjects

Marcellin, E.

Publications and source records attributed to Marcellin, E..

14 recordsLinked to original sources

bioRxiv2025

Co-Substrate Free Valorisation of Lignin Monomers by Assimilation of C1 and C2 By-Products

Lignin is an underutilised global resource with significant potential for the production of chemicals that are currently derived from fossil resources. However, biotechnological lignin valorisation faces various challenges including its recalcitrance and the toxicity of aromatic intermediates, products, and by-products like formaldehyde. While biochemical production from lignin-derived monomers has been demonstrated by disrupting native lignin degradation pathways, this approach required co-feeding additional carbon sources such as glucose for growth. This dependence on additional carbon sources can create competition with the food industry and undermine the economic sustainability of the bioprocess. Here, we report growth of a protocatechuate production strain of Pseudomonas putida EM42 on the by-products from p-coumarate and ferulate valorisation, achieving carbon efficiencies of up to 78 %. Additional flux balance analysis identified C1 assimilation pathways, including two novel pathways, beneficial for the growth on the formaldehyde by-product from ferulate degradation leading to improved carbon utilisation. This study demonstrates how by-product utilisation from lignin conversion can eliminate the need for co-feeding additional carbon sources, thereby potentially improving the efficiency of lignin valorisation.

bioengineering↗

bioRxiv2025

Abundant and active acetogens enhance the carbon dioxide sink of Blue Carbon ecosystems

Blue Carbon ecosystems, which include all tidal wetlands, mitigate climate change by capturing and storing carbon dioxide (CO2) from the atmosphere. Most carbon fixation in these systems is thought to be driven by plant and microbial photosynthesis, whereas chemosynthetic processes are assumed to play a minor role. However, these ecosystems often contain anoxic environments ideal for chemosynthetic microbes such as acetogens. Here, we show that acetogens are abundant and active mediators of carbon sequestration in tidal wetland soils by pairing gene-and genome-resolved metagenomic analysis with isolation and analysis of gas-fermenting acetogens in bioreactors. Metagenomic profiling revealed that diverse microbes can mediate carbon fixation, primarily through the Calvin-Benson-Bassham cycle and Wood-Ljungdahl pathways. These include various bacteria and archaea capable of reductive acetogenesis. On this basis, we grew bacterial enrichment cultures from tidal wetland soils using the gases hydrogen and CO2 as the sole energy and carbon sources. Bioreactor analysis revealed that these enrichments are dominated by clostridial acetogens that grow rapidly by converting CO2 into acetate and other products. Collectively, these results reveal Blue Carbon ecosystems harbour communities that can exclusively subsist by using CO2 as their sole electron acceptor and for carbon fixation, thereby providing evidence of a novel carbon sink pathway within these ecosystems beyond the known mechanisms of photosynthetic carbon fixation and soil sequestration. Additionally, the discovery and isolation of these chemosynthetic communities provide opportunities for developing further mechanisms of CO2 removal through industrial gas fermentation.

microbiology↗

bioRxiv2023

Autotrophic adaptive laboratory evolution of the acetogen Clostridium autoethanogenum delivers the gas-fermenting strain LAbrini with superior growth, products, and robustness

Microbes able to convert gaseous one-carbon (C1) waste feedstocks are increasingly important to transition to the sustainable production of renewable chemicals and fuels. Acetogens are interesting biocatalysts since gas fermentation using Clostridium autoethanogenum has already been commercialised. However, most acetogen strains need complex nutrients, display slow growth, and are not robust for routine bioreactor fermentations. In this work, we used three different and independent adaptive laboratory evolution (ALE) strategies to evolve the wild-type C. autoethanogenum to grow faster, without yeast extract and to be robust in operating continuous bioreactor cultures. Multiple evolved strains with improved phenotypes were isolated on a minimal medium with one strain, named "LAbrini" (LT1), exhibiting superior performance regarding the maximum specific growth rate, product profile, and robustness in continuous cultures. Whole-genome sequencing of the evolved strains identified 25 mutations. Of particular interest are two genes that acquired seven different mutations across the three ALE strategies, potentially as a result of convergent evolution. Reverse genetic engineering of sporulation-related mutations in genes CLAU_3129 (spo0A) and CLAU_1957 recovered all three superior features of our ALE strains through triggering significant proteomic rearrangements. This work provides a robust C. autoethanogenum strain to accelerate phenotyping and genetic engineering and to better understand acetogen metabolism, which we named "LAbrini".

bioengineering↗

bioRxiv2022

An integrated systems-biology platform for power-to-gas technology

Methanogenesis allows methanogenic archaea (methanogens) to generate cellular energy for their growth while producing methane. Hydrogenotrophic methanogens thrive on carbon dioxide and molecular hydrogen as sole carbon and energy sources. Thermophilic and hydrogenotrophic Methanothermobacter spp. have been recognized as robust biocatalysts for a circular carbon economy and are now applied in power-to-gas technology. Here, we generated the first manually curated genome-scale metabolic reconstruction for three Methanothermobacter spp.. We investigated differences in the growth performance of three wild-type strains and one genetically engineered strain in two independent chemostat bioreactor experiments. In the first experiment, with molecular hydrogen and carbon dioxide, we found the highest methane production rate for Methanothermobacter thermautotrophicus {Delta}H, while Methanothermobacter marburgensis Marburg reached the highest biomass growth rate. Systems biology investigations, including implementing a pan-model that contains combined reactions from all three microbes, allowed us to perform an interspecies comparison. This comparison enabled us to identify crucial differences in formate anabolism. In the second experiment, with sodium formate, we found stable growth with an M. thermautotrophicus {Delta}H plasmid-carrying strain with similar performance parameters compared to wild-type Methanothermobacter thermautotrophicus Z-245. Our findings reveal that formate anabolism influences the diversion of carbon to biomass and methane with implications for biotechnological applications of Methanothermobacter spp. in power-to-gas technology and for chemical production. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/522236v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1d468aeorg.highwire.dtl.DTLVardef@24a595org.highwire.dtl.DTLVardef@1f2dd60org.highwire.dtl.DTLVardef@1a3d8fc_HPS_FORMAT_FIGEXP M_FIG C_FIG Broader contextRenewable energy sources (e.g., wind and solar) provide carbon-free electric power. However, their intermittency and offset between peak production and demand generate the need to store this electric power. Furthermore, these technologies alone do not satisfy the demand for carbon-based commodities. Power-to-gas technology provides a means to store intermittent renewable electric power with concomitant carbon dioxide recycling into a chemical energy carrier, such as methane, on a centralized and decentralized scale. This is particularly important to establish equitable energy strategies for all countries, as is highlighted by the United Nations Sustainable Development Goals. With this work, we provide an integrated systems-biology platform for Methanothermobacter spp. to optimize biological power-to-gas technology and formulate strategies to produce other value-added products besides methane.

microbiology↗

bioRxiv2022

Molecular understanding of Eubacterium limosum chemostat methanol metabolism

Methanol is a promising renewable energy carrier that can be used as a favourable substrate for biotechnology, due to its high energy efficiency conversion and ease of integration within existing infrastructure. Some acetogenic bacteria have the native ability to utilise methanol, along with other C1 substrates such as CO2 and formate, to produce valuable chemicals. Continuous cultures favour economically viable bioprocesses, however, the performance of acetogens has not been investigated at the molecular level when grown on methanol. Here we present steady-state chemostat quantification of the metabolism of Eubacterium limosum, finding maximum methanol uptake rates up to 640{+/-}22 mmol/gDCW/d, with significant fluxes to butyrate. To better understand metabolism of acetogens under methanol growth conditions, we sampled chemostats for proteomics and metabolomics. Changes in protein expression and intracellular metabolomics highlighted key aspects of methanol metabolism, and highlighted bottleneck conditions preventing formation of the more valuable product, butanol. Interestingly, a small amount of formate in methylotrophic metabolism triggered a cellular state known in other acetogens to correlate with solventogenesis. Unfortunately, this was prevented by post-translation effects including an oxidised NAD pool. There remains uncertainty around ferredoxin balance at the methylene-tetrahydrofolate reductase (MTHFR) and at the Rnf level.

bioengineering↗

bioRxiv2022

Characterisation of acetogen formatotrophic potential using E. limosum

Formate is a promising energy carrier that could be used to transport renewable electricity. Some acetogenic bacteria, such as Eubacterium limosum, have the native ability to utilise formate as a sole substrate for growth, which has sparked interest in the biotechnology industry. However, formatotrophic metabolism in acetogens is poorly understood, and a systems-level characterization in continuous cultures is yet to be reported. Here we present the first steady-state dataset for E. limosum formatotrophic growth. At a defined dilution rate of 0.4 d-1, there was a high specific uptake rate of formate (280{+/-}56 mmol/gDCW/d), however, most carbon went to CO2 (150{+/-}11 mmol/gDCW/d). Compared to methylotrophic growth, protein differential expression data and intracellular metabolomics revealed several key features of formate metabolism. Upregulation of pta appears to be a futile attempt of cells to produce acetate as the major product. Instead, a cellular energy limitation resulted in the accumulation of intracellular pyruvate and upregulation of Pfl to convert formate to pyruvate. Therefore, metabolism is controlled, at least partially, at the protein expression level, an unusual feature for an acetogen. We anticipate that formate could be an important one-carbon substrate for acetogens to produce chemicals rich in pyruvate, a metabolite generally in low abundance during syngas growth.

bioengineering↗

bioRxiv2022

RNA-seq sample preparation kits strongly affect transcriptome profiles of a gas-fermenting bacterium

Transcriptome analysis via RNA sequencing (RNA-seq) has become a standard technique employed across various biological fields of study. This rapid adoption of the RNA-seq approach has been mediated, in part, by the development of different commercial RNA-seq library preparation kits compatible with standard next-generation sequencing (NGS) platforms. Generally, the essential steps of library preparation such as ribosomal RNA (rRNA) depletion and first-strand cDNA synthesis are tailored to a specific group of organisms (e.g. eukaryotes vs. prokaryotes) or genomic GC content. Therefore, the selection of appropriate commercial products is of crucial importance to capture the transcriptome of interest as closely to the native state as possible without introduction of technical bias. However, researchers rarely have the resources and time to test various commercial RNA-seq kits for their samples. This work reports a side-by-side comparison of RNA-seq data from Clostridium autoethanogenum obtained using three commercial rRNA removal and strand-specific library construction products by NuGEN Technologies, Qiagen, and Zymo Research and assesses their performance relative to published data. While all three vendors advertise their products as suitable for prokaryotes, we found significant differences in their performance regarding rRNA removal, strand-specificity, and, most importantly, transcript abundance distribution profiles. Notably, RNA-seq data obtained with Qiagen products were most similar to published data and delivered the best results in terms of library strandedness and transcript abundance distribution range. Our results highlight the importance of finding appropriate organism-specific workflows and library preparation products for RNA-seq studies. ImportanceRNA-seq is a powerful technique for transcriptome profiling while involving elaborate sample processing before library sequencing. Our work is important as we show that RNA-seq library preparation kits can strongly affect the outcome of the RNA-seq experiment. Although library preparation benefits from the availability of various commercial kits, choosing appropriate products for the specific samples can be challenging for new users or for users working with unconventional organisms. Evaluating the performance of different commercial products requires significant financial and time investment infeasible to most researchers. Therefore, users are often guided in their choice of kits by published data involving similar input samples. We conclude that important consideration should be given to selecting of sample processing workflows for any given organism.

systems biology↗

bioRxiv2022

Faster growth enhances low carbon fuel and chemical production through gas fermentation

Gas fermentation offers both fossil carbon-free sustainable production of fuels and chemicals and recycling of gaseous and solid waste using gas-fermenting microbes. Bioprocess development, systems-level analysis of biocatalyst metabolism, and engineering of cell factories are advancing the widespread deployment of the commercialised technology. Acetogens are particularly attractive biocatalysts but effects of the key physiological parameter - specific growth rate () - on acetogen metabolism and the gas fermentation bioprocess have not been established yet. Here, we investigate the -dependent bioprocess performance of the model-acetogen Clostridium autoethanogenum in CO and syngas (CO+CO2+H2) grown chemostat cultures and assess systems-level metabolic responses using gas analysis, metabolomics, transcriptomics, and metabolic modelling. We were able to obtain steady-states up to ~2.8 day-1 (~0.12 h-1) and show that faster growth supports both higher yields and productivities for reduced by-products ethanol and 2,3-butanediol. Transcriptomics data revealed differential expression of 1,337 genes with increasing and suggest that C. autoethanogenum uses transcriptional regulation to a large extent for facilitating faster growth. Metabolic modelling showed significantly increased fluxes for faster growing cells that were, however, not accompanied by gene expression changes in key catabolic pathways for CO and H2 metabolism. Cells thus seem to maintain sufficient "baseline" gene expression to rapidly respond to CO and H2 availability without delays to kick-start metabolism. Our work advances understanding of transcriptional regulation in acetogens and shows that faster growth of the biocatalyst improves the gas fermentation bioprocess.

systems biology↗

bioRxiv2021

Absolute proteome quantification in the gas-fermenting acetogen Clostridium autoethanogenum

Microbes that can recycle one-carbon (C1) greenhouse gases into fuels and chemicals are vital for the biosustainability of future industries. Acetogens are the most efficient known microbes for fixing carbon oxides CO2 and CO. Understanding proteome allocation is important for metabolic engineering as it dictates metabolic fitness. Here, we use absolute proteomics to quantify intracellular concentrations for >1,000 proteins in the model-acetogen Clostridium autoethanogenum grown on three gas mixtures. We detect prioritisation of proteome allocation for C1 fixation and significant expression of proteins involved in the production of acetate and ethanol as well as proteins with unclear functions. The data also revealed which isoenzymes are important. Integration of proteomic and metabolic flux data demonstrated that enzymes catalyse high fluxes with high concentrations and high in vivo catalytic rates. We show that flux adjustments were dominantly accompanied with changing enzyme catalytic rates rather than concentrations. Our work serves as a reference dataset and advances systems-level understanding and engineering of acetogens.

systems biology↗

bioRxiv2020

multiTFA: a Python package for multi-variate Thermodynamics-based Flux Analysis

SummaryWe achieve a significant improvement in thermodynamic-based flux analysis (TFA) by introducing multivariate treatment of thermodynamic variables and leveraging component contribution, the state-of-the-art implementation of the group contribution methodology. Overall, the method greatly reduces the uncertainty of thermodynamic variables. ResultsWe present multiTFA, a Python implementation of our framework. We evaluated our application using the core E. coli model and achieved a median reduction of 6.8 kJ/mol in reaction Gibbs free energy ranges, while three out of 12 reactions in glycolysis changed from reversible to irreversible. Availability and implementationOur framework along with documentation is available on https://github.com/biosustain/multitfa.

bioinformatics↗

bioRxiv2020

Enhancing CO2-valorization using Clostridium autoethanogenum for sustainable fuel and chemicals production

Acetogenic bacteria can convert waste gases into fuels and chemicals. Design of bioprocesses for waste carbon valorization requires quantification of steady-state carbon flows. Here, steady-state quantification of autotrophic chemostats containing Clostridium autoethanogenum grown on CO2 and H2 revealed that captured carbon (460 {+/-} 80 mmol/gDCW/day) had a significant distribution to ethanol (54 {+/-} 3 mol% with a 2.4 {+/-} 0.3 g/L titer). We were impressed with this initial result, but also observed limitations to biomass concentration and growth rate. Metabolic modelling predicted culture performance and indicated significant metabolic adjustments when compared to fermentation with CO as the carbon source. Moreover, modelling highlighted flux to pyruvate, and subsequently reduced ferredoxin, as a target for improving CO2 and H2 fermentation. Supplementation with a small amount of CO enabled co-utilisation with CO2, and enhanced CO2 fermentation performance significantly, while maintaining an industrially relevant product profile. Additionally, the highest specific flux through the Wood-Ljungdahl pathway was observed during co-utilization of CO2 and CO. Furthermore, the addition of CO led to superior CO2-valorizing characteristics (9.7 {+/-} 0.4 g/L ethanol with a 66 {+/-} 2 mol% distribution, and 540 {+/-} 20 mmol CO2/gDCW/day). Similar industrial processes are commercial or currently being scaled up, indicating CO-supplemented CO2 and H2 fermentation has high potential for sustainable fuel and chemical production. This work also provides a reference dataset to advance our understanding of CO2 gas fermentation, which can contribute to mitigating climate change.

bioengineering↗

bioRxiv2019

Native and synthetic methanol assimilation in Saccharomyces cerevisiae

Microbial fermentation for chemical production is becoming more broadly adopted as an alternative to petrochemical refining. Fermentation typically relies on sugar as a feedstock, however, one-carbon compounds like methanol are an attractive alternative as they can be derived from organic waste and natural gas. This study focused on engineering methanol assimilation in the yeast Saccharomyces cerevisiae. Three methanol assimilation pathways were engineered and tested: a synthetic xylulose monophosphate (XuMP), a hybrid methanol dehydrogenase-XuMP, and a bacterial ribulose monophosphate (RuMP) pathway, with the latter identified as the most effective at assimilating methanol. Additionally, 13C-methanol tracer analysis uncovered a native capacity for methanol assimilation in S. cerevisiae, which was optimized using Adaptive Laboratory Evolution. Three independent lineages selected in liquid methanol-yeast extract medium evolved premature stop codons in YGR067C, which encodes an uncharacterised protein that has a predicted DNA-binding domain with homology to the ADR1 transcriptional regulator. Adr1p regulates genes involved in ethanol metabolism and peroxisomal proliferation, suggesting YGR067C has a related function. When one of the evolved YGR067C mutations was reverse engineered into the parental CEN.PK113-5D strain, there were up to 5-fold increases in 13C-labelling of intracellular metabolites from 13C-labelled methanol when 0.1 % yeast extract was a co-substrate, and a 44 % increase in final biomass. Transcriptomics and proteomics revealed that the reconstructed YGR067C mutation results in down-regulation of genes in the TCA cycle, glyoxylate cycle, and gluconeogenesis, which would normally be up-regulated during growth on a non-fermentable carbon source. Combining the synthetic RuMP and XuMP pathways with the reconstructed Ygr067cp truncation led to further improvements in growth. These results identify a latent methylotrophic metabolism in S. cerevisiae and pave the way for further development of native and synthetic one-carbon assimilation pathways in this model eukaryote.

synthetic biology↗

bioRxiv2019

A TetR-family protein activates transcription from a new promoter motif associated with essential genes for autotrophic growth in acetogens

Acetogens can fix carbon (CO or CO2) into acetyl-CoA via the Wood-Ljungdahl pathway (WLP) that also makes them attractive cell factories for the production of fuels and chemicals from waste feedstocks. Although most biochemical details of the WLP are well understood and systems-level characterisation of acetogen metabolism has recently improved, key transcriptional features such as promoter motifs and transcriptional regulators are still unknown in acetogens. Here, we use differential RNA-sequencing to identify a previously undescribed promoter motif associated with essential genes for autotrophic growth of the model-acetogen Clostridium autoethanogenum. RNA polymerase was shown to bind to the new promoter motif using a DNA-binding protein assay and proteomics enabled the discovery of four candidates to potentially function directly in control of transcription of the WLP and other key genes of C1 fixation metabolism. Next, in vivo experiments showed that a TetR-family transcriptional regulator (CAETHG_0459) and the housekeeping sigma factor ({sigma}A) activate expression of a reporter protein (GFP) in-frame with the new promoter motif from a fusion vector in E. coli. Lastly, a protein-protein interaction assay with the RNA polymerase (RNAP) shows that CAETHG_0459 directly binds to the RNAP. Together, the data presented here advance the fundamental understanding of transcriptional regulation of C1 fixation in acetogens and provide a strategy for improving the performance of gas-fermenting bacteria by genetic engineering.

bioengineering↗

bioRxiv2019

Revisiting the evolution and taxonomy of Clostridia, a phylogenomic update

Clostridium is a large genus of obligate anaerobes belonging to the Firmicutes phylum of bacteria, most of which have a Gram-positive cell wall structure. The genus includes significant human and animal pathogens, causative of potentially deadly diseases such as tetanus and botulism. Despite their relevance and many studies suggesting that they are not a monophyletic group, the taxonomy of the group has largely been neglected. Currently, species belonging to the genus are placed in the unnatural order defined as Clostridiales, which includes the class Clostridia. Here we used genomic data from 779 strains to study the taxonomy and evolution of the group. This analysis allowed us to; (i) confirm that the group is composed of more than one genus (ii), detect major differences between pathogens classified as a single species within the group of authentic Clostridium spp. (sensu stricto), identify inconsistencies between taxonomy and toxin evolution that reflect on the pervasive misclassification of strains and, (iv) identify differential traits within central metabolism of members of what has been defined earlier and confirmed by us as cluster I. Our analysis shows that the current taxonomic classification of Clostridium species hinders the prediction of functions and traits, suggests a new classification for this fascinating class of bacteria and highlights the importance of phylogenomics for taxonomic studies.

microbiology↗