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Casini, I.

Publications and source records attributed to Casini, I..

4 recordsLinked to original sources

FROG Analysis Ensures the Reproducibility of Genome Scale Metabolic Models

Genome-scale metabolic models (GEMs) and other constraint-based models (CBMs) play a pivotal role in understanding biological phenotypes and advancing research in areas like metabolic engineering, human disease modelling, drug discovery, and personalized medicine. Despite their growing application, a significant challenge remains in ensuring the reproducibility of GEMs, primarily due to inconsistent reporting and inadequate model documentation of model results. Addressing this gap, we introduce FROG analysis, a community-driven initiative aimed at standardizing reproducibility assessments of CBMs and GEMs. The FROG framework encompasses four key analyses--Flux variability, Reaction deletion, Objective function, and Gene deletion--to produce standardized, numerically reproducible FROG reports. These reports serve as reference datasets, enabling model evaluators, curators, and independent researchers to verify the reproducibility of GEMs systematically. BioModels, a leading repository of systems biology models, has integrated FROG analysis into its curation workflow, enhancing the reproducibility and reusability of submitted GEMs. In our study evaluating 65 GEM submissions from the community, approximately 40% reproduced without intervention, 28% requiring minor adjustments, and 32% needing input from authors. The standardization introduced by FROG analysis facilitated the detection and resolution of issues, ultimately leading to the successful reproduction of all models. By establishing a standardized and comprehensive approach to evaluating GEM reproducibility, FROG analysis significantly contributes to making CBMs and GEMs more transparent, reusable, and reliable for the broader scientific community.

systems biology↗

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↗

Reprogramming acetogenic bacteria with CRISPR-targeted base editing via deamination

Acetogenic bacteria are rising in popularity as chassis microbes in biotechnology due to their capability of converting inorganic one-carbon (C1) gases to organic chemicals. To fully uncover the potential of acetogenic bacteria, synthetic-biology tools are imperative to either engineer designed functions or to interrogate the physiology. Here, we report a genome-editing tool at a one-nucleotide resolution, namely base editing, for acetogenic bacteria based on CRISPR-targeted deamination. This tool combines nuclease deactivated Cas9 with activation-induced cytidine deaminase to enable cytosine-to-thymine substitution without DNA cleavage, homology-directed repair, and donor DNA, which are generally the bottlenecks for applying conventional CRISPR-Cas systems in bacteria. We designed and validated a modularized base-editing tool in the model acetogenic bacterium Clostridium ljungdahlii. The editing principles were investigated, and an in-silico analysis revealed the capability of base editing across the genome. Moreover, genes related to acetate and ethanol production were disrupted individually by installing premature STOP codons to reprogram carbon flux towards improved acetate production. This resulted in engineered C. ljungdahlii strains with the desired phenotypes and stable genotypes. Our base-editing tool promotes the application and research in acetogenic bacteria and provides a blueprint to upgrade CRISPR-Cas-based genome editing in bacteria in general. SignificanceAcetogenic bacteria metabolize one-carbon (C1) gases, such as industrial waste gases, to produce fuels and commodity chemicals. However, the lack of efficient gene-manipulation approaches hampers faster progress in the application of acetogenic bacteria in biotechnology. We developed a CRISPR-targeted base-editing tool at a one-nucleotide resolution for acetogenic bacteria. Our tool illustrates great potential in engineering other A-T-rich bacteria and links designed single-nucleotide variations with biotechnology. It provides unique advantages for engineering industrially relevant bacteria without creating genetically modified organisms (GMOs) under the legislation of many countries. This base-editing tool provides an example for adapting CRISPR-Cas systems in bacteria, especially those that are highly sensitive to heterologously expressed Cas proteins and have limited ability of receiving foreign DNA.

synthetic biology↗

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↗