Biology subjects

Valgepea, K.

Publications and source records attributed to Valgepea, K..

14 recordsLinked to original sources

bioRxiv2026

Gas uptake stoichiometry governs carbon partitioning in syngas-fermenting Clostridium autoethanogenum

Given the current global environmental challenges, waste biomass is an attractive renewable resource for circular economies. Gasification of biomass yields syngas (CO, CO2, and H2) that is a suitable feedstock for gas fermentation in biomanufacturing of fuels and chemicals using acetogen microbes. While it is generally known that syngas composition influences both acetogen growth and process performance, we are lacking a consistent dataset quantifying these effects under controlled fermentation conditions. Here, we mapped the metabolic response of the model-acetogen Clostridium autoethanogenum to seven synthetic syngas mixtures during exponential batch growth in bioreactor fermentations. Notably, distinct gas compositions resulted in different fermentation profiles, affecting both growth and metabolite production. Maximum specific growth rates ranged within 0.05-0.13 h-1, with slower growth for low-CO mixtures. While acetate and ethanol production yields varied between 20-133 and 76-353 mmol per gram dry cell weight, respectively, minor production of 2,3-butanediol was detected. All syngas mixtures supported co-utilization of CO and H2, though gas uptake stoichiometry only moderately correlated with syngas content. Importantly, gas uptake stoichiometry strongly influenced carbon partitioning, with higher relative H2 uptake reducing CO2 loss or even realizing CO2 fixation together with increasing carbon flow towards metabolites. Interestingly, higher syngas H2 content favored ethanol and 2,3-butanediol production, while higher H2:CO uptake ratios increased total flux through the Wood-Ljungdahl pathway rather than selectively favoring reduced by-products. Our results are valuable for a better understanding of syngas composition effects on the acetogen biocatalyst and for process engineering towards optimizing gas fermentation performance.

microbiology↗

bioRxiv2026

Measure Catabolism: Real-time shifts in microbial metabolism through online pressure measurements

Microbial activity is often inferred from cell density measurements; however, biomass formation is merely an indirect, cumulative result of metabolism, known as anabolism. Microbial activity is more accurately indicated by energy conservation or catabolism. This is especially true under low or no-growth conditions, where anabolism remains constant, and shifts in catabolic fluxes go unnoticed with biomass measurements alone. In anaerobic and gas-based metabolic processes, net gas exchange is linked to energy conservation, and catabolism can then be quantified through headspace measurements. We introduce a sealed-vial, non-invasive workflow that uses high-resolution headspace pressure measurements to estimate gas exchange rates and catabolic reactions, enabling real-time visualisation of metabolic shifts throughout an entire batch cultivation cycle. The method was applied to carbon monoxide (CO) fermentations of three Clostridium autoethanogenum strains (JA1-1, LAbrini, and LAbrini_mut) cultivated in serum bottles. Two of them were indistinguishable by OD-derived max. Pressure-derived gas uptake rates resolved multiple exponential phases of gas consumption and identified specific shifts in the metabolism, consistent with transitions from mixotrophic to autotrophic growth. Small but significant differences in terminal headspace pressure were detected, providing an experimentally accessible end-state parameter for phenotypic characterisation that would be obscured by routine intrusive headspace sampling. Finally, pressure-derived catabolic rates further enabled estimates of relative product formation during the main autotrophic phase. The strains were successfully characterised and distinguished by identifying several exponential phases of gas consumption and their rates, as well as differences in the final absolute pressure threshold. This provided phenotypic characterisation and insights not obtainable from OD measurements alone. The work establishes a practical framework for catabolism-resolved microbial characterisation in sealed batch vials through high-resolution online pressure (gas exchange). The assumption that pressure measurements correlate with CO2 and catabolic rates is sensitive to solubility/buffering and temperature/vapour effects, but these limitations are addressable through controls and complementary analytics.

microbiology↗

bioRxiv2026

Genetic engineering of carbon monoxide dehydrogenases produces distinct autotrophic phenotypes in Clostridium autoethanogenum

Acetogens are promising microbes for sustainable biomanufacturing but improving acetogen gas fermentation requires efficient conversion of CO and CO2 into fuels and chemicals. Carbon monoxide dehydrogenase (CODH) enzymes couple carbon fixation to energy conservation in acetogens and serve as potential regulatory modules for tuning autotrophic metabolism. Intriguingly, the model-acetogen Clostridium autoethanogenum lost its unique truncation in the bifunctional CODH (acsA), essential for autotrophy, during autotrophic adaptive laboratory evolution while obtaining superior phenotypes. Additionally, protein expression of the monofunctional CODH cooS1 is high and conditionally-regulated in C. autoethanogenum. Here, we genetically engineered CODHs in C. autoethanogenum by replacing the stop codon in acsA with leucine (strain Leu_SNP) or serine (Ser_SNP), and deleting cooS1 ({Delta}cooS1). Phenotyping in autotrophic batch and chemostat cultures revealed altered growth profiles and significant redistribution of carbon and redox flows in SNP strains, whereas {Delta}cooS1 showed moderate and condition-dependent effects. Surprisingly, structural modelling identified no conformational differences between wild-type and mutant AcsA proteins. While transcriptomics showed limited transcriptional changes in {Delta}cooS1, it suggested potential transcriptional adjustments linked to reduced robustness and altered product profile of Leu_SNP. Our results demonstrate the impact of CODHs on autotrophy and offer targets for rational engineering of acetogen cell factories.

bioengineering↗

bioRxiv2025

Systems-level analysis identifies protein-RNA interactions contributing to autotrophy of Clostridium autoethanogenum

Burning fossil fuels drives climate change with detrimental consequences on humankind while waste accumulation from increasing consumption threatens biosustainability globally. Gas fermentation enables to recycle carbon oxides (CO and CO2) from industrial waste gases and gasified waste into value-added products using gas-fermenting microbes, namely acetogens. However, our limited understanding of gene function and metabolic regulation is hindering rational engineering of acetogen cell factories. In this work, we identified, for the first time in acetogens, genome-wide protein-RNA interactions contributing to autotrophy in the model-acetogen Clostridium autoethanogenum by combining steady-state chemostat cultivation, functional genomics, and computational methods. We first detected limited and uncoupled transcriptional and translational regulation between autotrophy and heterotrophy. Rigorous mapping of genome-wide transcriptional architecture revealed both differential usage and signal strength of transcriptional start and termination sites between genes and growth substrates. We then used computational tools to reconstruct protein-RNA interactions for differentially regulated genes predicting 14 trans-acting regulatory RNA-binding proteins (RBPs) involved in post-transcriptional regulation and contributing to autotrophy. Most RBPs, two of which are translationally regulated, perform RNA modifications and regulate mRNA stability while others target translation-related genes. Our work provides valuable knowledge for metabolic engineering of acetogens and potentially contributes towards understanding primordial life on Earth.

systems biology↗

bioRxiv2025

Different growth pattern during microbial electrosynthesis using C. ljungdahlii evolutionary adapted on iron

Clostridium ljungdahlii is an acetogen used for syngas fermentation and capable of microbial electrosynthesis. While C. ljungdahlii has potential for industrial application because of its broad spectrum of metabolites and substrates, including CO2 and CO, the efficiency of extracellular electron uptake in a bioelectrochemical system is low. Therefore, C. ljungdahlii was evolutionary adapted on iron as sole electron source with the aim to improve extracellular electron uptake. Over 38 batches, 95% of the culture was replaced with fresh medium biweekly to retain iron-attached C. ljungdahlii, leading to improved acetate production rates with each cycle. Eight isolated strains were tested on fructose, H2 and CO2, and iron to screen evolved mutants with desired mutations. Compared to the wild-type, growth on fructose was similar and growth on H2 and CO2 was, surprisingly, worse, with only minor differences between isolates. The isolated mutants produced acetate at a rate of 0.14 {+/-} 0.01 mM/d on iron, while the wild-type strain produced 0.75 {+/-} 0.14 mM/d. Whole genome sequencing of isolated mutants revealed 16 mutations, of which seven mutations were found in all isolates. Mostly, membrane proteins were mutated. In a BES reactor, acetate production ceased after day 1. The optical density (OD) of isolate #8 stopped increasing after day 2, reaching 0.122 {+/-} 0.005, followed by the production of formate and ethanol. The wild-type strain continued to grow until day 4, reaching an OD of 0.177 {+/-} 0.003. These results may indicate that C. ljungdahlii slows down growth and produces ethanol as an energy reserve as an evolutionary strategy for survival in an electron-limited environment.

microbiology↗

bioRxiv2025

Heterologous expression of pyruvate formate lyase enhances cell growth of Clostridium ljungdahlii during microbial electrosynthesis

Slow cell growth and low biomass yields are hurdles for gas fermentation by acetogens. Microbial electrosynthesis (MES) utilizes acetogens as biocatalysts to reduce CO2 and produce commodity chemicals using electricity. However, limited electron supply in a bioelectrochemical system (BES) aggravates the poor cell growth of acetogens resulting in low productivities. Formate is a soluble C1 feedstock that can be produced by CO2 reduction (1). Thus, assimilation of formate can unburden the amount of electrons required for MES. Acetobacterium woodii posseses multiple pyruvate formate lyase (PFL) genes that are upregulated during formatotrophic growth. In this study, Clostridium ljugdahlii was engineered to heterologously express a pfl geneset of A. woodii to increase the cell growth of C. ljungdahlii during microbial electrosynthesis. Different combinations of pfl A and pfl B from A. woodii were tested in C. ljungdahlii to find the best working combination under control of Pfdx-riboswitch expression system. Expression of pfl B1 and pflA of A. woodii showed higher maximum OD of C. ljugdahlii under H2:CO2 condition with supplementation of 80 mM sodium formate. More than 40 mM of sodium formate caused significantly longer lag phase but the lag phase could be shortened after adaptation in 80 mM of sodium formate. At the end, the engineered strain showed improved cell growth (ODmax 0.22 {+/-} 0.05) and acetate production (21.8 {+/-} 5.6 mM) during microbial electrosynthesis compared to the control strain (ODmax 0.10 {+/-}0.06 and 10.2 {+/-}2.5 mM acetate). These results will be useful for strain development for microbial electrosynthesis, as well as gas fermentation. HighlightsO_LIDifferent combinations of pyruvate formate lyases (pfl Bs) and pyruvate formate lyase acting enzymes (pfl As) from Acetobacterium woodii were tested to improve the cell growth of Clostridium ljungdahlii during gas fermentation and microbial electrosynthesis C_LIO_LIHeterologous expression of pfl B1 and pfl A using riboswitch expression system improved cell growth of C. ljungdahlii under H2:CO2 condition, even without inducer and formate supplementation. C_LIO_LIHigh concentraton of sodium formate caused longer lag phase, which was shortened by adaptation when pfl from A. woodii was heterologously expressed. C_LIO_LICell growth and acetate production of the engineered C. ljungdahlii strain improved during microbial electrosynthesis C_LI

microbiology↗

bioRxiv2023

Deletion of genes linked to the C1-fixing gene cluster affects growth, by-products, and proteome of Clostridium autoethanogenum

Gas fermentation has emerged as a sustainable route to produce fuels and chemicals by recycling inexpensive one-carbon (C1) feedstocks from gaseous and solid waste using gas-fermenting microbes. Currently, acetogens that utilise the Wood-Ljungdahl pathway to convert carbon oxides (CO and CO2) into valuable products are the most advanced biocatalysts for gas fermentation. However, our understanding of the functionalities of the genes involved in the C1-fixing gene cluster and its closely-linked genes is incomplete. Here, we investigate the role of two genes with unclear functions - hypothetical protein (hp; LABRINI_07945) and CooT nickel binding protein (nbp; LABRINI_07950) - directly adjacent and expressed at similar levels to the C1-fixing gene cluster in the gas-fermenting model-acetogen Clostridium autoethanogenum. Targeted deletion of either the hp or nbp gene using CRISPR/nCas9, and phenotypic characterisation in heterotrophic and autotrophic batch and autotrophic bioreactor continuous cultures revealed significant growth defects and altered by-product profiles for both {Delta}hp and {Delta}nbp strains. Variable effects of gene deletion on autotrophic batch growth on rich or minimal media suggest that both genes affect the utilisation of complex nutrients. Autotrophic chemostat cultures showed lower acetate and ethanol production rates and higher carbon flux to CO2 and biomass for both deletion strains. Additionally, proteome analysis revealed that disruption of either gene affects the expression of proteins of the C1-fixing gene cluster and ethanol synthesis pathways. Our work contributes to a better understanding of genotype-phenotype relationships in acetogens and offers engineering targets to improve carbon fixation efficiency in gas fermentation.

synthetic biology↗

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

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

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↗