Biology subjects

Reinmets, K.

Publications and source records attributed to Reinmets, K..

7 recordsLinked to original sources

bioRxiv2026

Targeted Deletions Uncover Previously Uncharacterised C1-fixing Cluster Genes as Essential for Autotrophy in Clostridium autoethanogenum

Recycling carbon oxides from industrial off-gases and gasified organic waste using gas fermentation offers a sustainable route for biomanufacturing of fuels and chemicals. Acetogens are attractive biocatalysts here and fix CO and CO2 via the Wood-Ljungdahl pathway (WLP). However, the incomplete functional annotation of the associated C1-fixing gene cluster limits rational engineering of acetogen cell factories. Here, we investigated five highly conserved but previously uncharacterised C1-fixing cluster genes to quantify their contributions to the growth of the model-acetogen Clostridium autoethanogenum using targeted CRISPR/Cas9-mediated single-gene deletions, physiological characterisation, comparative computational structural analyses, CRISPRi, and functional genomics. Importantly, we identified acsV, lpdA, cooC, and cooC2 as essential for autotrophy but not heterotrophy, whereas deletion of gcvH reduced autotrophic growth and shifted carbon flux from acetate towards 2,3-butanediol. Amino acid supplementation experiments and computational analyses indicate that lpdA and gcvH do not primarily function in glycine or serine biosynthesis but instead fulfil specialised roles in WLP-associated C1 and redox metabolism. Furthermore, genetic and physiological analyses showed that the two CooC-family ATPases, CooC and CooC2, have non-redundant roles in growth, with only cooC2 deletion impairing heterotrophy. Acetate supplementation tests revealed that {Delta}acsV cells retained metabolic activity despite abolished autotrophy, and computational analyses suggest a likely role for acsV in ATP-dependent corrinoid activation. Surprisingly, CRISPRi-mediated repression of acsV was translationally buffered, resulting in altered carbon partitioning without impairing autotrophy. Together, our findings provide insights into the physiological importance of the C1-fixing cluster genes in acetogens and support their metabolic engineering as gas-fermenting cell factories.

synthetic biology↗

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↗

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↗

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↗

bioRxiv2020

S-adenosylhomocysteine hydrolase regulates anterior patterning in Dugesia japonica

BackgroundBiological methylation requires S-adenosylmethionine (SAM) and participates in a range of processes from modulation of gene expression via histone modifications to neurotransmitter synthesis. An important factor in all methylation reactions is the concentration ratio of SAM to methylation byproduct S-adenosylhomocysteine (SAH). SAH hydrolase, also known as adenosylhomocysteinase, depletes SAH and thereby facilitates metabolite recycling and maintains the methylation permissive SAM/SAH ratio. While the importance of SAH hydrolase in sustaining methylation is obvious on the cellular level, the function of this metabolic process on the organismal scale is not clear. ResultsWe used planarian Dugesia japonica to investigate the role SAH hydrolase in physiological homeostasis on the body-wide scale. Remarkably, pharmacological inhibition of the SAH hydrolase results in regression of anterior tissues and is accompanied by extensive apoptosis throughout the planarian body. Moreover, exposure to the SAHH inhibitor AdOx leads to changes in brain morphology and spatial shift in the expression of Wnt-modulator Notum. Strikingly, planarians are able to overcome these destructive patterning defects through regeneration of the anterior tissues and adaptation to the used inhibitor. Transcriptome analysis indicates that resistance to the SAHH inhibitor is at least partly mediated by changes in folate cycle and lipid metabolism. ConclusionsSAH hydrolase plays a critical role in planarian homeostasis and anterior patterning potentially through modulation of Wnt signaling. Moreover, planarian adaptation to the SAHH inhibitor via metabolic reprogramming suggests potential targets for addressing methylation-related human conditions.

physiology↗