Biology subjects

Klask, C.-M.

Publications and source records attributed to Klask, C.-M..

3 recordsLinked to original sources

bioRxiv2021

Genetic evidence reveals the indispensable role of the rseC gene for autotrophy and the importance of a functional electron balance for nitrate reduction in Clostridium ljungdahlii

For Clostridium ljungdahlii, the RNF complex plays a key role for energy conversion from gaseous substrates such as hydrogen and carbon dioxide. In a previous study, a disruption of RNF-complex genes led to the loss of autotrophy, while heterotrophy was still possible via glycolysis. Furthermore, it was shown that the energy limitation during autotrophy could be lifted by nitrate supplementation, which resulted in an elevated cellular growth and ATP yield. Here, we used CRISPR-Cas12a to delete: 1) the RNF complex-encoding gene cluster rnfCDGEAB; 2) the putative RNF regulator gene rseC; and 3) a gene cluster that encodes for a putative nitrate reductase. The deletion of either rnfCDGEAB or rseC resulted in a complete loss of autotrophy, which could be restored by plasmid-based complementation of the deleted genes. We observed a transcriptional repression of the RNF-gene cluster in the rseC-deletion strain during autotrophy and investigated the distribution of the rseC gene among acetogenic bacteria. To examine nitrate reduction and its connection to the RNF complex, we compared autotrophic and heterotrophic growth of our three deletion strains with either ammonium or nitrate. The rnfCDGEAB- and rseC-deletion strains failed to reduce nitrate as a metabolic activity in non-growing cultures during autotrophy but not during heterotrophy. In contrast, the nitrate reductase deletion strain was able to grow in all tested conditions but lost the ability to reduce nitrate. Our findings highlight the important role of the rseC gene for autotrophy and contribute to understand the connection of nitrate reduction to energy metabolism. Significance StatementAcetogenic bacteria are widely known for their ability to convert gaseous substrates, such as hydrogen, carbon dioxide, and carbon monoxide, into short-chain fatty acids and alcohols, which can be utilized as sustainable platform chemicals and fuels. However, acetogenic bacteria conserve energy at the thermodynamic limit of life during autotrophy, and thus the production of more complex and energy-dense chemicals is limited due to low ATP yields. Therefore, it is key to decipher the interplay of the electron balancing reactions to understand and optimize the acetogenic metabolism. Recent findings with alternative electron acceptors that accelerated the cellular growth and ATP yield during autotrophy, such as nitrate, provide an opportunity to overcome energetic barriers in the acetogenic metabolism. The interrogation of the nitrate metabolism and the interplay between nitrate reduction and energy conservation in C. ljungdahlii, will contribute to fine-tuning of the acetogenic metabolism for biotechnological applications.

microbiology↗

bioRxiv2020

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↗

bioRxiv2019

An open-source multiple-bioreactor system for replicable gas-fermentation experiments: Nitrate feed results in stochastic inhibition events, but improves ethanol production of Clostridium ljungdahlii with CO2 and H2

The pH-value in fermentation broth has a large impact on the metabolic flux and growth behavior of acetogens. A decreasing pH level throughout time due to undissociated acetic acid accumulation is anticipated under uncontrolled pH conditions such as in bottle experiment. As a result, the impact of changes in the metabolism (e.g., due to a genetic modification) might remain unclear or even unrevealed. In contrast, pH-controlled conditions can be easily achieved in commercially available bioreactors. However, their acquisition is costly and their operation is time consuming, and therefore the experiment is often limited to a single bioreactor run. Here, we present a self-built, relatively cheap, and easy to handle open-source multiple-bioreactor system (MBS) consisting of six pH-controlled bioreactors at a 1-L scale. The functionality of the MBS was tested in three experiments by cultivating the acetogen Clostridium ljungdahlii with CO2 and H2 at steady-state conditions (=chemostat). The experiments were addressing the questions: (1) does the MBS provide replicable data for gas-fermentation experiments?; (2) does feeding acetate alter the production rate of ethanol; and (3) does feeding nitrate influence the product spectrum under controlled pH conditions with CO2 and H2? We applied four different periods in each experiment ranging from pH 6.0 to pH 4.5. Our data show high reproducibility for gas-fermentation experiments with C. ljungdahlii, using the MBS. We found that feeding acetate did not improve ethanol production, but rather impaired growth and reduced acetate production. Using nitrate as sole N-source, on the other hand, enhanced biomass production even at a low pH. However, we observed differences in growth, acetate, and ethanol production rates between triplicate bioreactors (n=3). We explained the different performances because of stochastic inhibition events, which we observed through the accumulation of nitrite, and which led to complete crashes at different operating times. One of these bioreactors recovered after the crash and showed enhanced ethanol production rates while simultaneously producing less acetate. The MBS offers a great opportunity to perform bench-scale bioreactor experiments at steady-state conditions with replicates, which is especially attractive for academia.

microbiology↗