Interrogating the energy conservation of Clostridium ljungdahlii by genetic manipulation and bench-scale fermentation

Acetogene Bakterien haben ein großes Potenzial für den Einsatz in einer modernen und nachhaltig-orientierten Industrie, um gasförmige Substrate wie Kohlenmonoxid (CO), Kohlendioxid (CO2) und Wasserstoff (H2) in wertvolle Biochemikalien umzuwandeln. Die Gasfermentation mit acetogenen Bakterien hat nur einen geringen oder sogar negativen CO2-Fußabdruck und bietet damit eine interessante Alternative zu bestehenden petro-chemischen Produktionswegen. Die Hauptprodukte der acetogenen Gasfermentation sind Acetat und Ethanol, wobei letzteres direkt als Biokraftstoff verwendet werden kann. Kommerzielle Gasfermentationsanlagen mit acetogenen Bakterien werden bereits von der Firma Lanzatech betrieben, was die industrielle Relevanz dieser Technologie unterstreicht. Obwohl acetogene Bakterien als vielversprechende Biokatalysatoren gelten, ist ihr Energiestoffwechsel jedoch stark limitiert. Die Hauptwährung des Energiestoffwechsels innerhalb einer Bakterienzelle ist ATP. Dieses ATP wird für das Zellwachstum und für viele weitere Stoffwechselwege benötigt, z.B. auch für solche die zur Herstellung wertvoller und industriell relevanter Produkte genutzt werden. Alle bekannten acetogenen Bakterien nutzen den Wood-Ljungdahl pathway (WLP) zur Fixierung von Kohlenstoff. Der WLP hat jedoch keinen Netto-ATP-Gewinn. Ein Mol ATP wird investiert, um CO2 zu fixieren, während ein Mol ATP durch die Dephosphorylierung von Acetylphosphat zu Acetat regeneriert wird. Die einzige Möglichkeit, wie ein acetogenes Bakterium unter autotrophen Bedingungen zusätzliches ATP erzeugen kann, basiert auf Membrankomplexen wie dem Rhodobacter Nitrogen Fixation-like complex (RNF-Komplex). Dieser Komplex erzeugt einen Protonen- oder Natriumionengradienten über die Bakterienmembran, der dann von einer ATPase zur Erzeugung von ATP genutzt wird. Der ATP-Gewinn dieses chemiosmotischen Mechanismus ist jedoch ebenfalls gering. So kann das acetogene Bakterium Clostridium ljungdahlii beim Wachstum mit H2 und CO2 maximal 0.63 ATP/mol H2 erzeugen. Diese geringe Menge an ATP reicht gerade aus, um Wachstum und einen funktionierenden Grundstoffwechsel zu ermöglichen. Einerseits ist der geringe ATP-Gewinn ein Vorteil für die mikrobielle Biokraftstoffproduktion, da die Elektronen überwiegend für das Produkt und nicht für die Biomasse verwendet werden. Andererseits ist die Energiebeschränkung bei acetogenen Bakterien eine der größten Hürden, die es zu überwinden gilt, da diese maßgeblich die Produktion hochwertiger und ATP-fordernder Fermentationsprodukte einschränkt. Diese sind aber für eine breite Anwendung der Gasfermentation mit acetogenen Bakterien in der Industrie erforderlich, wodurch weitere Forschung mit diesen Mikroben notwendig ist. In dieser Dissertation wird der RNF-Komplex des acetogenen Bakteriums C. ljungdahlii und sein Einfluss auf den autotrophen Energiestoffwechsel untersucht. Für die genetische Manipulation von Genen, die mit dem RNF-Komplex und dem Energiestoffwechsel assoziiert sind, wurde eine CRISPR-Cas12a-Plasmid-Technik entwickelt und eingesetzt. Hiermit wurde erstmals eine vollständige Deletion der gesamten RNF-Komplex-Gene in C. ljungdahlii erreicht, welche die essenzielle Rolle dieses Komplexes für die Autotrophie bestätigte. Darüber hinaus konnte durch die Manipulation des Gens rseC, welches eine entscheidene Rolle in der transkriptionellen Kontrolle der RNF-Komplex-Genexpression spielt, ein neuer und bisher unbekannter Faktor für einen funktionellen RNF-Komplex in C. ljungdahlii aufgedeckt werden. Zudem liegt ein weiterer Fokus dieser Dissertation auf dem Nitrat-Stoffwechsel von C. ljungdahlii. Dieser wurde erst kürzlich beschrieben und ist auf noch nicht vollständig aufgeklärter Weise eng mit dem Energiestoffwechsel verbunden. Wenn Nitrat zusammen mit CO2 verwertet wird, entsteht zusätzliches ATP für den Metabolismus. Die potenzielle Nutzung dieses erhöhten ATP-Pools wird mit der Implementierung eines Stoffwechselwegs zur Produktion des Biopolymers Cyanophycin in dieser Dissertation angesprochen. Cyanophycin besteht hauptsächlich aus den Aminosäuren Arginin und Aspartat und könnte ein interessantes Vorstufenprodukt für die Lebensmittel- und Futtermittelindustrie sein. Des Weiteren wurden neue Erkenntnisse durch die Untersuchung der Nitratreduktion in selbstgebauten Bioreaktoren gewonnen. Bioreaktorexperimente mit acetogenen Bakterien sind essenziell, um das mikrobielle Verhalten unter kontrollierten Bedingungen, wie z.B. pH-Kontrolle, kontinuierliche Begasung oder kontinuierliche Mediumzufuhr, zu untersuchen. Experimentelle Daten aus Bioreaktoren sind ein wesentlicher Schritt für den Skalierungsprozess der Gasfermentation von acetogenen Bakterien und damit ein Schlüsselfaktor für die Anwendbarkeit dieser Technologie.Acetogenic bacteria have great potential for the use in modern industries to convert gaseous substrates, such as carbon monoxide (CO), carbon dioxide (CO2), and hydrogen (H2) into valuable biochemicals. Gas fermentation with acetogenic bacteria provides an alternative route with a reduced CO2 footprint when compared to existing petrochemical-based production processes. The main products of the acetogenic gas fermentation are acetate and ethanol, with the latter being a drop-in biofuel. Commercial gas fermentation plants with acetogenic bacteria are operated by the company Lanzatech, which underlines the industrial relevance of this technology. Although acetogenic bacteria are promising biocatalysts, their general metabolism is suffering from energy limitations. The main currency of the energy metabolism of a bacterial cell is ATP. The ATP is required for cell growth and for many metabolic pathways, such as those that can be used to produce valuable and industrial relevant products. All known acetogenic bacteria use the Wood-Ljungdahl pathway (WLP) to fix carbon. However, the WLP has no net ATP gain. One mole of ATP is invested to fix CO2, while one mole of ATP is regenerated through dephosphorylation of acetyl phosphate to acetate. The only way an acetogenic bacterium can acquire surplus ATP during autotrophy is based on membrane complexes such as the Rhodobacter Nitrogen Fixation-like complex (RNF complex). This complex generates a proton or sodium ion gradient across the bacterial membrane, which is then used by an ATPase to generate ATP. However, the ATP gain of this chemiosmotic mechanism is low. For instance, the acetogenic bacterium Clostridium ljungdahlii can generate a maximum of 0.63 ATP/mol H2 when growing with H2 and CO2. This small amount of ATP is just enough to enable growth and a functional metabolism. On the one hand, the low ATP gain is an advantage for biofuel production because electrons are predominantly used for the product rather than for biomass. On the other hand, the energy limitation in acetogenic bacteria is one of the highest burdens to overcome, and still limits the production of high-value and ATP-demanding fermentation products, which are required for a broad application of the acetogenic gas fermentation in industry. Therefore, more research with these microbes is highly required. This dissertation investigates the RNF complex of the acetogenic bacterium C. ljungdahlii and its impact on autotrophic energy metabolism. A CRISPR-Cas12a technique was developed and used for the genetic manipulation of genes that are associated with the RNF complex and the energy metabolism. A full deletion of all RNF complex genes was achieved in C. ljungdahlii for the first time and confirmed the essential role of this complex for autotrophy. Furthermore, the manipulation of the gene rseC, which has a potential role in the transcriptional control of the RNF-complex gene expression, unraveled a novel and unknown factor for a functional RNF complex in C. ljungdahlii. In addition, this dissertation focuses on nitrate metabolism of C. ljungdahlii, which was recently characterized in detail. Nitrate reduction in C. ljungdahlii is tightly connected to energy metabolism. The mechanism behind this is not understood yet. However, when C. ljungdahlii co-utilizes nitrate and CO2, more ATP is generated and available for its metabolism. The potential use of this increased ATP pool is addressed with the implementation of a pathway to produce the biopolymer cyanophycin. Cyanophycin mainly consists of the amino acids arginine and aspartate and might be a suitable precursor for the feed and food industry. In addition, new insights were made by studying the nitrate reduction of C. ljungdahlii in self-built bioreactors. Bioreactor experiments with acetogenic bacteria are essential to investigate the microbial behavior under controlled conditions (e.g., pH-control, continuous gassing, continuous medium feed). Experimental data from bioreactor experiments are one critical step for the upscaling process of gas fermentation of acetogenic bacteria, and therefore a key factor to show the applicability of this technology

Nitrate Feed Improves Growth and Ethanol Production of Clostridium ljungdahlii With CO2 and H-2, but Results in Stochastic Inhibition Events

The pH-value in fermentation broth is a critical factor for 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 experiments. 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 achieved in bioreactors. Here, we present a self-built, comparatively cheap, and user-friendly multiple-bioreactor system (MBS) consisting of six pH-controlled bioreactors at a 1-L scale. We tested the functionality of the MBS by cultivating the acetogen Clostridium ljungdahlii with CO2 and H2 at steady-state conditions (=chemostat). The experiments (total of 10 bioreactors) were addressing the two questions: (1) does the MBS provide replicable data for gas-fermentation experiments?; and (2) 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. On the one hand, our data showed high reproducibility for gas-fermentation experiments with C. ljungdahlii under standard cultivation conditions using the MBS. On the other hand, feeding nitrate as sole N-source improved growth by up to 62% and ethanol production by 2-3-fold. However, we observed differences in growth, and acetate and ethanol production rates between all nitrate bioreactors. We explained the different performances with a pH-buffering effect that resulted from the interplay between undissociated acetic acid production and ammonium production and because of stochastic inhibition events, which led to complete crashes at different operating times

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 in addition, contribute to understand the connection of nitrate reduction to energy metabolism

Reprogramming Acetogenic Bacteria with CRISPR-Targeted Base Editing via Deamination

Acetogenic bacteria are rising in popularity as chassis microbes for 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 and the potential for off-target events. Moreover, genes related to acetate and ethanol production were disrupted individually by installing premature STOP codons to reprogram carbon flux toward 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