The climate crisis
Planet earth is facing a rapidly accelerating climate change, caused by the emission of fossil fuel combustion[1]. The thereby generated greenhouse gases promote the temperature increase of the planet and the perturbation of the complex climate systems on earth. In order to ensure a habitable earth for the future, the international community took measures to counteract this in the Paris climate act[2]. Even though these actions have a noticeable effect on CO2 emissions, no technology has been developed yet which reduces CO2 emissions drastically enough to meet the limit of temperature increase set by the UN.
>Therefore, new technologies are required to decrease the immense CO2 emissions.
As biologists, we learn from early on that nature created sophisticated pathways for CO2 capture. The most prominent example is photosynthesis. However, there are several other ways of CO2 fixation in many domains of life. The Wood-Ljungdahl pathway is the only linear CO2 fixation pathway, but has not been studied intensively in the past due to low yields on an industrial scale. However, a paper caught our attention, stating that some high yield biofuel producing bacteria show genetic remnants of this pathway and might be able to use it as the main metabolic route when the missing fragments are provided[3]. We therefore teamed up with the department for Genomic and Applied Microbiology of the University of Göttingen, as they specialized on the identification and sequencing of new clostridial bacteria, the hosts of the Wood-Ljungdahl pathway.
To get a thorough understanding of the pathway and the significance of our project, let’s first introduce the Wood-Ljungdahl pathway.
The Wood-Ljungdahl pathway is the only linear carbon fixation pathway and combines the ability of carbon fixation from the atmosphere with the energy production by a proton gradient. Already in 1932 the first acetogenic Clostridia were discovered and their ability to turn H2 and CO2 first into acetyl-CoA and then into acetic acid was shown[5]. Acetogenic bacteria are not the only bacterial class with this pathway, however, they are the only class which are utilizing the full potential of carbon fixation in the form of CO2 and producing energy by hydrogen ions[6].
In general, the pathway is divided into two branches, which are fused together in the final step. In the methyl-branch CO2 is fixated in the form of formate by the addition of hydrogen. Afterwards, formate is bound to tetrahydrofolate (THF) and processed via a series of enzymatic reactions. Eventually, the methyl group is transferred to a corrinoid-iron-sulfur protein which gains one methyl-group from one CO2. Via the carbonyl-branch CO2 is converted to CO to generate a free carbonyl-group. The final enzyme of each of these branches form one dimer of the acetyl-CoA synthetase (ACS) and the CO dehydrogenase (CODH)-complex. At this point, the carbonyl-group and the methyl-group are fused and transferred onto coenzyme A to form acetyl-CoA. In most acetogenic bacteria acetyl-CoA is then transformed into acetate, giving them their name[7].
As it can be seen from figure 1, the methyl- and carbonyl-group generation require several steps of hydrogen transfer by cofactors and consumes ATP. However, no ATP is regained, giving the pathway a negative energy balance. In order to allow growth based on the Wood-Ljungdahl pathway, bacteria have developed highly sophisticated energy production systems, utilizing ferredoxin and membrane bound enzymes coupled to the pathway to generate energy from a proton gradient[8].
In the genome of Clostridia, the pathway genes are located in one or two large clusters.
Some acetogenic bacteria, most prominently Clostridium autoethanogenum, were used for industrial application. However, most of them fail due to very low yields of valuable products[9]. It is therefore of great interest to combine the capability of carbon fixation with high yield production.
The energy conservation mechanisms (Ech- or, Rnf-complex, electron bifurcation) as mentioned above are highly complex in these bacteria and only first attempts were undergone to transform it to another organism[10]. Our approach of transforming the Wood-Ljungdahl pathway was therefore limited to industrial biofuel producers possessing these systems. A recent paper indicates that in some biofuel producing Clostridia, genetic remnants of the Wood-Ljungdahl pathway and the energy conservation mechanisms are present. However, this is so far the only study showing this and additionally merely a superficial analysis[11].
For the aimed transformation step, we had to identify a donor organism and a recipient to ensure a successful expression. As a donor system we relied on the most thoroughly studied organisms as this would provide a maximum of available information. Eventually, we focused on Clostridium ljungdahlii (C. ljungdahlii) and Acetobacterium woodii (A. woodii) as donor organisms as they are well studied, have a functional WLP and show a suitable genomic organization[12]. For C. ljungdahlii, the WLP is organized in one 18 kb cluster while it is split into two clusters for A. woodii. As we are aiming to restore the fragmented pathway of the recipient strain, we choose two different organisms as potential donor systems in order to increase the chance of compatibility of the enzymes.
For the first steps of cloning we had to identify target genes which had to be cloned. This was in particular difficult as only a single paper showed superficial presence of important pathway genes. To get started we decided for a single gene to keep the size for the cloning procedures small. For this purpose we choose the acetyl-CoA synthetase. This enzyme is a key enzyme in fusing the carbonyl- and the methyl-branch of the Wood-Ljungdahl pathway and it was indicated to be the only gene absent in C. beijerinckii[11]. However, by browsing the genome, it was foreseeable that more proteins would likely be missing. We therefore amplified the acsB gene (acetyl-CoA synthetase) from the genome of A. woodii and C. ljungdahlii by PCR. As the proteins of the Wood-Ljungdahl pathway directly interact and important proteins as the CODH and ACS form complexes, we aimed to eventually transform the entire pathway into a new target organism. Due to the difficult genetic accessibility of Clostridia, we decided to order synthesized gene fragments. The detailed design of these complexes can be reviewed in the results section.
Cloning in Clostridia is not a trivial task. Due to the low GC content of the genome, even simple techniques such as PCR can oppose difficulties. Even synthesizing the genes by commercial companies was difficult. Furthermore, we had to identify a plasmid system which can be expressed in E. coli for plasmid amplification and works additionally in Clostridia. As shown by iGEM Nottingham 2019, the ClosTron plasmid system fulfills these requirements. Since only a few groups world wide work on clostridial cloning, we chose three promoters from iGEM Nottingham as they were able to show functional expression and we therefore could omit the task of identifying new promoters. We choose the lactose inducible promoter since we could transform the plasmid without induction and therefore decrease the chance of toxicity. In addition, we used the thiolase and ferredoxin promoters as they were shown as very strong promoters for the final expression.
As target organism, the bacterium which should finally express the Wood-Ljungdahl pathway, we were looking for four central aspects: The organism should be well studied, it should produce biofuel, it should contain an energy conservation system like Rnf, Ech, or electron bifurcation, and it should contain at least fragments of the Wood-Ljungdahl pathway. The well-studied C. beijerinckii provided all of these prerequisites and is closely related to the donor organism C. ljungdahlii. Furthermore, it was the first and so far only organism in which a partial existence of the Wood-Ljungdahl pathway was described[11]. During our research, we then got in touch with industry related researchers (for details see: Human practices) who recommended Clostridium saccharoperbutylacetonicum (C. saccharoperbutylacetonicum) to us. This rather new organism made its way into industrial biofuel production due to its high butanol yield[13]. By further investigation of the genome, we found that C. saccharoperbutylacetonicum provides the majority of Wood-Ljungdahl genes as well. Additionally, it possesses an Rnf energy conservation system, which is the same that C. ljungdahlii utilizes. We therefore switched to this organism to increase both the cloning success probability and the final butanol production.
Finding a suitable medium for Clostridia is not as simple as it is for other well established organisms such as E. coli. Clostridia require for growth a wide range of metal-cofactors and vitamins, which need to be supplied in form of trace element solutions. Furthermore, the appropriate medium needs to be adapted to the individual Clostridium and the purpose for which it is used. For normal cultivation of sugar metabolizing Clostridia such as C. saccharoperbutylacetonicum, complex media such as Clostridial Growth Medium (CGM) are suitable. When growth on CO2 and H2 is desired, a suitable minimal medium, which does not contain any sugars but provides the required amount of trace metals and vitamins, is required. Furthermore, as transformations into Clostridia proved to be challenging, a minimal medium was applied after transformation to decrease the growth speed and therefore restrict toxic effects.
Sources
[1] IPCC (2014): Ar5 climate change 2014: Mitigation of climate change. available from: https://www.ipcc.ch/report/ar5/wg3/ [accessed 10/10/2022].
[2] Umweltbundesamt (2022): IPCC-Bericht: Sofortige globale Trendwende nötig [online]. available from: https://www.umweltbundesamt.de/themen/ipcc-bericht-sofortige-globale-trendwende-noetig [accessed 30/08/2022].
[3] Sandoval-Espinola, W. J.; Chinn, M. S.; Thon, M. R.; Bruno-Barcena, J. M. (2017): Evidence of mixotrophic carbon-capture by n-butanol-producer Clostridium beijerinckii. Sci Rep 7, 12759.
[4] Ragsdale, S. W. (2008): Enzymology of the wood-ljungdahl pathway of acetogenesis. Annals of the New York Academy of Sciences, 1125(1),129-136. DOI: 10.1196/annals.1419.015.
[5] Fischer, F. (1932): Biologische Gasreaktionen. II. Uber die Bildung von Essigsaure bei der Biologischen Umsetzung von Kohlenoxyd und Kohlensa mit Wasserstoff zn Methan. Biochem. Z 245, 2-12.
[6] Fuchs, G. (1994): Variations of the Acetyl-CoA Pathway in Diversely Related Microorganisms That Are Not Acetogens. in Acetogenesis (ed. Drake, H. L.) 507-520 (Springer US). DOI: 10.1007/978-1-4615-1777-1_19
[7] Köpke, M.; Straub, M.; Dürre, P. (2013): Clostridium difficile Is an Autotrophic Bacterial Pathogen. PLOS ONE, 8(4), DOI: 10.1371/journal.pone.0062157.
[8] Schuchmann, K.; Müller, V. (2014): Autotrophy at the thermodynamic limit of life: a model for energy conservation in acetogenic bacteria. Nat. Rev. Microbiol. 12, 809-821. DOI: 10.1038/nrmicro3365.
[9] Katsyv, A.; Müller, V. (2020): Overcoming Energetic Barriers in Acetogenic C1 Conversion. Frontiers in Bioengineering and Biotechnology. DOI: 10.3389/fbioe.2020.621166.
[10] Schwarz, F. M.; Müller, V. (2020): Whole-cell biocatalysis for hydrogen storage and syngas conversion to formate using a thermophilic acetogen. Biotechnol Biofuels 13, 32. DOI: 10.1186/s13068-020-1670-x.
[11] Sandoval-Espinola, W. J.; Chinn, M. S.; Thon, M. R.; Bruno-Barcena, J. M. (2017): Evidence of mixotrophic carbon-capture by n-butanol-producer Clostridium beijerinckii. Sci Rep 7, 12759. DOI: 10.1038/s41598-017-12962-8;
[12] Wiechmann, A., Müller, V. (2017): Synthesis of Acetyl-CoA from Carbon Dioxide in Acetogenic Bacteria. In: Geiger, O. (eds) Biogenesis of Fatty Acids, Lipids and Membranes. Handbook of Hydrocarbon and Lipid Microbiology . Springer, Cham. DOI: 10.1007/978-3-319-43676-0_4-2.
[13] Wang, P.; Feng, J.; Guo, L.; Fasina, O.; Wang, Y. (2019): Engineering Clostridium saccharoperbutylacetonicum for high level Isopropanol-Butanol-Ethanol (IBE) production from acetic acid pretreated switchgrass using the CRISPR-Cas9 system. ACS Sustainable Chemistry & Engineering 7 (21), 18153-18164. DOI: 10.1021/acssuschemeng.9b05336.