Lessons from Clostridial Genetics: Toward Engineering Acetogenic Bacteria

Lee, Joungmin

doi:10.1007/s12257-021-0062-9

Cited by 4 publications

(2 citation statements)

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“…Next, we determined the presence of restriction endonuclease activities, which are known to be the major barriers to DNA transfer in various clostridial strains [ 24 – 26 ]. However, no evidence of DNA digestion was observed when pKLJM005 was treated with the crude extract of AWRP cells and commercially available restriction enzyme buffers, indicating that recalcitrance of AWRP may not result from the existence of typical type II restriction endonucleases (data not shown).…”

Section: Resultsmentioning

confidence: 99%

Development of a genetic engineering toolbox for syngas-utilizing acetogen Clostridium sp. AWRP

Kwon,

Lee,

Kwon

et al. 2024

Microb Cell Fact

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Background Clostridium sp. AWRP (AWRP) is a novel acetogenic bacterium isolated under high partial pressure of carbon monoxide (CO) and can be one of promising candidates for alcohol production from carbon oxides. Compared to model strains such as C. ljungdahlii and C. autoethanogenum, however, genetic manipulation of AWRP has not been established, preventing studies on its physiological characteristics and metabolic engineering. Results We were able to demonstrate the genetic domestication of AWRP, including transformation of shuttle plasmids, promoter characterization, and genome editing. From the conjugation experiment with E. coli S17-1, among the four replicons tested (pCB102, pAMβ1, pIP404, and pIM13), three replicated in AWRP but pCB102 was the only one that could be transferred by electroporation. DNA methylation in E. coli significantly influenced transformation efficiencies in AWRP: the highest transformation efficiencies (102–103 CFU/µg) were achieved with unmethylated plasmid DNA. Determination of strengths of several clostridial promoters enabled the establishment of a CRISPR/Cas12a genome editing system based on Acidaminococcus sp. BV3L6 cas12a gene; interestingly, the commonly used CRISPR/Cas9 system did not work in AWRP, although it expressed the weakest promoter (C. acetobutylicum Pptb) tested. This system was successfully employed for the single gene deletion (xylB and pyrE) and double deletion of two prophage gene clusters. Conclusions The presented genome editing system allowed us to achieve several genome manipulations, including double deletion of two large prophage groups. The genetic toolbox developed in this study will offer a chance for deeper studies on Clostridium sp. AWRP for syngas fermentation and carbon dioxide (CO2) sequestration.

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Section: Resultsmentioning

confidence: 99%

Development of a genetic engineering toolbox for syngas-utilizing acetogen Clostridium sp. AWRP

Kwon,

Lee,

Kwon

et al. 2024

Microb Cell Fact

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“…For example, acetone and isopropanol production have been realised in C. autoethanogenum (Liew et al, 2022), and efficient 3-hydroxybutyrate (3-HB) production was accomplished in C. ljungdahlii (Lo et al, 2022). However, genomic manipulation of acetogens is still challenging (Charubin et al, 2018;Lee, 2021), and the production of ATP-demanding products remains difficult to achieve due to the energy-limited metabolism of these microorganisms.…”

Section: Introductionmentioning

confidence: 99%

Synthetic microbial communities for syngas-driven odd-chain elongation

Parera Olm

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The last century has seen an unprecedented rise in carbon dioxide (CO 2 ) emissions into the atmosphere, causing average global temperatures to rise to levels never seen before (Intergovernmental Panel on Climate Change [IPCC], 2018[IPCC], , 2023. Human activities with a large environmental footprint such as burning fossil fuels, deforestation, and industrial processes have contributed to this increase. The rise in CO 2 emissions is recognized as a major driver of climate change, which has already resulted in profound societal and environmental impacts. Among others, these include rising sea levels, intensified heatwaves, diminished rainforests, heightened risks of species extinction, and compromised food security. Unfortunately, current projections indicate that the emission reduction commitments outlined by countries under the 2015 Paris Climate Agreement (United Nations Framework Convention on Climate Change, 2015) are unlikely to be fulfilled (Climate Action Tracker).Considering the projected growth of the human population in the next decades (United Nations Department of Economic and Social Affairs, Population Division, 2022), it is expected that, if no actions are taken, the future scenario regarding climate change will worsen, although it is important to note that anthropogenic emissions alone are not the sole contributor (IPCC, 2018(IPCC, , 2023. Natural climate fluctuations and geological events also play a role in climate change. Nevertheless, population growth is a factor that strongly influences the demand for resources and energy consumption, making anthropogenic emissions a key factor in the equation. It is widely recognized that humanity is exceeding the planet's ecological limits (Global Footprint Network). This concept is often referred to as ecological overshoot, where human demand for resources and ecological services exceeds the regenerative capacity of Earth.It is therefore crucial to implement sustainable practices across all sectors (IPCC, 2023). These include transitioning to cleaner energy sources, improving forest-and crop-land management, and reducing food waste, all attainable strategies that should be incentivised and supported with effective policies. In this thesis, we address one of these necessary practices: the sustainable production of chemicals.11 General introduction and Thesis outline The syngas fermentation era and the rise of acetogensRecent years have witnessed a growing interest on C1 feedstocks (i.e., syngas, methanol, formate) for microbial conversion to biochemicals. This approach holds promise in reducing reliance on fossil fuels and minimizing carbon emissions. Furthermore, advancements in biotechnology and microbial engineering have expanded our understanding of C1 utilization pathways, paving the way for innovative approaches in converting these feedstocks into valuable chemicals (

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