Abstract
BackgroundClostridium spp. can synthesize valuable chemicals and fuels by utilizing diverse waste-stream substrates, including starchy biomass, lignocellulose, and industrial waste gases. However, metabolic engineering in Clostridium spp. is challenging due to the low efficiency of gene transfer and genomic integration of entire biosynthetic pathways.ResultsWe have developed a reliable gene transfer and genomic integration system for the syngas-fermenting bacterium Clostridium ljungdahlii based on the conjugal transfer of donor plasmids containing large transgene cassettes (> 5 kb) followed by the inducible activation of Himar1 transposase to promote integration. We established a conjugation protocol for the efficient generation of transconjugants using the Gram-positive origins of replication repL and repH. We also investigated the impact of DNA methylation on conjugation efficiency by testing donor constructs with all possible combinations of Dam and Dcm methylation patterns, and used bisulfite conversion and PacBio sequencing to determine the DNA methylation profile of the C. ljungdahlii genome, resulting in the detection of four sequence motifs with N6-methyladenosine. As proof of concept, we demonstrated the transfer and genomic integration of a heterologous acetone biosynthesis pathway using a Himar1 transposase system regulated by a xylose-inducible promoter. The functionality of the integrated pathway was confirmed by detecting enzyme proteotypic peptides and the formation of acetone and isopropanol by C. ljungdahlii cultures utilizing syngas as a carbon and energy source.ConclusionsThe developed multi-gene delivery system offers a versatile tool to integrate and stably express large biosynthetic pathways in the industrial promising syngas-fermenting microorganism C. ljungdahlii. The simple transfer and stable integration of large gene clusters (like entire biosynthetic pathways) is expanding the range of possible fermentation products of heterologously expressing recombinant strains. We also believe that the developed gene delivery system can be adapted to other clostridial strains as well.
Highlights
The microbial conversion of industrial waste streams into bulk chemicals and fuels is a promising approach to reduce greenhouse gas emissions and offset the effects of climate change [1]
We describe the development of an efficient conjugation and genomic integration protocol that allows the transfer of large gene cassettes, e.g. coding for entire metabolic pathways, to the host species C. ljungdahlii and facilitates the production of different bulk chemicals and fuels from a syngas-fermenting bacterial strain
The availability of a complete C. ljungdahlii genome sequence will facilitate this process [35], several technical challenges must be addressed before efficient metabolic engineering is possible, including the low efficiency of transformation with large gene cassettes [36]
Summary
The microbial conversion of industrial waste streams into bulk chemicals and fuels is a promising approach to reduce greenhouse gas emissions and offset the effects of climate change [1]. The feedstock is often starchy plant material that provides sugars as a carbon and energy source, e.g., C. acetobutylicum can produce acetone using starch as a substrate [3]. C. ljungdahlii and C. autoethanogenum among others can utilize syngas as a sole carbon and energy source [4, 5] These species use the reductive acetyl-CoA pathway, known as the Wood–Ljungdahl pathway, to reduce CO, CO2, and H2 to the key metabolite acetyl-CoA, which is processed further into biomass and the main fermentation products ethanol and acetate [6,7,8]. Clostridium spp. can synthesize valuable chemicals and fuels by utilizing diverse waste-stream substrates, including starchy biomass, lignocellulose, and industrial waste gases. Metabolic engineering in Clostridium spp. is challenging due to the low efficiency of gene transfer and genomic integration of entire biosynthetic pathways
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