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Background   Clostridium  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.    Results  We 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 N 6 -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.    Conclusions  The 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.    Electronic supplementary material  The online version of this article (10.1186/s13068-019-1448-1) contains supplementary material, which is available to authorized users.

Development of a metabolic pathway transfer and genomic integration system for the syngas-fermenting bacterium Clostridium ljungdahlii