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Development of a Metabolic Pathway Transfer and Genomic Integration Philipps et al. Biotechnol Biofuels (2019) 12:112 https://doi.org/10.1186/s13068-019-1448-1 Biotechnology for Biofuels RESEARCH Open Access Development of a metabolic pathway transfer and genomic integration system for the syngas-fermenting bacterium Clostridium ljungdahlii Gabriele Philipps1, Sebastian de Vries1,2 and Stefan Jennewein1* Abstract Background: Clostridium spp. can synthesize valuable chemicals and fuels by utilizing diverse waste-stream sub- strates, including starchy biomass, lignocellulose, and industrial waste gases. However, metabolic engineering in Clostridium spp. is challenging due to the low efciency of gene transfer and genomic integration of entire biosyn- thetic 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 cas- settes (> 5 kb) followed by the inducible activation of Himar1 transposase to promote integration. We established a conjugation protocol for the efcient generation of transconjugants using the Gram-positive origins of replica- tion repL and repH. We also investigated the impact of DNA methylation on conjugation efciency by testing donor constructs with all possible combinations of Dam and Dcm methylation patterns, and used bisulfte conversion and PacBio sequencing to determine the DNA methylation profle 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 confrmed by detecting enzyme proteo- typic 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 ofers 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. Keywords: Synthetic biology, Metabolic engineering, Acetogen, Conjugation, Inducible promoter, Himar1 transposase, Genomic integration, Syngas fermentation, Isopropanol, Acetone, Bio-fuels *Correspondence: [email protected] 1 Department for Industrial Biotechnology, Fraunhofer IME, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstr. 6, 52074 Aachen, Germany Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Philipps et al. Biotechnol Biofuels (2019) 12:112 Page 2 of 14 Introduction between Gram-negative and Gram-positive bacteria [13] Te microbial conversion of industrial waste streams and from bacteria to eukaryotes [14]. Tis method has into bulk chemicals and fuels is a promising approach to been used for DNA transfer to C. beijerinckii NCIMB reduce greenhouse gas emissions and ofset the efects 8052 (formerly Clostridium acetobutylicum NCIB 8052) of climate change [1]. Te bacterial genus Clostridium [15] and C. perfringens [16] and is the standard method includes several species that can produce valuable chemi- for DNA transfer to C. difcile [17]. Although conjuga- cal building blocks, such as acetone, organic acids, and tion is an efcient strategy for gene transfer to Clostrid­ various short/medium-chain alcohols [2]. For industrial- ium spp., the donor plasmid is susceptible to the host’s scale production, the feedstock is often starchy plant restriction–modifcation system [18]. Te decline in con- material that provides sugars as a carbon and energy jugation efciency caused by unmethylated recognition source, e.g., C. acetobutylicum can produce acetone using sites within the donor plasmid sequence has been dem- starch as a substrate [3]. However, one drawback is that onstrated for C. difcile [17]. Plasmid instability is also starch crops cultivated for industrial purposes compete an important issue, especially in an industrial context with food and feed crops. A more attractive feedstock where the bulk use of antibiotics to maintain selection is synthesis gas (syngas), a mixture of carbon monoxide, pressure is too expensive [19, 20]. Tese issues can be carbon dioxide, and hydrogen, which is emitted in large addressed by targeted genomic integration, as has been amounts e.g., by the steel industry and can be produced demonstrated using the ClosTron system based on group by the gasifcation of municipal waste [4]. C. ljungdahlii II introns [21]. Various transposon systems have also and C. autoethanogenum among others can utilize syn- been used to achieve non-targeted integration, includ- gas as a sole carbon and energy source [4, 5]. Tese spe- ing the conjugative transposon Tn916 [22–24] and non- cies use the reductive acetyl-CoA pathway, also known as conjugative transposons such as Tn5 [25, 26]. While most the Wood–Ljungdahl pathway, to reduce CO, CO2, and approaches use transposon mutagenesis as a means for H2 to the key metabolite acetyl-CoA, which is processed reverse genetics to disrupt genes [23–25, 27], it has also further into biomass and the main fermentation products been applied to integrate entire biosynthetic pathways ethanol and acetate [6–8]. into several Gram-negative bacteria species [26, 28]. A Te ability of certain Clostridium species to convert useful alternative is the mariner-type transposon Himar1 syngas into acetyl-CoA, a central biochemical building from Haematobia irritans [29], which has the capabil- block, could be exploited to produce additional chemi- ity to deliver biosynthetic pathways as large as 29.6 and cals by extending and modifying the corresponding 57.5 kb into the genome [30]. Te Himar1 transposase metabolic pathways. However, the genetic manipulation facilitates the random and even multiple integration of of Gram-positive bacteria remains much more challeng- DNA sequences fanked by ~ 27-bp inverted terminal ing than the amenable processes developed for model repeats (ITRs) without additional cofactors [29], making Gram-negative species such as Escherichia coli, due to the system applicable in any host including Clostridium the physiological and biochemical distinctions between spp. [31]. Te integration occurs by a ‘cut and paste’ these groups of bacteria. Successful metabolic engi- mechanism in the 2-bp TA target sequence of Himar1, neering in Gram-positive bacteria requires a strategy to which is duplicated upon insertion [32]. Te Staphylococ­ achieve the introduction of multiple transgenes encoding cus xylosus xylose-inducible promoter–repressor system appropriate enzymes and to ensure their stable integra- [33] is functional in C. acetobutylicum [34] and can be tion and expression, while overcoming the restriction– used to induce the transposase. After conjugal transfer of methylation system of the host. the donor plasmid, the addition of xylose induces Himar1 Multi-gene transformation in Gram-positive bacte- transposase expression and promotes the integration of ria can be achieved using traditional approaches such as one or more copies of the cassette bounded by the ITRs electroporation, but the efciency of gene transfer tends for increased transgene stability, with multiple integra- to be inversely proportional to the plasmid size, as shown tion events enhancing the gene dosage and ultimately the in other species such as E. coli [9, 10]. Optimal param- anticipated product yield. eters must be established empirically or systematically Here we describe the development of an efcient con- for each species [11]. An alternative strategy is conjuga- jugation and genomic integration protocol that allows the tion, a natural DNA transfer process that requires only transfer of large gene cassettes, e.g. coding for entire met- the presence of a dedicated origin of transfer (oriT) abolic pathways, to the host species C. ljungdahlii and on the donor plasmid, with all other functions (tra and thus facilitates the production of diferent bulk chemicals mob genes) provided in trans [12]. Conjugation is ver- and fuels from a syngas-fermenting bacterial strain. Te satile because it mobilizes large plasmids efciently and industrial scaling of such a process would allow the recy- can transfer DNA between species, including transfer cling of syngas, reducing the environmental harm caused Philipps et al. Biotechnol Biofuels (2019) 12:112 Page 3 of 14 Helper strain Donor strain Recipient strain Genomic integraon strain (E. coli) (E. coli) (C. ljungdahlii) (C. ljungdahlii) + 2% Xylose Genome
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