Philipps Gabriele, de Vries Sebastian, Jennewein Stefan
Department for Industrial Biotechnology, Fraunhofer IME, Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstr. 6, 52074 Aachen, Germany.
2Present Address: Department of Intensive Care Medicine, University Hospital, RWTH Aachen University, Pauwelsstr. 30, 52074 Aachen, Germany.
Biotechnol Biofuels. 2019 May 8;12:112. doi: 10.1186/s13068-019-1448-1. eCollection 2019.
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 spp. is challenging due to the low efficiency of gene transfer and genomic integration of entire biosynthetic pathways.
We have developed a reliable gene transfer and genomic integration system for the syngas-fermenting bacterium based on the conjugal transfer of donor plasmids containing large transgene cassettes (> 5 kb) followed by the inducible activation of transposase to promote integration. We established a conjugation protocol for the efficient generation of transconjugants using the Gram-positive origins of replication and . 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 genome, resulting in the detection of four sequence motifs with N-methyladenosine. As proof of concept, we demonstrated the transfer and genomic integration of a heterologous acetone biosynthesis pathway using a 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 cultures utilizing syngas as a carbon and energy source.
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 . 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.
某些菌种能够通过利用多种废物流底物来合成有价值的化学品和燃料，这些底物包括淀粉生物质、木质纤维素和工业废气。然而，由于基因转移效率低以及整个生物合成途径的基因组整合困难，对这些菌种进行代谢工程改造具有挑战性。
我们基于含有大转基因盒（>5 kb）的供体质粒的接合转移，随后诱导转座酶激活以促进整合，开发了一种用于合成气发酵细菌的可靠基因转移和基因组整合系统。我们建立了一种接合方案，用于使用革兰氏阳性复制起点和高效产生接合子。我们还通过测试具有Dam和Dcm甲基化模式所有可能组合的供体构建体，研究了DNA甲基化对接合效率的影响，并使用亚硫酸氢盐转化和PacBio测序来确定该菌种基因组的DNA甲基化谱，从而检测到四个带有N - 甲基腺苷的序列基序。作为概念验证，我们展示了使用由木糖诱导型启动子调控的转座酶系统转移和基因组整合异源丙酮生物合成途径。通过检测酶蛋白型肽以及利用合成气作为碳源和能源的培养物中丙酮和异丙醇的形成，证实了整合途径的功能。
所开发的多基因递送系统为在具有工业应用前景的合成气发酵微生物中整合和稳定表达大型生物合成途径提供了一种通用工具。大基因簇（如整个生物合成途径）的简单转移和稳定整合正在扩大异源表达重组菌株可能发酵产物的范围。我们还认为，所开发的基因递送系统也可适用于其他梭菌菌株。
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