報告題目：Development of platform technologies which facilitate construction of microbial cell factories

報  告 人： Akihiko Kondo教授,日本神戶大學

報告時間： 10月27日14:00-16:00

報告地點：閔行校區生物藥學樓3號樓105

聯  系 人：趙心清  xqzhao@sjtu.edu.cn

報告摘要🔼：

We have developed the platform technologies such as metabolic pathway design systems, genome editing, a large gene cluster synthesis system and multi-omics analysis systems and are going to integrate to set up the automated systems for efficient construction of microbial cell factories.

As pathway design tools, have developed the computational platform, such as M-path and BioProV, to explore synthetic metabolic pathways including putative enzymatic reactions and compounds [1]. M-path and BioProV are iterative random algorithm, which makes efficient use of chemical and enzymatic databases to find potential synthetic metabolic pathways and can readily control the search space and perform well compared with exhaustively enumerating possible pathways.

We have developed a genome editing tool that enables targeted point mutagenesis by tethering the DNA deaminase activity to nuclease-deficient CRISPR/Cas9 system, [2, 3]. An AID orthologue PmCDA1 was attached to nuclease-deficient mutant of Cas9 (D10A and H840A) to perform highly efficient and target-specific nucleotide editing. This hybrid system, termed Target-AID, induced cytosine point mutation in 3-5 bases range at the distal site within target sequence. The toxicity associated with Cas9 has been greatly diminished, enabling application of this technique to wider range of organisms including yeast, bacteria, animals and plants.

We have also developed an efficient DNA assembly method, namely, Ordered Gene Assembly in B. subtilis (OGAB) method. OGAB method can assemble more than 50 DNA fragments in one-step using B. subtilis [4]. Thanks to this high processability, even in construction of long DNA (~100 kb), material DNA fragments can be kept in chemical DNA synthesis-friendly and sequencing-friendly small size (< 2 kb). Since there is no in vitro DNA synthesis step that may cause unexpected mutation(s), long DNA by OGAB method using sequence-confirmed material DNA thus contains essentially no mutation. We are now constructing user friendly DNA system by integrating new automation system, such like a liquid handling robot that is specifically developed for OGAB method

These technologies might lead to new pipelines through which functional genomes are cleated with much faster speed to construct microbial cell factories to produce variety of biofuels and chemicals.

[1] Bioinformatics, 31(6), 905 (2015).   [2] Science, 353 (6305), aaf8729 (2016).

[3] Nature Biotechnology, 35(5), 441(2017).    [4] Sci. Rep. 5, 10655 (2015).