Presentation Information
[1AFOB-12]Development of microbiota modification techniques: from synthetic biology to synthetic ecology
○Kenji Okano1, Tomoki Tanaka1, Miku Kato1, Hiroaki Iwaki1 (1. Kansai University (Japan))
Keywords:
Synthetic ecology,Bacteriophage
[Purpose] Various microorganisms have been isolated from nature, and their functions have been elucidated and applied to industry. In nature, microbes do not exist alone, but as a part of giant ecosystem formed by microbial interactions. For example, the human gut hosts about 1,000 species and 100 trillion microorganisms, forming a complex microbiome. Next-generation sequencing enables comprehensive microbial genome analysis, revealing microbiome composition. Comparing healthy and diseased individuals has helped identify microbes linked to disease onset and suppression. The next challenge is to reveal the role of these microbes and regulate microbiome function. However, analyzing the functional role of individual species within such a vast ecosystem is difficult. Just as gene disruption reveals gene function, could microbial inactivation clarify individual roles within the microbiome? This study aimed to develop a technology to selectively eliminate target microorganisms in microbiome.
[Method & Rsults] To meet this purpose, we focused on bacteriophages. Phages are viruses that infect bacteria in a host-specific manner, and their host specificity can be observed at the species or strain level. Additionally, phages replicate within the host and lyse it, making them "replicating bactericidal agents." Leveraging these properties, we succeeded to specifically kill only the target bacteria in an artificial microbiome consisting of Escherichia coli, Pseudomonas putida, Bacillus subtilis, and Lactiplantibacillus. plantarum. Further study revealed that phages can modify artificial microbiome consisting of seven Bacteroides species at species-specific manner.
To apply this approach to real microbiome, it is necessary to quickly obtain phages that target the desired microorganisms. The isolation of phages from environmental samples is somewhat random, and there is no guarantee that phages infecting the target microbes will be isolated. Interestingly, some phages integrate their genome into the host microorganism's genome, indicating that the blueprint for the phage infecting that microorganism is recorded within its genome. Therefore, if phages could be synthesized based on this blueprint, it would be possible to obtain them without searching for them. To demonstrate the feasibility of phage synthesis from prophage, the DNA fragments comprising λ phage were amplified from the genome of the λ lysogenized strain and they were introduced into a λ non-infective strain. By expressing homologous recombination enzymes in the λ non-infective strain, the DNA fragments are assembled in vivo and phage was synthesized form resulting full-length phage DNA. By using the artificially synthesized phages, the artificial microbiome could be successfully modified just like natural phages.
This phage synthesis technique was further utilized to implement non-natural functions in phages. Addition of genome-editing function inhibited the emergence of phage-resistant strain and swapping of tail-fiber genes between phages can alter the host-specificity of phages.
[Method & Rsults] To meet this purpose, we focused on bacteriophages. Phages are viruses that infect bacteria in a host-specific manner, and their host specificity can be observed at the species or strain level. Additionally, phages replicate within the host and lyse it, making them "replicating bactericidal agents." Leveraging these properties, we succeeded to specifically kill only the target bacteria in an artificial microbiome consisting of Escherichia coli, Pseudomonas putida, Bacillus subtilis, and Lactiplantibacillus. plantarum. Further study revealed that phages can modify artificial microbiome consisting of seven Bacteroides species at species-specific manner.
To apply this approach to real microbiome, it is necessary to quickly obtain phages that target the desired microorganisms. The isolation of phages from environmental samples is somewhat random, and there is no guarantee that phages infecting the target microbes will be isolated. Interestingly, some phages integrate their genome into the host microorganism's genome, indicating that the blueprint for the phage infecting that microorganism is recorded within its genome. Therefore, if phages could be synthesized based on this blueprint, it would be possible to obtain them without searching for them. To demonstrate the feasibility of phage synthesis from prophage, the DNA fragments comprising λ phage were amplified from the genome of the λ lysogenized strain and they were introduced into a λ non-infective strain. By expressing homologous recombination enzymes in the λ non-infective strain, the DNA fragments are assembled in vivo and phage was synthesized form resulting full-length phage DNA. By using the artificially synthesized phages, the artificial microbiome could be successfully modified just like natural phages.
This phage synthesis technique was further utilized to implement non-natural functions in phages. Addition of genome-editing function inhibited the emergence of phage-resistant strain and swapping of tail-fiber genes between phages can alter the host-specificity of phages.
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