Sequence-guided approaches for the cultivation and engineering of the gut microbiome

Jérémy Christophe Daniel Armetta

Research output: Book/ReportPh.D. thesis

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Abstract

Microbial life can be found everywhere, from the bottom of the oceans to the top of mountains. Consequently, humans constantly interact with incredibly complex communities of bacteria, archaea, fungi, and viruses that have taken up residence on the skin, in oral cavities or in the gut. Generally referred to as the human microbiota or microbiome, these microorganisms protect the body and influence its development,
neurology, immunity, or metabolism at each stage of life. One of the most promising options to efficiently intervene in the gut microbiome lies in the use of microbial cell therapies, in particular next-generation probiotics. Unfortunately, the massive taxonomic, functional, and phenotypic diversity of the gut microbiome remains hardly accessible due to laborious, expensive, and time-consuming cultivation processes. The difficulty to target and access individual strains impedes not only the resolution of causal links between strains/communities and disease states, but also the discovery of novel strains for microbiome therapies. As a result, current solutions focus on a handful of strains with limited therapeutic effects and features, hampering the scope and efficiency of live therapeutics.

This PhD thesis contributes to the fields of engineering and cultivation of the gut microbiome by addressing two limitations: the translatability of in vitro performance for advanced microbial therapeutics to a robust in vivo efficacy, and the isolation of microorganisms displaying a beneficial characteristic for the development of nextgeneration probiotics. To achieve a better translatability of performances in vivo, we
leveraged metagenomics, machine learning and flow cytometry to build the Schantzetta promoter library. This library is spanning a wide expression range with a high degree of robustness in both in vitro and in vivo conditions. Sequence-guided approaches are further demonstrated to improve upon the enrichment, and isolation of gut microbes in an integrated design. First, I introduce a method to selectively enrich
for taxa and functions of interest combining whole metagenomics together with culturomics. Using insights from the sequencing, we tailored growth conditions combining selective pressures to foster the growth of target microorganisms. Second, I present a streamlined high-throughput isolation pipeline that seamlessly integrates affordable laboratory automation, metagenomics, and anaerobic cultivation to support the recovery and identification of gut microbes. Third, I demonstrate the benefits of additive manufacturing and CNC machining to build three devices enabling a cost-efficient and high-throughput cultivation of anaerobes.

Overall, the work presented in this thesis illustrates the use of sequence-guided approaches for the development of next-generation probiotics and demonstrate direct applications for the cultivation, isolation, and engineering of the gut microbiome.
Original languageEnglish
PublisherTechnical University of Denmark
Number of pages259
Publication statusPublished - 2022

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