Periodic Reporting for period 2 - PRO_PHAGE (Impact and interaction of prophage elements in bacterial host strains of biotechnological relevance)
Reporting period: 2019-07-01 to 2020-12-31
By combining an explorative approach with subsequent molecular analysis of selected targets, PRO_PHAGE will deliver novel insights into this genetic resource and will reveal the risks and potential for metabolic engineering by pursuing four major objectives. 1) Based on a comprehensive bioinformatic analysis, the impact of phage elements is studied by HT phenotyping of selected strains. 2) A second objective addresses the regulatory interaction of the phage and the host by focusing on host-encoded xenogeneic silencing proteins and their role in the integration of foreign DNA. 3) The spontaneous activation of phage elements is studied at the genomic scale to decipher molecular triggers and their impact on host gene expression. For this purpose, we already developed a novel workflow combining fluorescence-activated cell sorting and NGS enabling the analysis of microbial population dynamics at unprecedented resolution. 4) Finally, the insights obtained are benchmarked for metabolic engineering approaches in order to generate robust and flexible chassis strains for industrial production.
growth experiment. Our study presents the prophage-free variant of V. natriegens as a promising platform strain for future biotechnological applications.
Following on the question of how ‘foreign’/viral genetic material is tolerated by the host and how it is integrated into host regulatory networks, we systematically addressed the function of xenogeneic silencing (XS) proteins. In actinobacteria, Lsr2-like nucleoid-associated proteins function as XS of horizontally acquired genomic regions, including viral elements, virulence gene clusters in Mycobacterium tuberculosis, and genes involved in cryptic specialized metabolism in Streptomyces species. Consequently, a detailed mechanistic understanding of Lsr2 binding in vivo is relevant for the use as a potential drug target and for the identification of novel bioactive compounds. In our study, we followed an in vivo approach to investigate the rules underlying xenogeneic silencing and countersilencing of the Lsr2-like XS CgpS from Corynebacterium glutamicum. Our results demonstrated that CgpS distinguishes between self and foreign DNA by recognizing a distinct drop in GC profile in combination with a short, sequence-specific motif at the nucleation site. Following a synthetic counter-silencer approach, we studied the potential and constraints of transcription factors to counteract CgpS silencing, thereby facilitating the integration of new genetic traits into host regulatory networks.
The principle of XS was further harnessed for the establishment of inducible expression systems representing key modules in regulatory circuit design and metabolic engineering approaches. However, established systems are often limited in terms of applications due to high background expression levels and inducer toxicity. In a recent study of this project, we successfully established a promoter library based on the principle of XS and counter-silencing. For selected candidates, background expression levels were confirmed to be significantly reduced in comparison to established heterologous expression systems. Finally, this principle was implemented in a metabolic toggle switch allowing the dynamic redirection of carbon flux between biomass and L-valine production in a C. glutamicum production strain.
Further preliminary results of this project highlight bacteriophages and prophage elements as a rich source for the identification of novel antibacterial proteins as well as for the establishment of efficient molecular tools targeting key regulatory hubs of the host cell. Therefore, we expect further interesting results with medical and biotechnological relevance in the remaining time of the project.