A toolbox for optimized resource allocation in unicellular and multicellular bioproduction systems

Tbox4BioProd's objectives are to provide a toolbox of broad-based genetic parts and devices for genome engineering, dynamic regulation, cross-species compatibility, biosensing, establishing and maintaining microbial consortia, and long-term evolutionary stability. The project is divided into 5 complementary tasks, 4 of which started in 2023.

Activities and results:

In task 1, we aimed to develop standardized genetic tools and protocols for genome engineering that are marker-less, amenable to multiplexing, and cross-species compatible, enabling easy adaptation for new non-model organisms of interest. In 2023, we finalized the setup of a marker-less, CRISPR-based multiplex genome engineering strategy in Bacillus subtilis, which we anticipate to be cross-species compatible.

In task 2, we aimed to develop genetic modules for dynamic feedback control to reduce cellular burden of genetic circuits and metabolic pathways production. The tools developed will have broad host-range compatibility to facilitate portability between different species of biotechnological interest, thus reducing the prototyping time and accelerating applications.

In task 3, we aimed to develop inducible genetic switches responding to inducers of interest. We tested various libraries of ribosome binding sites and degradation tags and performed screens to identify non-leaky, switchable variants. This work is under finalization. We also developed cross-species inducible promoters responding to IPTG and operating in B. subtilis and Lactobacillus gasseri (DOI: 10.1021/acssynbio.3c00438).

In task 4, we aimed to engineer mono-species and multispecies co-dependent communities for improved production. We evaluated B. subtilis synthetic consortia exhibiting obligate mutualistic interactions based on amino-acid cross-feeding. Thirty-six auxotrophic B. subtilis strains were grown in pairwise cocultures, and 70 consortia showed a biomass accumulation greater than 50% of that of the wild type. A directed evolution experiment was performed to optimize the fitness of one mono-species consortium in continuous culture for 380 generations. Variants with increased fitness were selected and are being characterized to uncover the mechanisms responsible for the division of labor and cross-feeding improvement.