Bioproduction, bioprotection, and biocontainment in multi-Kingdom microbial systems with 3D spatial control

Engineered living materials (ELMs) are a class of hybrid materials that include engineered microbes encapsulated by a polymer matrix. The biotic and abiotic components define the ELMs design space and can be altered to improve performance and function. While current synthetic materials in the field display robust biocompatibility with both native and engineered living systems, we have a limited understanding of how to leverage 3D form factors to spatially organize and control microbial dynamics within the material. Motivated by this knowledge gap, we employed extrusion-based 3D printing to fabricate multi-material hydrogel constructs for the encapsulation of both single and dual-microbial systems. Core-shell cubic constructs enabled the spatial organization of a constitutive multi-kingdom system of levodopa (L-DOPA)-producing E. coli and betaxanthins-producing S. cerevisiae. This deliberate spatial organization in 3D materials can introduce precise control over bioproduction, bioprotection, and biocontainment, features that are critical to the efficacy of current ELMs. The relative spatial organization of the organisms, as well as the surface area-to-volume ratio were investigated to determine how these design elements impact microbial behavior (metabolite production, growth, expression and cell distribution) over time. We demonstrated that F127-BUM core-shell geometries enable the hierarchical 3D printing of multi-kingdom constructs, offering customizable control over bioproduction, bioprotection, and biocontainment. With the optimization of these core-shell structures for continuous bioproduction, these ELMs could be deployed as compact, sustainable bioreactors in remote environments.