Bacterial biofilms can be programmed to produce living materials with self-healing and evolvable functionalities. However, the wider use of artificial biofilms has been hindered by limitations on processability and functional protein secretion capacity. We describe a highly flexible and tunable living functional materials platform based on the TasA amyloid machinery of the bacterium Bacillus subtilis. We demonstrate that genetically programmable TasA fusion proteins harboring diverse functional proteins or domains can be secreted and can assemble into diverse extracellular nano-architectures with tunable physicochemical properties. Our engineered biofilms have the viscoelastic behaviors of hydrogels and can be precisely fabricated into microstructures having a diversity of three-dimensional (3D) shapes using 3D printing and microencapsulation techniques. Notably, these long-lasting and environmentally responsive fabricated living materials remain alive, self-regenerative, and functional. This new tunable platform offers previously unattainable properties for a variety of living functional materials having potential applications in biomaterials, biotechnology, and biomedicine.

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We herein introduce a strategy that leverages and integrates the attributes of whole-cell catalysis with enhanced stability of extracellular immobilized enzymes for rapid, robust, recyclable enzyme cascade reactions in a scalable fashion. We demonstrated the utility of the integrative strategy for catalytic synthesis of trehalose from soluble starch with two-step sequential bioconversion enzymatic reactions, implemented by coupling the enzymatic immobilization of β-amylase (BA), based upon E. coli biofilm curli display technique, with intracellular expression of trehalose synthase (TreS) within the same cells. This integrative strategy, compared with a strategy based on cells coupled with isolated BA, enabled a 103.5 ± 18.7% increase in the maximum trehalose formation rate by efficiently reducing the average distance of BA to intracellluar TreS enzyme. In addition, the maximum yield of starch into trehalose reached as high as 59.0 ± 1.3% at a relatively high starch concentration (10% w/v) with 15 g/L of engineered cells. We further showed that the productivity of trehalose and the percentages of cell viability remained 89.1 ± 4.4% and 85.2 ± 3.6%, respectively, even after 8 continuous rounds of biocatalysis. In addition, this strategy exhibited superb operational stability even under harsh conditions, for example, solutions rich in high amount of organic solvents. The strategy demonstrated here opens up research opportunities of combining extracellular catalysis with intracellular reactions for rapid and robust production of various value-based products.

Programming living cells to organize inorganic nano-objects (NOs) in a spatiotemporally precise fashion would advance new techniques for creating ordered ensembles of NOs and new bio-abiotic hybrid materials with emerging functionalities. Bacterial cells often grow in cellular communities called biofilms. Here, a strategy is reported for programming dynamic biofilm formation for the synchronized assembly of discrete NOs or hetero-nanostructures on diverse interfaces in a dynamic, scalable, and hierarchical fashion. By engineering Escherichia coli to sense blue light and respond by producing biofilm curli fibers, biofilm formation is spatially controlled and the patterned NOs' assembly is simultaneously achieved. Diverse and complex fluorescent quantum dot patterns with a minimum patterning resolution of 100 µm are demonstrated. By temporally controlling the sequential addition of NOs into the culture, multilayered heterostructured thin films are fabricated through autonomous layer-by-layer assembly. It is demonstrated that biologically dynamic self-assembly can be used to advance a new repertoire of nanotechnologies and materials with increasing complexity that would be otherwise challenging to produce.