High energy photons (λ < 400 nm) are frequently used to initiate free radical polymerizations to form polymer networks, but are only effective for transparent objects. This phenomenon poses a major challenge to additive manufacturing of particle‐reinforced composite networks since deep light penetration of short‐wavelength photons limits the homogeneous modification of physicochemical and mechanical properties. Herein, the unconventional, yet versatile, multiexciton process of triplet–triplet annihilation upconversion (TTA‐UC) is employed for curing opaque hydrogel composites created by direct‐ink‐write (DIW) 3D printing. TTA‐UC converts low energy red light (λmax = 660 nm) for deep penetration into higher‐energy blue light to initiate free radical polymerizations within opaque objects. As proof‐of‐principle, hydrogels containing up to 15 wt.% TiO2 filler particles and doped with TTA‐UC chromophores are readily cured with red light, while composites without the chromophores and TiO2 loadings as little as 1–2 wt.% remain uncured. Importantly, this method has wide potential to modify the chemical and mechanical properties of complex DIW 3D‐printed composite polymer networks.
Engineered living materials (ELMs) combine living cells with polymeric matrices to yield unique materials with programmable functions. While the cellular platform and the polymer network determine the material properties and applications, there are still gaps in the ability to seamlessly integrate the biotic (cellular) and abiotic (polymer) components into singular materials, then assemble them into devices and machines. Herein, the additive-manufacturing of ELMs wherein bioproduction of metabolites from the encapsulated cells enhanced the properties of the surrounding matrix is demonstrated. First, aqueous resins are developed comprising bovine serum albumin (BSA) and poly(ethylene glycol diacrylate) (PEGDA) with engineered microbes for vat photopolymerization to create objects with a wide array of 3D form factors. The BSA-PEGDA matrix afforded hydrogels that are mechanically stiff and tough for use in load-bearing applications. Second, the continuous in situ production of l-DOPA, naringenin, and betaxanthins from the engineered cells encapsulated within the BSA-PEGDA matrix is demonstrated. These microbial metabolites bioaugmented the properties of the BSA-PEGDA matrix by enhancing the stiffness (l-DOPA) or resistance to enzymatic degradation (betaxanthin). Finally, the assembly of the 3D printed ELM components into mechanically functional bolts and gears to showcase the potential to create functional ELMs for synthetic living machines is demonstrated.
Engineered living materials (ELMs) have broad applications for enabling on-demand bioproduction of compounds ranging from small molecules to large proteins. However, most formulations and reports lack the capacity for storage beyond a few months. In this study, we develop an optimized procedure to maximize stress resilience of yeast-laden ELMs through the use of desiccant storage and 10% trehalose incubation before lyophilization. This approach led to over 1-year room temperature storage stability across a range of strain genotypes. In particular, we highlight the superiority of exogenously added trehalose over endogenous, engineered production in yielding robust preservation resilience that is independent of cell state. This simple, effective protocol enables sufficient accumulation of intracellular trehalose over a short period of contact time across a range of strain backgrounds without requiring the overexpression of a trehalose importer. A variety of microscopic analysis including µ-CT and confocal microscopy indicate that cells form spherical colonies within F127-BUM ELMs that have variable viability upon storage. The robustness of the overall procedure developed here highlights the potential for widespread deployment to enable on-demand, cold-chain independent bioproduction.
Plant-derived phenylpropanoids, in particular phenylpropenes, have diverse industrial applications ranging from flavors and fragrances to polymers and pharmaceuticals. Heterologous biosynthesis of these products has the potential to address low, seasonally dependent yields hindering ease of widespread manufacturing. However, previous efforts have been hindered by the inherent pathway promiscuity and the microbial toxicity of key pathway intermediates. Here, in this study, we establish the propensity of a tripartite microbial co-culture to overcome these limitations and demonstrate to our knowledge the first reported de novo phenylpropene production from simple sugar starting materials. After initially designing the system to accumulate eugenol, the platform modularity and downstream enzyme promiscuity was leveraged to quickly create avenues for hydroxychavicol and chavicol production. The consortia was found to be compatible with Engineered Living Material production platforms that allow for reusable, cold-chain-independent distributed manufacturing. This work lays the foundation for further deployment of modular microbial approaches to produce plant secondary metabolites.
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