Enhancing long‐term storage and stability of engineered living materials through desiccant storage and trehalose treatment

Abstract

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.

Additive Manufacturing of Engineered Living Materials with Bio‐Augmented Mechanical Properties and Resistance to Degradation

“…When the samples cultured over 21 d, high cell density were observed in µ CT (Figure 3b), which showed the S. cerevisiae cells were able to grow in the BSA‐PEGDA network, similar in time but different in spatial organization from another ELM formulation we previously studied. [ 22 ]…”

“…When the samples cultured over 21 d, high cell density were observed in µCT (Figure 3b), which showed the S. cerevisiae cells were able to grow in the BSA-PEGDA network, similar in time but different in spatial organization from another ELM formulation we previously studied. [22] Scanning electron microscopy (SEM) analysis of the dehydrated cylindrical samples were additionally used to assess the cell distribution and proliferation throughout the printed structure. Cross-sectional images were taken from different regions of a printed cylinder (Figure 3c) of ELM-SC-BXN and ELM-EC-LDOPA after 3 days of culturing, which showed the presence of spherical-shaped yeast cells [23] and rod-shaped bacterial cells, [24] respectively (Figure 3).…”

A tripartite microbial co-culture system for de novo biosynthesis of diverse plant phenylpropanoids

“…To bypass the challenges of liquid culture-based production flexibility and co-culture stability, the use of ELMs has been proposed 31 , 33 , 34 . Here, we sought to apply the principles of encapsulation-based production to 2- and 3- part consortia for ferulic acid and eugenol production, respectively.…”

“…These ELM-based approaches can uniquely support stable co-cultures by spatially encapsulating individual microbes to avoid growth-based competitions that can occur during outgrowth and repeated culturing. Additionally, ELM bioproduction can enable on-demand production without cold-chain requirements, with over one year of shelf stability for encapsulated fungal bioproduction systems 34 , thus enabling resource-limited biomanufacturing 35 . On-demand phenylpropene production would be advantageous as these compounds have limited stability at elevated temperatures 36 , 37 .…”

Engineered Living Material Bioreactors with Tunable Mechanical Properties using Vat Photopolymerization

“…This exotherm arises from the thermal polymerization of unreacted acrylates in the samples. [26,27] However, when these samples are treated with a post-print cure, wherein the constructs are irradiated with 400 nm in a custom photocuring chamber (Quans, 400 nm, 1 mW cm −2 ) for 60 min, the exothermic peak was not present for any of the samples (Figure 2d-e). When glycerol was present in the resin formulation this peak was absent, which suggests that glycerol may promote efficient network formation by aggregating the PEGDA chains to facilitate the reaction of the chain ends.…”