3D Printed Engineered Living Materials with Genetically Programmed Mechanical Properties and Bioproduction Performance for the Design of Functional Objects and Therapeutic Delivery Platforms

Thesis (Ph.D.)--University of Washington, 2024The synergy of synthetic biology and materials science yields innovative strategies to find alternative approaches for environmental, medical, and manufacturing challenges. Among these approaches, Engineered Living Materials (ELMs) stand out as a promising platform. ELMs, are a distinctive class of smart materials, which are synthetic living systems where genetically modified microorganisms are integrated into a polymer network, forming functional objects. The material properties and applications are determined by the cellular platform and the encapsulating polymer network. Nevertheless, gaps still exist in the seamless integration of biotic (cellular) and abiotic (polymer) components into a singular material, followed by their assembly into devices and machines. Herein, two different biocompatible polymer networks were developed including (i) a protein-based composite, bovine serum albumin (BSA) – poly (ethylene glycol) diacrylate (PEGDA), and (ii) a synthetic matrix comprising PEGDA-glycerol. These photocurable polymer networks were designed for processing ELMs in light-based 3D printing technologies. The relationships between embedded microorganisms and surrounding polymer matrices were investigated with respect to microbial viability, microbial proliferation behavior, bioproduction capacity, and mechanical properties of ELMs. Subsequently, the interactions between engineered microbial metabolites (L-dopa, betaxanthin, and proteinase A) and protein-based (BSA-PEGDA) polymer matrix were utilized to program mechanical stiffness and degradation time points as desired of 3D printed ELM objects. In an alternative strategy, the polymer concentration of the synthetic matrix (PEGDA-Glycerol) was adjusted to tune the toughness and moduli of 3D printed ELM bioreactors. Finally, an innovative approach toward ELMs for advanced drug delivery was developed using metabolically engineered probiotic strains and 3D printed medical stents. These ELM stents were designed to detect inflammatory biomarkers and initiate responses through the secretion of anti-inflammatory small molecules. This strategy presents a substantial opportunity for facilitating long-term, localized delivery

Optimizing encapsulation of black carrot extract using complex coacervation technique : Maximizing the bioaccessibility and release kinetics in different food matrixes

The encapsulation of bioactive compounds at the micro-scale presents an advanced approach for the food and nutraceutical industries. However, the challenge lies in determining optimal encapsulation parameters for each specific bioactive compound and encapsulation method. In addressing this, our study demonstrates the application of statistical programs to streamline the optimization of wet-lab experimental designs. Focusing on the encapsulation technique of complex coacervates for black carrot phenolic extract (BCPE), the response surface methodology was employed to optimize encapsulation parameters in terms of coating material, the core-to-coating material ratio, and the pH of the encapsulation environment. The optimum conditions predicted by RSM to produce BCPE-loaded complex coacervates were found to be a pH of 3.02, a core-to-coating ratio of 10g/100g, and a coating material composition of 59.10g/100 mL maltodextrin, and 0.90g/100 mL whey protein isolate. According to the RSM pattern with 84% desirability, encapsulation efficiency was found 86.08%. In addition, the effect of different food matrixes was examined on the in vitro bioaccessibility of spray-dried BCPE loaded complex coacervated powder (BCPE-CCp). To explore the impact of protein and carbohydrate richness, along with food temperature on capsule stability, the BCPE-CCp was incorporated into skim milk, apple juice, and chocolate beverages (1g/100 mL). Notably, heat treatment had no significant effect on the in vitro bioaccessibility of BCPE-CCp in terms of total phenolic compound and antioxidant activity (p < 0.05), indicating its suitability for hot formulations. Furthermore, the release of BCPE in a protein-rich environment was observed to be higher than in a carbohydrate-rich food matrix under both gastric and intestinal conditions. These findings provide valuable insights into the stability and release dynamics of BCPE-CCp in different food settings, supporting its adaptability for diverse formulations, including those involving elevated temperatures.Peer reviewe

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

Abstract 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