Living plant cells can be engineered to produce heterologous proteins in response to a wide variety of external stimuli including chemicals, light, temperature, and biotic and abiotic stress. They are ideal controllable living systems for incorporation into nonliving matrices (polymers, gels, etc.) using either immobilization or bioprinting technologies for several reasons. First, their relatively low metabolic uptake rates reduce concerns about nutrient and oxygen mass transfer limitations within the matrices even for reasonably high seeding densities. Second, their slow growth rates allow them to be used over long time periods before reaching physical space constraints. Third, they are relatively robust and able to withstand short term perturbations in nutrient concentrations, temperature, pH, and shear stress.<br/><br/>As a step towards 3D printing of transgenic plant cells for use in a stimulus-responsive bioproduction system we have developed a process for immobilization and use of transgenic rice (<i>Oryza sativa</i>) cell aggregates in hydrogels. The rice cells have been engineered to produce recombinant butyrylcholinesterase (BChE) as a prophylactic/therapeutic against organophosphate nerve agent poisoning, cocaine toxicity, and neurodegenerative diseases like Alzheimer’s. BChE is a large (~340 kDa), multimeric (homotetrimer) glycoprotein (10 N-linked glycosylation sites per monomer), presenting a challenge for heterologous production. In our transgenic rice system, BChE production is controlled by a sugar-responsive promoter, the rice alpha-amylase 3D promoter, thus sugar concentration is the primary control stimulus, with high sugar levels promoting growth and sugar-free conditions leading to BChE production. Immobilized transgenic rice cells sustained viability for 14 days in the matrix, undergoing a growth phase from days 0–6, a BChE production phase in sugar-free medium from days 6–12, and a growth/recovery phase from days 12–14. The rice cells in the hydrogel also produced a significant amount of active BChE, comparable to the levels produced in liquid cultures, and a substantial fraction was secreted into the media, allowing for easier product separation. This work serves as a foundation for further investigation towards engineering stimulus-responsive living plant cells for intensified bioproduction, representing a simple, efficient, robust, modular, and potentially field-deployable bioreactor system for the manufacture of biologics and other products.

Engineered living plant cells for intensified bioproduction

ACS Spring 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.