SMART-Assisted Fabrication of Engineered Pichia pastoris Microbiofactories for Stable In Vitro Antigen Production and Storage

Abstract

The rapid growth of recombinant protein vaccines underscores the need for manufacturing platforms that are efficient, stable, and scalable. We present a biomanufacturing approach integrating bioengineered Pichia pastoris with sodium alginate bioinks using the patented Sprayed Multi Adsorbed-Droplet Reposing Technology (SMART, US20250288524A1) to fabricate uniform microbiofactories. Engineered Pichia pastoris X33 strains expressing the truncated Epstein–Barr virus nuclear antigen 1 (EBNA1, aa 379–641) from the pPICZαA-EBNA1 vector were encapsulated and induced with methanol for antigen production. SMART-assisted encapsulation generated microcarriers averaging ∼50 μm in diameter with a postencapsulation yeast recovery of 87% as assessed by the Alamar Blue viability assay. Across three independent biological replicates, hydrogel-encapsulated yeast consistently produced higher EBNA1 titers than free yeast, corresponding to a 1.70 ± 0.37-fold enhancement, as quantified by ELISA and confirmed by Western blot. Following lyophilization, in a single-experiment proof-of-concept storage study, the microbiofactories sustained EBNA1 production at 392 ± 11 ng/mL over 28 days at ambient temperature (24 °C), compared to 154 ± 18 ng/mL for free yeast; the sustained ELISA signal at day 28 constitutes functional evidence of metabolically active yeast upon reconstitution. These findings demonstrate the in vitro proof-of-concept feasibility of SMART-assisted hydrogel encapsulation as an ambient-temperature-stable platform for microbial antigen production. This approach provides a foundation for future development of biomanufacturing systems that may help reduce cold-chain dependence, pending further scale-up and immunological validation.