Work

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

Year: 2026

Type: article

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.

Source: ACS Omega

DOI: 10.1021/acsomega.6c05614

Authors: Shao-Ching Feng, Prashant Pandey, Leela Raghava Jaidev Chakka, Yu Zhang, Mohammed Maniruzzaman

Institution: University of Mississippi

Language: English

Topic: Fungal and yeast genetics research

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

SDG: Good health and well-being

Open Access status: gold

Estimated APC paid: $1,935