SMART-Assisted Fabrication of Engineered Pichia pastoris Microbiofactories for Stable In Vitro Antigen Production and Storage
- 1University of Mississippi , , , ,
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.
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BibTeX
@article{feng2026smartassisted,
title = {{SMART-Assisted Fabrication of Engineered Pichia pastoris Microbiofactories for Stable In Vitro Antigen Production and Storage}},
author = {Shao-Ching Feng and Prashant Pandey and Leela Raghava Jaidev Chakka and Yu Zhang and Mohammed Maniruzzaman},
journal = {ACS Omega},
year = {2026},
month = oct,
doi = {10.1021/acsomega.6c05614},
url = {https://pubs.acs.org/acsodf/article/doi/10.1021/acsomega.6c05614/5454859/SMART-Assisted-Fabrication-of-Engineered-Pichia},
note = {CC BY-NC-ND 4.0},
}
RIS
TY - JOUR
TI - SMART-Assisted Fabrication of Engineered Pichia pastoris Microbiofactories for Stable In Vitro Antigen Production and Storage
AU - Shao-Ching Feng
AU - Prashant Pandey
AU - Leela Raghava Jaidev Chakka
AU - Yu Zhang
AU - Mohammed Maniruzzaman
PY - 2026
DA - 2026/10/01
JO - ACS Omega
DO - 10.1021/acsomega.6c05614
UR - https://pubs.acs.org/acsodf/article/doi/10.1021/acsomega.6c05614/5454859/SMART-Assisted-Fabrication-of-Engineered-Pichia
AB - 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.
SN - 2470-1343
ER -