Date of Award
10-25-2022
Document Type
Dissertation
Degree Name
Molecular Biosciences, Ph.D.
First Advisor
Jianfeng Xu
Committee Members
Asela Wijeratne; Fabricio Medina-Bolivar; Maureen Dolan; Vibha Srivastava
Call Number
LD 251 .A566d 2022 K37
Abstract
Plant cell culture is emerging as a promising alternative bioproduction platform for valuable proteins due to its low cost of production, safety (free from animal pathogen contamination) and ease of scale-up. However, there are common challenges for commercialization, including low protein yields, difficulty in cryopreservation, and non-human glycosylation, etc. This research project addresses the overarching research hypothesis that strategically modifying the plant cell genome using CRISPR/Cas9 technology and rationally designing protein molecules by fusing with a functional module enhance the production of protein in plant cell culture and improve protein function. There are two specific aims addressed in this project: Aim1: Explore cellular engineering of plant cells to improve protein production; Aim 2: Rationally design and engineer protein molecules in plant cells for enhanced bioproduction and function. Specifically, Aim 1 investigated cellular engineering of plant cells to create cell wall deficient (CWD) cell lines. Bright Yellow-2 (BY-2) cell lines with substantially reduced cellulose content were created by either chemical treatment with a cellulose synthesis inhibitor or CRISPR/Cas9 genome editing technology. The viability, morphology, transcriptomics, and bioproductivity of the created CWD cells were characterized. CWD BY-2 cells generated by either chemical treatment or genome editing formed large clumps, and their cellulose content was drastically reduced by 80 % and 40 %, respectively. Aim 2 leveraged two unique post-translational modifications –“Hydroxyproline (Hyp)-O-glycosylation” and “Glycosylphosphati-dylinositol (GPI) anchor”– to strategically design and engineer proteins in plant cells to improve production and function. A Hyp-O-glycosylation module composed of 20 repeats of the “Ser-Pro” motif was engineered to human erythropoietin (EPO) to substantially increase the secreted protein yields in BY-2 cell culture up to 8.61 μg/mL. GPI-anchored proteins, including reporter protein, therapeutic protein and enzyme, was engineered into BY-2 cells and tobacco plants. GPI anchor was found to facilitate the intracellular trafficking and glycosylation (Hyp-O-linked and N-/O-linked glycosylation) of the anchored proteins. This study establishes proof-of-concept for top-down plant cell wall engineering to create CWD plant cell lines, and provides a new biomolecular engineering strategy to improve and expand plant-based production.
Rights Management
This work is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 4.0 International License.
Recommended Citation
Karki, Uddhab, "Enhancing Production of Functional Proteins in Plant Cell Culture Through Cellular Engineering and Rational Protein Design" (2022). Student Theses and Dissertations. 232.
https://arch.astate.edu/all-etd/232
