Encapsulation enhances protoplast fusant stability

Authors

Jordan Gulli, Georgia Institute of Technology
Eugene Kroll, Georgia Institute of Technology
Frank Rosenzweig, Georgia Institute of Technology

Document Type

Article

Publication Title

Biotechnology and bioengineering

Publication Date

2-2020

Volume

117

Issue

6

Disciplines

Biology | Life Sciences

Abstract

A barrier to cost-efficient biomanufacturing is the instability of engineered genetic elements, such as plasmids. Instability can also manifest at the whole-genome level, when fungal dikaryons revert to parental species due to nuclear segregation during cell division. Here, we show that by encapsulating Saccharomyces cerevisiae-Pichia stipitis dikaryons in an alginate matrix, we can limit cell division and preserve their expanded metabolic capabilities. As a proxy to cellulosic ethanol production, we tested the capacity of such cells to carry out ethanologenic fermentation of glucose and xylose, examining substrate use, ploidy, and cell viability in relation to planktonic fusants, as well as in relation to planktonic and encapsulated cell cultures consisting of mixtures of these species. Glucose and xylose consumption and ethanol production by encapsulated dikaryons were greater than planktonic controls. Simultaneous co-fermentation did not occur; rather the order and kinetics of glucose and xylose catabolism by encapsulated dikaryons were similar to cultures where the two species were encapsulated together. Over repeated cycles of fed-batch culture, encapsulated S. cerevisiae-P. stipitis fusants exhibited a dramatic increase in genomic stability, relative to planktonic fusants. Encapsulation also increased the stability of antibiotic-resistance plasmids used to mark each species and preserved a fixed ratio of S. cerevisiae to P. stipitis cells in mixed cultures. Our data demonstrate how encapsulating cells in an extracellular matrix restricts cell division and, thereby, preserves the stability and biological activity of entities ranging from genomes to plasmids to mixed populations, each of which can be essential to cost-efficient biomanufacturing.

Keywords

cell encapsulation, cellulosic fermentation, dikaryon, genomic stability, Pichia stipitis, protoplast fusion, Saccharomyces cerevisiae

DOI

https://doi.org/10.1002/bit.27318

Rights

© 2020 The Authors

Creative Commons License

This work is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 4.0 International License.

Recommended Citation

Gulli J, Kroll E, Rosenzweig F. Encapsulation enhances protoplast fusant stability. Biotechnology and Bioengineering. 2020;117:1696–1709. https://doi.org/10.1002/bit.27318