Biotechnology and Bioprocess Engineering 2019; 24(3): 568-578  
Inhibition of Autolysosome Formation Improves rrhGAA Production Driven by RAmy3D Promoter in Transgenic Rice Cell Culture
Jong Kwang Hong1,†, Hong-Yeol Choi2,†, Hae-Rim Park2, Dong-Il Kim2,*, and Dong-Yup Lee1,3*
1Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), Singapore 138668, Singapore
2Department of Biological Engineering, Inha University, Incheon 22212, Korea
3School of Chemical Engineering, Sungkyunkwan University, Suwon 16419, Korea
Correspondence to: Dong-Il Kim
Tel: +82-32-860-7515; Fax: +82-32-872-4046

Dong-Yup Lee
Tel: +82-31-290-7253; Fax: +82-31-290-7272

These authors contributed equally to this article.
Received: January 7, 2019; Revised: February 10, 2019; Accepted: February 23, 2019; Published online: June 30, 2019.
© The Korean Society for Biotechnology and Bioengineering. All rights reserved.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Although plant cell cultures produce low yields of recombinant proteins compared to other production systems, a dramatic increase of the heterologous protein production in transgenic rice cells was achieved with the alpha-amylase isozyme 3D (RAmy3D) promoter system. However, this expression system has inherent limitations in that gene expression is initiated by sucrose/glucose deprivation, concurrently triggering starvation-derived autophagy and rapid cell death. Decreased viability and culture longevity subsequently prevent further increment of production. In this study, we introduced autophagy inducers and inhibitors in the rrhGAA-producing transgenic rice cell cultures in order to explore their effects on production controlled by the RAmy3D promoter. The autophagy inducers rapamycin and CCI-779 increased autophagosome and autolysosome while concanamycin A1 and bafilomycin A successfully decreased autolysosome. Interestingly, autophagy inhibitors improved viability, DCW loss, and rrhGAA production, while autophagy inducers deteriorated these profiles compared to the control. As the production conditions under the death phase may facilitate protein degradation, and subsequently exacerbate functional activity, the size variant distribution and enzyme activity of the purified rrhGAAs were evaluated. However, no significant difference in rrhGAA degradation as well as GAA activity was observed compared to the control condition, thus indicating that the autophagy regulation is an efficient approach to increase protein yield in rice cell culture system for rrhGAA production.
Keywords: transgenic rice cell culture, alpha-amylase isozyme 3D (RAmy3D) promoter, human acid alpha-glucosidase (rrhGAA), autophagy regulators

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