Optimization of Culture Conditions for Secretory Production of 3-Hydroxybutyrate Oligomers Using Recombinant Escherichia coli

Tetsuo Sakurai; Shoji Mizuno; Yuki Miyahara; Ayaka Hiroe; Seiichi Taguchi; Takeharu Tsuge

doi:10.3389/fbioe.2022.829134

Optimization of Culture Conditions for Secretory Production of 3-Hydroxybutyrate Oligomers Using Recombinant Escherichia coli

Front Bioeng Biotechnol. 2022 Feb 25:10:829134. doi: 10.3389/fbioe.2022.829134. eCollection 2022.

Authors

Tetsuo Sakurai^{1

2}, Shoji Mizuno^{1

2}, Yuki Miyahara^{1

2}, Ayaka Hiroe^{3

2}, Seiichi Taguchi³, Takeharu Tsuge^{1

2}

Affiliations

¹ Department of Materials Science and Engineering, Tokyo Institute of Technology, Yokohama, Japan.
² MIRAI, Japan Science and Technology Agency (JST), Saitama, Japan.
³ Department of Chemistry for Life Sciences and Agriculture, Faculty of Life Sciences, Tokyo University of Agriculture, Setagaya, Japan.

Abstract

Poly(3-hydroxybutyrate) [P(3HB)] is the most representative polyhydroxyalkanoate (PHA), which is a storage polyester for prokaryotic cells. P(3HB)-producing recombinant Escherichia coli secretes diethylene glycol (DEG)-terminated 3HB oligomers (3HBO-DEG) through a PHA synthase-mediated chain transfer and alcoholysis reactions with externally added DEG. The purpose of this study was to optimize the culture conditions for the secretory production of 3HBO-DEG with jar fermenters. First, the effects of culture conditions, such as agitation speed, culture temperature, culture pH, and medium composition on 3HBO-DEG production, were investigated in a batch culture using 250-ml mini jar fermenters. Based on the best culture conditions, a fed-batch culture was conducted by feeding glucose to further increase the 3HBO-DEG titer. Consequently, the optimized culture conditions were reproduced using a 2-L jar fermenter. This study successfully demonstrates a high titer of 3HBO-DEG, up to 34.8 g/L, by optimizing the culture conditions, showing the feasibility of a new synthetic strategy for PHA-based materials by combining secretory oligomer production and subsequent chemical reaction.

Keywords: 3-hydroxybutyrate; alcoholysis; chain transfer reaction; class IV PHA synthase; fed-batch culture; oligomer; secretion.