Biotechnology and Bioprocess Engineering 2023; 28(3): 419-427  
Production of Tagatose by Whole-cell Bioconversion from Fructose Using Corynebacterium glutamicum
Eun Jung Jeon, Young-Mi Lee, Eun Jung Choi, Seong-Bo Kim, and Ki Jun Jeong
Eun Jung Jeon, Ki Jun Jeong
Department of Chemical and Biomolecular Engineering (BK Plus Program), Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Korea
Eun Jung Jeon
Synthetic Biology Research Center, Synthetic Biology and Bioengineering Research Institute, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Korea
Young-Mi Lee, Eun Jung Choi, Seong-Bo Kim
CJ Cheiljedang Food Research Institute, Suwon 16495, Korea
Seong-Bo Kim
Korea Bio-Living Engineering Major, Global Leaders College, Yonsei University, Seoul 03722, Korea
Ki Jun Jeong*
Institute for the BioCentury, Korea Advanced Institute of Science and Technology (KAIST), Daejeon 34141, Korea
Tel: +82-42-350-3934; Fax: +82-42-350-3910
E-mail: kjjeong@kaist.ac.kr
Received: October 5, 2022; Revised: November 22, 2022; Accepted: December 7, 2022; Published online: June 30, 2023.
© 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 (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Tagatose is a rarely occurring sugar found in food and sweet fruits. It is a potential sweetener with low calories but a sweetness similar to that of sucrose. In this study, we developed a whole-cell biocatalyst using Corynebacterium glutamicum for direct bioconversion of fructose to tagatose. First, we constructed a biological conversion pathway for the conversion of fructose to tagatose by expressing tagatose 4-epimerase (TN) from Thermotoga neapolitana in C. glutamicum. Next, to increase the expression level of the enzyme, we engineered copy number of plasmid. Plasmid library was constructed by randomizing the cgrI antisense RNA region in the plasmid, and the plasmid with high-copy number was isolated using fluorescence-activated single cell sorting (FACS)-based high-throughput screening. The isolated plasmid, pHCP7, had 2-fold higher copy numbers than the original plasmid. A higher expression level and conversion yield could be achieved using pHCP7. Finally, we examined a novel tagatose 4-epimerase TN(KNF4E) isolated from Kosmotoga olearia. The gene expression level was further increased (33.9% of total fraction) through codon optimization and expression in pHCP7, and a conversion yield as high as 21.7% was achieved.
Keywords: Corynebacterium glutamicum, tagatose, fructose, whole-cell biocatalyst, high copy plasmids

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