Production of 12-hydroxy dodecanoic acid methyl ester using a signal peptide sequence-optimized transporter AlkL and a novel monooxygenase

Hee-Wang Yoo; Joonwon Kim; Mahesh D Patil; Beom Gi Park; Sung-Yeon Joo; Hyungdon Yun; Byung-Gee Kim

doi:10.1016/j.biortech.2019.121812

Production of 12-hydroxy dodecanoic acid methyl ester using a signal peptide sequence-optimized transporter AlkL and a novel monooxygenase

Bioresour Technol. 2019 Nov:291:121812. doi: 10.1016/j.biortech.2019.121812. Epub 2019 Jul 15.

Authors

Hee-Wang Yoo¹, Joonwon Kim², Mahesh D Patil³, Beom Gi Park², Sung-Yeon Joo², Hyungdon Yun³, Byung-Gee Kim⁴

Affiliations

¹ Interdisciplinary Program of Bioengineering, Seoul National University, Seoul 08826, Republic of Korea; Institute of Molecular Biology and Genetics, Seoul National University, Seoul 08826, Republic of Korea.
² Institute of Molecular Biology and Genetics, Seoul National University, Seoul 08826, Republic of Korea; School of Chemical and Biological Engineering, Seoul National University, Seoul, Republic of Korea.
³ Department of Systems Biotechnology, Konkuk University, Seoul, Republic of Korea.
⁴ Interdisciplinary Program of Bioengineering, Seoul National University, Seoul 08826, Republic of Korea; Institute of Molecular Biology and Genetics, Seoul National University, Seoul 08826, Republic of Korea; School of Chemical and Biological Engineering, Seoul National University, Seoul, Republic of Korea; Institute of Engineering Research, Seoul National University, Seoul 08826, Republic of Korea. Electronic address: byungkim@snu.ac.kr.

PMID: 31376668
DOI: 10.1016/j.biortech.2019.121812

Abstract

In this study, a signal peptide of AlkL was replaced with other signal peptides to improve the soluble expression and thereby facilitate the transport of dodecanoic acid methyl ester (DAME) substrate into the E. coli. Consequently, AlkL with signal peptide FadL (AlkL_f) showed higher transport activity toward DAME. Furthermore, the promoter optimization for the efficient heterologous expression of the transporter AlkL_f and alkane monooxygenase (AlkBGT) system was conducted and resulted in increased ω-oxygenation activity of AlkBGT system. Moreover, bioinformatic studies led to the identification of novel monooxygenase from Pseudomonas pelagia (Pel), which exhibited 20% higher activity towards DAME as substrate compared to AlkB. Finally, the construction of a chimeric transporter and the expression of newly identified monooxygenase enabled the production of 44.8 ± 7.5 mM of 12-hydroxy dodecanoic acid methyl ester (HADME) and 31.8 ± 1.7 mM of dodecanedioic acid monomethyl ester (DDAME) in a two-phase reaction system.

Keywords: AlkBGT; Dodecanoic acid methyl ester; Signal peptide; Substrate uptake; ω-hydroxylation.

MeSH terms

Escherichia coli
Membrane Transport Proteins*
Metabolic Engineering*
Mixed Function Oxygenases
Protein Sorting Signals

Substances

Membrane Transport Proteins
Protein Sorting Signals
Mixed Function Oxygenases