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Increased Production of Recombinant O-Phospho-L-Serine Sulfhydrylase from the Hyperthermophilic Archaeon Aeropyrum pernix K1 Using Escherichia coli

[ Vol. 8 , Issue. 1 ]

Author(s):

Takashi Nakamura*, Emi Takeda, Tomoko Kiryu, Kentaro Mori, Miyu Ohori, Eiki Kikugawa and Kazuhiko Ishikawa   Pages 15 - 23 ( 9 )

Abstract:


Background: O-phospho-L-serine sulfhydrylase from the hyperthermophilic archaeon Aeropyrum pernix K1 (ApOPSS) is thermostable and tolerant to organic solvents. It can produce nonnatural amino acids in addition to L-cysteine.

Objective: We aimed to obtain higher amounts of ApOPSS compared to those reported with previous methods for the convenience of research and for industrial production of L-cysteine and non-natural amino acids.

Methods: We performed codon optimization of cysO that encodes ApOPSS, for optimal expression in Escherichia coli. We then examined combinations of conditions such as the host strain, plasmid, culture medium, and isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration to improve ApOPSS yield.

Results and Discussion: E. coli strain Rosetta (DE3) harboring the expression plasmid pQE-80L with the codon-optimized cysO was cultured in Terrific broth with 0.01 mM IPTG at 37°C for 48 h to yield a 10-times higher amount of purified ApOPSS (650 mg·L-1) compared to that obtained by the conventional method (64 mg·L-1). We found that the optimal culture conditions along with codon optimization were essential for the increased ApOPSS production. The expressed ApOPSS had a 6-histidine tag at the N-terminal, which did not affect its activity. This method may facilitate the industrial production of cysteine and non-natural amino acids using ApOPSS.

Conclusion: We conclude that these results could be used in applied research on enzymatic production of L-cysteine in E. coli, large scale production of non-natural amino acids, an enzymatic reaction in organic solvent, and environmental remediation by sulfur removal.

Keywords:

Aeropyrum pernix K1, O-phospho-L-serine sulfhydrylase, cysteine synthesis, high expression level, codon optimization, terrific broth, low IPTG concentration.

Affiliation:

Laboratory of Molecular Biochemistry, Nagahama Institute of Bio-Science and Technology, 1266 Tamura-cho, Nagahama, Shiga 526-0829, Laboratory of Molecular Biochemistry, Nagahama Institute of Bio-Science and Technology, 1266 Tamura-cho, Nagahama, Shiga 526-0829, Laboratory of Molecular Biochemistry, Nagahama Institute of Bio-Science and Technology, 1266 Tamura-cho, Nagahama, Shiga 526-0829, Laboratory of Molecular Biochemistry, Nagahama Institute of Bio-Science and Technology, 1266 Tamura-cho, Nagahama, Shiga 526-0829, Laboratory of Molecular Biochemistry, Nagahama Institute of Bio-Science and Technology, 1266 Tamura-cho, Nagahama, Shiga 526-0829, Laboratory of Molecular Biochemistry, Nagahama Institute of Bio-Science and Technology, 1266 Tamura-cho, Nagahama, Shiga 526-0829, National Institute of Advanced Industrial Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577

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