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.
Graphical Abstract
[http://dx.doi.org/10.1007/s00253-006-0587-z] [PMID: 17021879]
[http://dx.doi.org/10.1038/nbt807] [PMID: 12640465]
[http://dx.doi.org/10.1177/106002808802200721] [PMID: 3046890]
[http://dx.doi.org/10.1021/jm9704098] [PMID: 9397180]
[PMID: 29285203]
[http://dx.doi.org/10.1128/JB.185.7.2277-2284.2003] [PMID: 12644499]
[http://dx.doi.org/10.1016/S0014-5793(03)00913-X] [PMID: 12965218]
[http://dx.doi.org/10.1107/S0907444902017900] [PMID: 12554945]
[http://dx.doi.org/10.1016/j.jmb.2005.05.064] [PMID: 16005886]
[http://dx.doi.org/10.1016/j.jmb.2012.05.009] [PMID: 22580223]
[http://dx.doi.org/10.1007/s00792-016-0862-6] [PMID: 27377295]
[http://dx.doi.org/10.1080/09168451.2015.1020753] [PMID: 25779754]
[http://dx.doi.org/10.1016/j.bej.2009.10.015]
[http://dx.doi.org/10.1246/cl.170822]
[http://dx.doi.org/10.1021/cr300131h] [PMID: 23547530]
[http://dx.doi.org/10.1021/cn400075h] [PMID: 23705903]
[http://dx.doi.org/10.1016/j.pep.2006.01.002] [PMID: 16546401]
[http://dx.doi.org/10.1016/j.pep.2016.07.012] [PMID: 27449918]
[http://dx.doi.org/10.1016/j.pep.2013.10.016] [PMID: 24211770]
[http://dx.doi.org/10.1016/j.jbiotec.2004.08.004] [PMID: 15607230]
[http://dx.doi.org/10.1016/S0958-1669(96)80051-6] [PMID: 8939627]
[http://dx.doi.org/10.1371/journal.pone.0007002] [PMID: 19759823]
[http://dx.doi.org/10.1016/0003-2697(76)90527-3] [PMID: 942051]
[http://dx.doi.org/10.1042/bj1040627] [PMID: 6048802]
[http://dx.doi.org/10.1007/BF00332244] [PMID: 2659969]
[http://dx.doi.org/10.1007/s10295-016-1773-3] [PMID: 27130461]
[http://dx.doi.org/10.1007/s002530000334] [PMID: 10919319]
[PMID: 29213997]
[http://dx.doi.org/10.1016/j.jgeb.2018.03.006] [PMID: 30733745]
[http://dx.doi.org/10.3390/v9070159] [PMID: 28644404]
[http://dx.doi.org/10.1107/S0907444906052024] [PMID: 17327666]
[http://dx.doi.org/10.1021/bi050485+] [PMID: 15952768]
[http://dx.doi.org/10.1111/j.1742-4658.2007.06063.x] [PMID: 17894825]