Development of a regulatable low-temperature protein expression system using the psychrotrophic bacterium, Shewanella livingstonensis Ac10, as the host.

Soichiro Kawai,Jun Kawamoto,Tatsuo Kurihara,Takuya Ogawa

doi:10.1080/09168451.2019.1638754

Soichiro Kawai, Jun Kawamoto + Show 2 more

Open Access

https://doi.org/10.1080/09168451.2019.1638754

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Abstract

A low-temperature protein expression system is useful for the production of thermolabile proteins. We previously developed a system that enables constitutive protein production at low temperatures, using the psychrotrophic bacterium Shewanella livingstonensis Ac10 as the host. To increase the utility of this system, in the present study, we introduced a repressible promoter of the trp operon of this bacterium into the system. When ß-lactamase was produced under the control of this promoter at 18°C and 4°C, the yields were 75 and 33 mg/L-culture, respectively, in the absence of L-Trp, and the yields were decreased by 72% and 77%, respectively, in the presence of L-Trp. We also found that 3-indoleacrylic acid, a competitive inhibitor of the Escherichia coli trp repressor, increased the expression of the reporter gene. This repressible gene expression system would be useful for regulatable recombinant protein production at low temperatures.

Highlights

A number of recombinant protein expression systems have been constructed using various organisms including bacteria [1], eukaryotic microorganisms [2], and mammalian cells [3] as the hosts
We introduced a gene regulatory system of the trp operon into the heterologous protein production system operating at low temperatures using S. livingstonensis Ac10
S. livingstonensis Ac10-Rifr was grown under aerobic conditions in Luria-Bertani (LB) medium consisting of 10 g/L tryptone (BD Difco, Detroit, MI), 10 g/L NaCl (Nacalai Tesque, Kyoto, Japan), and 5 g/L yeast extract (BD Difco) for 24 h at 18 °C, and transferred to modified DSMZ medium 79

Summary

Introduction

A number of recombinant protein expression systems have been constructed using various organisms including bacteria [1], eukaryotic microorganisms [2], and mammalian cells [3] as the hosts. We introduced a gene regulatory system of the trp operon into the heterologous protein production system operating at low temperatures using S. livingstonensis Ac10. The recombinant S. livingstonensis Ac10-Rifr strains harboring each of the promoter-assay plasmids were cultivated aerobically in liquid modified DSMZ medium with 0.5% w/v casamino acids supplementation containing 30 μg/mL of chloramphenicol in the presence or absence of 0.1% L-Trp at 18 °C and 4 °C.

Results

Conclusion