Screening of a novel strong promoter by RNA sequencing and its application to H2 production in a hyperthermophilic archaeon

Title
Screening of a novel strong promoter by RNA sequencing and its application to H2 production in a hyperthermophilic archaeon
Author(s)
이성혁; 김민식; 정해창; 이진원; 이정현; 이현숙; 강성균
KIOST Author(s)
Lee, Seong Hyuk(이성혁)Jung, Hae Chang(정해창)Lee, Jung Hyun(이정현)Lee, Hyun Sook(이현숙)Kang, Sung Gyun(강성균)
Publication Year
2015-04-16
Abstract
A strong promoter increases transcription of the genes of interest and enhances the production of various valuable substances. For a hyperthermophilic archaeon Thermococcus onnurineus NA1, which can produce H2 by carbon monoxide oxidation, we searched for a novel endogenous strong promoter by transcriptome analysis using high-throughput RNA sequencing. Based on the relative transcript abundance, we selected one promoter to encode a hypothetical gene, of which homologs were found only in several Thermococcales strains. This promoter, PTN0510, was introduced into the front of CO-responsible hydrogenase gene cluster encoding a carbon monoxide dehydrogenase (CODH), a hydrogenase and a Na+/H+ antiporter. In the resulting mutant strain, KS0510, transcription and translation level of the gene cluster increased by 4- to 14-folds and 1.5- to 1.9-folds, respectively, in comparison with those of wild-type strain. Additionally, H2 production rate of KS0510 mutant was 4.8-fold higher than that of wild-type strain. The PTN0510 was identified to be much stronger than the well-known two strong promoters, gdh and slp promoters from Thermococcus strains, through RT-qPCR and western blotting analyses and kinetics of H2 production. In this study, we demonstrated that the RNA-seq approach is a good strategy to mine a novel strong promoter of use to a Thermococcus strain when developed as a biotechnologically promising strain to produce vwe searched for a novel endogenous strong promoter by transcriptome analysis using high-throughput RNA sequencing. Based on the relative transcript abundance, we selected one promoter to encode a hypothetical gene, of which homologs were found only in several Thermococcales strains. This promoter, PTN0510, was introduced into the front of CO-responsible hydrogenase gene cluster encoding a carbon monoxide dehydrogenase (CODH), a hydrogenase and a Na+/H+ antiporter. In the resulting mutant strain, KS0510, transcription and translation level of the gene cluster increased by 4- to 14-folds and 1.5- to 1.9-folds, respectively, in comparison with those of wild-type strain. Additionally, H2 production rate of KS0510 mutant was 4.8-fold higher than that of wild-type strain. The PTN0510 was identified to be much stronger than the well-known two strong promoters, gdh and slp promoters from Thermococcus strains, through RT-qPCR and western blotting analyses and kinetics of H2 production. In this study, we demonstrated that the RNA-seq approach is a good strategy to mine a novel strong promoter of use to a Thermococcus strain when developed as a biotechnologically promising strain to produce v
URI
https://sciwatch.kiost.ac.kr/handle/2020.kiost/25644
Bibliographic Citation
한국미생물학회 국제학술대회, pp.190, 2015
Publisher
한국미생물학회
Type
Conference
Language
English
Publisher
한국미생물학회
Related Researcher
Research Interests

marine biotechnology,molecular microbiology,해양생명공학,분자미생물학

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