title: Screening of a novel strong promoter by RNA sequencing and its application to H2 production in a hyperthermophilic archaeon.
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 from 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, P TN0510 , 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 the wild-type strain. Additionally, H2 production rate of KS0510 mutant was 4.8-fold higher than that of the wild-type strain. The P TN0510 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 novel strong promoters of use to a Thermococcus strain when developed as a biotechnologically promising strain to produce valuable products such as enzymes and metabolites through metabolic engineering.
authors.affiliation: Korea Institute of Ocean Science and Technology, Ansan, Korea.
authors.forename: Seong Hyuk
authors.initials: SH
authors.lastname: Lee
authors.forename: Min-Sik
authors.initials: MS
authors.lastname: Kim
authors.forename: Hae Chang
authors.initials: HC
authors.lastname: Jung
authors.forename: Jinwon
authors.initials: J
authors.lastname: Lee
authors.forename: Jung-Hyun
authors.initials: JH
authors.lastname: Lee
authors.forename: Hyun Sook
authors.initials: HS
authors.lastname: Lee
authors.forename: Sung Gyun
authors.initials: SG
authors.lastname: Kang
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D002248:Carbon Monoxide / Q000378:metabolism; D015870:Gene Expression; D020869:Gene Expression Profiling; D005820:Genetic Testing; D006859:Hydrogen / Q000378:metabolism*; D006864:Hydrogenase / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism; D005810:Multigene Family; D011401:Promoter Regions, Genetic; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017423:Sequence Analysis, RNA; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism; D019712:Thermococcus / Q000235:genetics* / Q000378:metabolism*
chemical_list: D009097:Multienzyme Complexes; D017923:Sodium-Hydrogen Exchangers; D002248:Carbon Monoxide; D006859:Hydrogen; D006864:Hydrogenase; D000445:Aldehyde Oxidoreductases; C019823:carbon monoxide dehydrogenase