Screening of a novel strong promoter by RNA sequencing and its application to H2 production in a hyperthermophilic archaeon.
Appl Microbiol Biotechnol, 2015/5;99(9):4085-92.
Lee SH[1], Kim MS, Jung HC, Lee J, Lee JH, Lee HS, Kang SG
Affiliations
PMID: 25690310DOI: 10.1007/s00253-015-6444-1
Impact factor: 5.56
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.
MeSH terms
Aldehyde Oxidoreductases; Blotting, Western; Carbon Monoxide; Gene Expression; Gene Expression Profiling; Genetic Testing; Hydrogen; Hydrogenase; Molecular Sequence Data; Multienzyme Complexes; Multigene Family; Promoter Regions, Genetic; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Sequence Analysis, RNA; Sodium-Hydrogen Exchangers; Thermococcus
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