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

Lee, Seong; Kim, Min-Sik; Jung, Hae; Lee, Jinwon; Lee, Jung-Hyun; Lee, Hyun; Kang, Sung

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 H 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, 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, H production rate of KS0510 mutant was 4.8-fold higher than that of the wild-type strain. The P 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 H 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.

RNA sequencing; HYDROGEN production; THERMOPHILIC microorganisms; OXIDATION of carbon monoxide; PROMOTERS (Genetics); HYDROGENASE; METABOLITES; BIOTECHNOLOGY

Applied Microbiology & Biotechnology, 2015, Vol 99, Issue 9, p4085

0175-7598

Academic Journal

10.1007/s00253-015-6444-1