title: Isolation of fully synthetic promoters for high-level gene expression in Corynebacterium glutamicum.
abstract: Corynebacterium glutamicum is an important industrial organism that is widely used in the production of amino acids, nucleotides and vitamins. To extend its product spectrum and improve productivity, C. glutamicum needs to undergo further engineering, including the development of applicable promoter system. Here, we isolated new promoters from the fully synthetic promoter library consisting of 70-bp random sequences in C. glutamicum. Using green fluorescent protein (GFP) as a reporter, highly fluorescent cells were screened from the library by fluorescent activated cell sorting (FACS). Twenty potential promoters of various strengths were isolated and characterized through extensive analysis of DNA sequences and mRNA transcripts. Among 20 promoters, 6 promoters which have different strengths were selected and their activities were successfully demonstrated using two model proteins (antibody fragment and endoxylanase). Finally, the strongest promoter (P(H36)) was employed for the secretory production of endoxylanase in fed-batch cultivation, achieving production levels of 746 mg/L in culture supernatant. This is the first report of synthetic promoters constructed in C. glutamicum, and our screening strategy together with the use of synthetic promoters of various strengths will contribute to the future engineering of C. glutamicum.
authors: Department of Chemical and Biomolecular Engineering, KAIST, 335 Gwahagno, Yuseong-gu, Daejeon, 305-701, Republic of Korea.
authors: Sung Sun
authors: SS
authors: Yim
authors: Seul Ji
authors: SJ
authors: An
authors: Misuk
authors: M
authors: Kang
authors: Jinho
authors: J
authors: Lee
authors: Ki Jun
authors: KJ
authors: Jeong
mesh_terms: D000906:Antibodies / Q000032:analysis / Q000235:genetics; D019677:Artificial Gene Fusion; D048230:Corynebacterium glutamicum / Q000235:genetics*; D043364:Endo-1,4-beta Xylanases / Q000032:analysis / Q000235:genetics; D005434:Flow Cytometry; D015870:Gene Expression; D020869:Gene Expression Profiling; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D060847:Metabolic Engineering / Q000379:methods*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics
chemical_list: D000906:Antibodies; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D043364:Endo-1,4-beta Xylanases
keywords: Corynebacterium glutamicum; FACS; secretion; synthetic promoter