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.

Construction of Synthetic Promoter-Based Expression Cassettes for the Production of Cadaverine in Recombinant Corynebacterium glutamicum

“…In many cases, how to combine target genes with suitable promoters is the first key step to determine gene expression levels that can ultimately affect the production capacities. Recently, a series of synthetic promoters were isolated and characterized for gene expression in C. glutamicum [13]. Up to our knowledge, it was the first report of comparing and examining different kinds of promoters and their contributions to gene expression levels in C. glutamicum.…”

“…C. glutamicum KCTC 1857 (Korean Collection for Type Cultures, Korea), a mutant strain of C. glutamicum ATCC 13032 by a Japanese company, Kyowa Hakko Kogyo Co., Ltd. (Tokyo, Japan), was used as the host strain for the production of cadaverine [20]. Plasmids pCES208L10GFP, pCES208L26GFP, pCES208I16GFP, pCES208I64GFP, pCES208H30GFP, and pCES208H36GFP have been previously described [13].…”

“…Therefore, herein, we have developed gene expression cassettes for C. glutamicum employing six synthetic promoters having different strengths, which were previously isolated and characterized [13], to express the E. coli ldcC gene for the production of cadaverine. Six LDC expression vectors and accompanying recombinant C. glutamicum strains were constructed and examined to determine which expression cassette is the most efficient for high-level production of cadaverine.…”

“…In this respect, a lot of studies have been conducted focusing on searching and developing efficient engineering tools of C. glutamicum including native or non-native promoters, expression vectors, promoter-prove vectors, and reporter genes [9][10][11][12]. However, lack of knowledge on how to constitute promoters and target genes for efficient production of products still remains one of main barriers to develop engineered C. glutamicum strains having desired features [13].…”

Combinatorial optimization of CO2 transport and fixation to improve succinate production by promoter engineering

“…Many promoter engineering strategies, such as Ep-PCR (Alper et al, 2005;Kagiya et al, 2005;Nevoigt et al, 2006), randomization of nonconserved sequences (Jensen and Hammer, 1998;Siegl et al, 2013;Yim et al, 2013), hybrid promoter engineering (Blazeck et al, 2011(Blazeck et al, , 2012Brown et al, 2014), TFBS modification (Hartner et al, 2008;Murphy et al, 2007), energy matrix (Kinney et al, 2010) were adopted for promoter library building. In the above methods, the first four methods can obtain a wide range of promoter strength as long as the library is big enough.…”