title: Development of a potential stationary-phase specific gene expression system by engineering of SigB-dependent cg3141 promoter in Corynebacterium glutamicum.
abstract: Corynebacterium glutamicum is a non-pathogenic, non-sporulating Gram-positive soil bacterium that has been used for the industrial production of various proteins and chemicals. To achieve enhanced and economical production of target molecules, the development of strong auto-inducible promoters is desired, which can be activated without expensive inducers and has significant advantages for industrial-scale use. Here, we developed a stationary-phase gene expression system by engineering a sigma factor B (SigB)-dependent promoter that can be activated during the transition phase between exponential and stationary growth phases in C. glutamicum. First, the inducibilities of three well-known SigB-dependent promoters were examined using super-folder green fluorescent protein as a reporter protein, and we found that promoter of cg3141 (P cg3141 ) exhibited the highest inducibility. Next, a synthetic promoter library was constructed by randomizing the flanking and space regions of P cg3141 , and the stationary-phase promoters exhibiting high strengths were isolated via FACS-based high-throughput screening. The isolated synthetic promoter (P4-N14) showed a 3.5-fold inducibility and up to 20-fold higher strength compared to those of the original cg3141 promoter. Finally, the use of the isolated P4-N14 for fed-batch cultivation was verified with the production of glutathione S-transferase as a model protein in a lab-scale (5-L) bioreactor.
authors.affiliation: Department of Chemical and Biomolecular Engineering, BK21 Plus Program, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea.
authors.forename: Min Jeong
authors.initials: MJ
authors.lastname: Kim
authors.affiliation: Department of Chemical and Biomolecular Engineering, BK21 Plus Program, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea.
authors.forename: Sung Sun
authors.initials: SS
authors.lastname: Yim
authors.affiliation: Department of Chemical and Biomolecular Engineering, BK21 Plus Program, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea.
authors.forename: Jae Woong
authors.initials: JW
authors.lastname: Choi
authors.affiliation: Department of Chemical and Biomolecular Engineering, BK21 Plus Program, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea. kjjeong@kaist.ac.kr.|Institute for the BioCentury, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon, 34141, Republic of Korea. kjjeong@kaist.ac.kr.
authors.forename: Ki Jun
authors.initials: KJ
authors.lastname: Jeong
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D048230:Corynebacterium glutamicum / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D015723:Gene Library; D005818:Genetic Engineering / Q000379:methods*; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D012808:Sigma Factor / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; C079011:SigB protein, Bacteria; D012808:Sigma Factor; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase
keywords: Corynebacterium glutamicum; FACS; Sigma factor B; Stationary-phase promoter; cg3141