title: Overexpression of endogenous 1-deoxy-d-xylulose 5-phosphate synthase (DXS) in cyanobacterium Synechocystis sp. PCC6803 accelerates protein aggregation.
abstract: 1-Deoxy-d-xylulose 5-phosphate synthase (DXS) is a rate-limiting enzyme in the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, which is responsible for the production of precursors of all isoprenoids. In a previous study, we had examined the overexpression of an endogenous DXS in a Synechocystis sp. PCC6803 mutant (DXS_ox), and found that the dxs mRNA level was 4-fold higher than that in the wild-type (WT) strain. However, the DXS protein level was only 1.5-fold higher, leading to the assumption that the level might be regulated by post-transcriptional events. In this study, we have additionally introduced an exogenous isoprene synthase (IspS; which can release MEP pathway products from the cell as gaseous isoprene) into the WT and DXS_ox strains (WT-isP and DXSox-isP strains, respectively), and their detailed DXS expression profiles were investigated from the induction phase through to the late-logarithmic phase. In the induction phase, the isoprene productivity of the DXSox-isP strain was slightly but significantly (1.4- to 1.8-fold) higher than that of the WT-isP strain, whereas the levels were comparable in the other phases. Interestingly, the ratios of soluble:insoluble DXS protein were remarkably low in the DXSox-isP strain during the induction phase to the early-logarithmic phase, resulting in a moderate level of soluble DXS. All our results suggested that the high translation rate of DXS disturbs the refolding process of DXS. To enhance the concentration of the active DXS in cyanobacteria, the enhancement of the DXS maturation system or the introduction of exogenous and robust DXS proteins might be necessary.
authors.affiliation: Department of Bioscience and Food Production Science, Interdisciplinary Graduate School of Science and Technology, Shinshu University, 8304 Minamiminowa, Nagano 399-4511, Japan.
authors.forename: Kai
authors.initials: K
authors.lastname: Kudoh
authors.affiliation: Faculty of Agriculture, Shinshu University, 8304 Minamiminowa, Nagano 399-4511, Japan.
authors.forename: Shingo
authors.initials: S
authors.lastname: Hotta
authors.affiliation: Faculty of Agriculture, Shinshu University, 8304 Minamiminowa, Nagano 399-4511, Japan.
authors.forename: Midori
authors.initials: M
authors.lastname: Sekine
authors.affiliation: Faculty of Agriculture, Shinshu University, 8304 Minamiminowa, Nagano 399-4511, Japan.
authors.forename: Rintaro
authors.initials: R
authors.lastname: Fujii
authors.affiliation: Faculty of Agriculture, Shinshu University, 8304 Minamiminowa, Nagano 399-4511, Japan.
authors.forename: Arisu
authors.initials: A
authors.lastname: Uchida
authors.affiliation: Faculty of Agriculture, Shinshu University, 8304 Minamiminowa, Nagano 399-4511, Japan.
authors.forename: Genma
authors.initials: G
authors.lastname: Kubota
authors.affiliation: Innovation Medical Research Institute, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Kawano
authors.affiliation: Faculty of Agriculture, Shinshu University, 8304 Minamiminowa, Nagano 399-4511, Japan; JST, PRESTO, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan; Department of Interdisciplinary Genome Sciences and Cell Metabolism, Institute for Biomedical Sciences, Interdisciplinary Cluster for Cutting Edge Research, Shinshu University, 8304 Minamiminowa, Kamiina, Nagano 399-4598, Japan. Electronic address: m_ihara@shinshu-u.ac.jp.
authors.forename: Masaki
authors.initials: M
authors.lastname: Ihara
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D002070:Butadienes; D004896:Erythritol / Q000031:analogs & derivatives / Q000378:metabolism; D005740:Gases / Q000378:metabolism; D045782:Hemiterpenes / Q000096:biosynthesis; D060847:Metabolic Engineering; D010420:Pentanes; D010428:Pentosephosphates / Q000096:biosynthesis; D066329:Protein Aggregates; D012333:RNA, Messenger / Q000032:analysis; D012995:Solubility; D013403:Sugar Phosphates / Q000378:metabolism; D046939:Synechocystis / Q000235:genetics* / Q000378:metabolism*; D013729:Terpenes / Q000378:metabolism; D014166:Transferases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*
chemical_list: C109460:1-deoxylulose 5-phosphate; C114232:2-C-methylerythritol 4-phosphate; D002070:Butadienes; D005740:Gases; D045782:Hemiterpenes; D010420:Pentanes; D010428:Pentosephosphates; D066329:Protein Aggregates; D012333:RNA, Messenger; D013403:Sugar Phosphates; D013729:Terpenes; C005059:isoprene; D014166:Transferases; C109461:deoxyxylulose-5-phosphate synthase; D019883:Alkyl and Aryl Transferases; C093854:isoprene synthase; D004896:Erythritol
keywords: 1-Deoxyxylulose 5-phosphate synthase; Isoprene; MEP pathway; Metabolic engineering; Protein solubility