title: Prerequisite for highly efficient isoprenoid production by cyanobacteria discovered through the over-expression of 1-deoxy-d-xylulose 5-phosphate synthase and carbon allocation analysis.
abstract: Cyanobacteria have recently been receiving considerable attention owing to their potential as photosynthetic producers of biofuels and biomaterials. Here, we focused on the production of isoprenoids by cyanobacteria, and aimed to provide insight into metabolic engineering design. To this end, we examined the over-expression of a key enzyme in 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, 1-deoxy-d-xylulose 5-phosphate synthase (DXS) in the cyanobacterium Synechocystis sp. PCC6803. In the DXS-over-expression strain (Dxs_ox), the mRNA and protein levels of DXS were 4-times and 1.5-times the levels in the wild-type (WT) strain, respectively. The carotenoid content of the Dxs_ox strain (8.4 mg/g dry cell weight [DCW]) was also up to 1.5-times higher than that in the WT strain (5.6 mg/g DCW), whereas the glycogen content dramatically decreased to an undetectable level. These observations suggested that the carotenoid content in the Dxs_ox strain was increased by consuming glycogen, which is a C-storage compound in cyanobacteria. We also quantified the total sugar (145 and 104 mg/g DCW), total fatty acids (31 and 24 mg/g DCW) and total protein (200 and 240 mg/g DCW) content in the WT and Dxs_ox strains, respectively, which were much higher than the carotenoid content. In particular, approximately 54% of the proteins were phycobiliproteins. This study demonstrated the major destinations of carbon flux in cyanobacteria, and provided important insights into metabolic engineering. Target yield can be improved through optimization of gene expression, the DXS protein stabilization, cell propagation depression and restriction of storage compound synthesis.
authors: Faculty of Agriculture, Shinshu University, 8403 Minamiminowa, Nagano 399-4511, Japan.
authors: Kai
authors: K
authors: Kudoh
authors: Faculty of Agriculture, Shinshu University, 8403 Minamiminowa, Nagano 399-4511, Japan; Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan.
authors: Yusuke
authors: Y
authors: Kawano
authors: Faculty of Agriculture, Shinshu University, 8403 Minamiminowa, Nagano 399-4511, Japan.
authors: Shingo
authors: S
authors: Hotta
authors: Faculty of Agriculture, Shinshu University, 8403 Minamiminowa, Nagano 399-4511, Japan.
authors: Midori
authors: M
authors: Sekine
authors: Faculty of Agriculture, Shinshu University, 8403 Minamiminowa, Nagano 399-4511, Japan.
authors: Takafumi
authors: T
authors: Watanabe
authors: Faculty of Agriculture, Shinshu University, 8403 Minamiminowa, Nagano 399-4511, Japan; Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan. Electronic address: m_ihara@shinshu-u.ac.jp.
authors: Masaki
authors: M
authors: Ihara
mesh_terms: D001426:Bacterial Proteins / Q000032:analysis; D002241:Carbohydrates / Q000032:analysis; D002244:Carbon / Q000378:metabolism; D002338:Carotenoids / Q000096:biosynthesis; D004896:Erythritol / Q000031:analogs & derivatives / Q000378:metabolism; D015870:Gene Expression; D008055:Lipids / Q000032:analysis; D010428:Pentosephosphates / Q000378:metabolism; D013403:Sugar Phosphates / Q000378:metabolism; D046939:Synechocystis / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D013729:Terpenes / Q000378:metabolism*; D014166:Transferases / Q000235:genetics / Q000378:metabolism*
chemical_list: C109460:1-deoxylulose 5-phosphate; C114232:2-C-methylerythritol 4-phosphate; D001426:Bacterial Proteins; D002241:Carbohydrates; D008055:Lipids; D010428:Pentosephosphates; D013403:Sugar Phosphates; D013729:Terpenes; D002338:Carotenoids; C031625:xylulose-5-phosphate; D002244:Carbon; D014166:Transferases; D004896:Erythritol
keywords: 1-Deoxy-d-xylulose 5-phosphate synthase; 2-C-Methyl-d-erythritol 4-phosphate pathway; Carbon flux; Carotenoid; Cyanobacteria; Isoprenoid; Metabolic engineering