title: Genetic engineering and metabolite profiling for overproduction of polyhydroxybutyrate in cyanobacteria.
abstract: Genetic engineering and metabolite profiling for the overproduction of polyhydroxybutyrate (PHB), which is a carbon material in biodegradable plastics, were examined in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Transconjugants harboring cyanobacterial expression vectors that carried the pha genes for PHB biosynthesis were constructed. The overproduction of PHB by the engineering cells was confirmed through microscopic observations using Nile red, transmission electron microscopy (TEM), or nuclear magnetic resonance (NMR). We successfully recovered PHB from transconjugants prepared from nitrogen-depleted medium without sugar supplementation in which PHB reached approximately 7% (w/w) of the dry cell weight, showing a value of 12-fold higher productivity in the transconjugant than that in the control strain. We also measured the intracellular levels of acetyl-CoA, acetoacetyl-CoA, and 3-hydroxybutyryl-CoA (3HB-CoA), which are intermediate products for PHB. The results obtained indicated that these products were absent or at markedly low levels when cells were subjected to the steady-state growth phase of cultivation under nitrogen depletion for the overproduction of bioplastics. Based on these results, efficient factors were discussed for the overproduction of PHB in recombinant cyanobacteria.
authors.affiliation: College of Agriculture, Ibaraki University, 3-21-1 Ami, Ibaraki 300-0393, Japan.
authors.forename: Sayaka
authors.initials: S
authors.lastname: Hondo
authors.affiliation: College of Agriculture, Ibaraki University, 3-21-1 Ami, Ibaraki 300-0393, Japan.
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Takahashi
authors.affiliation: Japan Science and Technology Agency (JST), Precursory Research for Embryonic Science and Technology (PRESTO), 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan; RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Yokohama 230-0045, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Osanai
authors.affiliation: Japan Science and Technology Agency (JST), Precursory Research for Embryonic Science and Technology (PRESTO), 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan; Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkodai, Kobe 657-8501, Japan.
authors.forename: Mami
authors.initials: M
authors.lastname: Matsuda
authors.affiliation: Japan Science and Technology Agency (JST), Precursory Research for Embryonic Science and Technology (PRESTO), 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan; Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkodai, Kobe 657-8501, Japan.
authors.forename: Tomohisa
authors.initials: T
authors.lastname: Hasunuma
authors.affiliation: College of Agriculture, Ibaraki University, 3-21-1 Ami, Ibaraki 300-0393, Japan.
authors.forename: Akio
authors.initials: A
authors.lastname: Tazuke
authors.affiliation: College of Agriculture, Ibaraki University, 3-21-1 Ami, Ibaraki 300-0393, Japan.
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Nakahira
authors.affiliation: College of Agriculture, Ibaraki University, 3-21-1 Ami, Ibaraki 300-0393, Japan.
authors.forename: Shigeru
authors.initials: S
authors.lastname: Chohnan
authors.affiliation: College of Agriculture, Ibaraki University, 3-21-1 Ami, Ibaraki 300-0393, Japan.
authors.forename: Morifumi
authors.initials: M
authors.lastname: Hasegawa
authors.affiliation: College of Agriculture, Ibaraki University, 3-21-1 Ami, Ibaraki 300-0393, Japan; Japan Science and Technology Agency (JST), Precursory Research for Embryonic Science and Technology (PRESTO), 4-1-8 Honcho Kawaguchi, Saitama 332-0012, Japan. Electronic address: asam@mx.ibaraki.ac.jp.
authors.forename: Munehiko
authors.initials: M
authors.lastname: Asayama
mesh_terms: D000105:Acetyl Coenzyme A / Q000032:analysis / Q000378:metabolism; D000214:Acyl Coenzyme A / Q000032:analysis / Q000378:metabolism; D003227:Conjugation, Genetic; D005818:Genetic Engineering; D006885:Hydroxybutyrates / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D055442:Metabolome; D046529:Microscopy, Electron, Transmission; D009584:Nitrogen / Q000172:deficiency; D010078:Oxazines; D046939:Synechocystis / Q000235:genetics* / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D000214:Acyl Coenzyme A; D006885:Hydroxybutyrates; D010078:Oxazines; C010667:acetoacetyl CoA; C030372:3-hydroxybutyryl-coenzyme A; D000105:Acetyl Coenzyme A; D009584:Nitrogen; C044808:nile red
keywords: Acetyl-CoA; Cyanobacteria; Metabolite analysis; Nitrogen depletion; Polyhydroxybutyrate