title: A simple method for isolation and construction of markerless cyanobacterial mutants defective in acyl-acyl carrier protein synthetase.
abstract: Cyanobacterial mutants defective in acyl-acyl carrier protein synthetase (Aas) secrete free fatty acids (FFAs) into the external medium and hence have been used for the studies aimed at photosynthetic production of biofuels. While the wild-type strain of Synechocystis sp. PCC 6803 is highly sensitive to exogenously added linolenic acid, mutants defective in the aas gene are known to be resistant to the externally provided fatty acid. In this study, the wild-type Synechocystis cells were shown to be sensitive to lauric, oleic, and linoleic acids as well, and the resistance to these fatty acids was shown to be enhanced by inactivation of the aas gene. On the basis of these observations, we developed an efficient method to isolate aas-deficient mutants from cultures of Synechocystis cells by counter selection using linoleic acid or linolenic acid as the selective agent. A variety of aas mutations were found in about 70 % of the FFA-resistant mutants thus selected. Various aas mutants were isolated also from Synechococcus sp. PCC 7002, using lauric acid as a selective agent. Selection using FFAs was useful also for construction of markerless aas knockout mutants from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002. Thus, genetic engineering of FFA-producing cyanobacterial strains would be greatly facilitated by the use of the FFAs for counter selection.
authors.affiliation: Department of Biological Chemistry, Chubu University, Kasugai, 487-8501, Japan.|Japan Science and Technology Agency, CREST, Kawaguchi, Japan.|Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601, Japan.
authors.forename: Kouji
authors.initials: K
authors.lastname: Kojima
authors.affiliation: Department of Biological Chemistry, Chubu University, Kasugai, 487-8501, Japan.|Japan Science and Technology Agency, CREST, Kawaguchi, Japan.
authors.forename: Sumie
authors.initials: S
authors.lastname: Keta
authors.affiliation: Japan Science and Technology Agency, CREST, Kawaguchi, Japan.|Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601, Japan.
authors.forename: Kazuma
authors.initials: K
authors.lastname: Uesaka
authors.affiliation: Japan Science and Technology Agency, CREST, Kawaguchi, Japan.|Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601, Japan.
authors.forename: Akihiro
authors.initials: A
authors.lastname: Kato
authors.affiliation: Japan Science and Technology Agency, CREST, Kawaguchi, Japan.|Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601, Japan.
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Takatani
authors.affiliation: Japan Science and Technology Agency, CREST, Kawaguchi, Japan.|Center for Gene Research, Nagoya University, Nagoya, 464-8602, Japan.
authors.forename: Kunio
authors.initials: K
authors.lastname: Ihara
authors.affiliation: Japan Science and Technology Agency, CREST, Kawaguchi, Japan.|Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601, Japan.
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Omata
authors.affiliation: Department of Biological Chemistry, Chubu University, Kasugai, 487-8501, Japan. makiko@isc.chubu.ac.jp.|Japan Science and Technology Agency, CREST, Kawaguchi, Japan. makiko@isc.chubu.ac.jp.
authors.forename: Makiko
authors.initials: M
authors.lastname: Aichi
mesh_terms: D019730:Carbon-Sulfur Ligases / Q000172:deficiency*; D024881:Drug Resistance, Bacterial; D017353:Gene Deletion; D007850:Lauric Acids / Q000633:toxicity; D019787:Linoleic Acid / Q000633:toxicity; D009154:Mutation; D012641:Selection, Genetic; D046940:Synechococcus / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D046939:Synechocystis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics
chemical_list: D007850:Lauric Acids; C030358:lauric acid; D019787:Linoleic Acid; D019730:Carbon-Sulfur Ligases; C018749:long-chain-fatty-acid-(acyl-carrier-protein) ligase
keywords: Acyl-ACP synthetase; Biofuel; Counter selection; Cyanobacteria; Free-fatty acid; Sensitivity to FFAs