title: Identification of a Cyanobacterial RND-Type Efflux System Involved in Export of Free Fatty Acids.
abstract: An RND (resistance-nodulation-division)-type transporter having the capacity to export free fatty acids (FFAs) was identified in the cyanobacterium Synechococcus elongatus strain PCC 7942 during characterization of a mutant strain engineered to produce FFAs. The basic strategy for construction of the FFA-producing mutant was a commonly used one, involving inactivation of the endogenous acyl-acyl carrier protein synthetase gene (aas) and introduction of a foreign thioesterase gene ('tesA), but a nitrate transport mutant NA3 was used as the parental strain to achieve slow, nitrate-limited growth in batch cultures. Also, a nitrogen-regulated promoter PnirA was used to drive 'tesA to maximize thioesterase expression during the nitrate-limited growth. The resulting mutant (dAS2T) was, however, incapable of growth under the conditions of nitrate limitation, presumably due to toxicity associated with FFA overproduction. Incubation of the mutant culture under the non-permissive conditions allowed for isolation of a pseudorevertant (dAS2T-pr1) capable of growth on nitrate. Genome sequence and gene expression analyses of this strain suggested that expression of an RND-type efflux system had rescued growth on nitrate. Targeted inactivation of the RND-type transporter genes in the wild-type strain resulted in loss of tolerance to exogenously added FFAs including capric, lauric, myristic, oleic and linolenic acids. Overexpression of the genes in dAS2T, on the other hand, enhanced FFA excretion and cell growth in nitrate-containing medium, verifying that the genes encode an efflux pump for FFAs. These results demonstrate the importance of the efflux system in efficient FFA production using genetically engineered cyanobacteria.
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601 Japan.
authors.forename: Akihiro
authors.initials: A
authors.lastname: Kato
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601 Japan Japan Science and Technology Agency, CREST.
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Takatani
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601 Japan.
authors.forename: Kazuhide
authors.initials: K
authors.lastname: Use
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601 Japan Japan Science and Technology Agency, CREST.
authors.forename: Kazuma
authors.initials: K
authors.lastname: Uesaka
authors.affiliation: Institute for Advanced Biosciences, Keio University, Yamagata, 997-0052 Japan Japan Science and Technology Agency, CREST Present address: Laboratory for Metabolomics, RIKEN Center for Integrative Medical Sciences, Yokohama, 230-0045 Japan.
authors.forename: Kazutaka
authors.initials: K
authors.lastname: Ikeda
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601 Japan Japan Science and Technology Agency, CREST Present address: Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing 210014, Jiangsu, PR China.
authors.forename: Yajun
authors.initials: Y
authors.lastname: Chang
authors.affiliation: Department of Biological Chemistry, Chubu University, Kasugai, 487-8501 Japan Japan Science and Technology Agency, CREST Present address: 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.
authors.forename: Makiko
authors.initials: M
authors.lastname: Aichi
authors.affiliation: Center for Gene Research, Nagoya University, Nagoya, 464-8602 Japan Japan Science and Technology Agency, CREST.
authors.forename: Kunio
authors.initials: K
authors.lastname: Ihara
authors.affiliation: Institute for Advanced Biosciences, Keio University, Yamagata, 997-0052 Japan Japan Science and Technology Agency, CREST.
authors.forename: Kenji
authors.initials: K
authors.lastname: Nakahigashi
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601 Japan Japan Science and Technology Agency, CREST.
authors.forename: Shin-Ichi
authors.initials: S
authors.lastname: Maeda
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601 Japan Japan Science and Technology Agency, CREST omata@agr.nagoya-u.ac.jp.
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Omata
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001692:Biological Transport; D005230:Fatty Acids, Nonesterified / Q000378:metabolism*; D017343:Genes, Plant; D009154:Mutation / Q000235:genetics; D009566:Nitrates / Q000378:metabolism; D010802:Phylogeny; D046940:Synechococcus / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D005230:Fatty Acids, Nonesterified; D009566:Nitrates
keywords: Biofuel production; Cyanobacteria; Free fatty acid; RND-type efflux system