Identification of gene disruptions for increased poly-3-hydroxybutyrate accumulation in Synechocystis PCC 6803
Inverse metabolic engineering (IME) is a combinatorial approach for identifying genotypes associated with a particular phenotype of interest. In this study, gene disruptions that increase the biosynthesis of poly‐3‐hydroxybutyrate (PHB) in the photosynthetic bacterium Synechocystis PCC6803 were iden...
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| Veröffentlicht in: | Biotechnology progress 2009-09, Vol.25 (5), p.1236-1243 |
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| creator | Tyo, Keith E. J. Jin, Yong-Su Espinoza, Freddy A. Stephanopoulos, Gregory |
| description | Inverse metabolic engineering (IME) is a combinatorial approach for identifying genotypes associated with a particular phenotype of interest. In this study, gene disruptions that increase the biosynthesis of poly‐3‐hydroxybutyrate (PHB) in the photosynthetic bacterium Synechocystis PCC6803 were identified. A Synechocystis mutant library was constructed by homologous recombination between the Synechocystis genome and a mutagenized genomic plasmid library generated through transposon insertion. Using a fluorescence‐activated cell sorting‐based high throughput screen, high PHB accumulating mutants from the library grown in different nutrient conditions were isolated and characterized. While several mutants isolated from the screen had increased PHB accumulation, transposon insertions in only two ORFs could be linked to increased PHB production. Disruptions of sll0461, coding for gamma‐glutamyl phosphate reductase (proA), and sll0565, a hypothetical protein, resulted in increased accumulation in standard growth media and acetate supplemented media. These genetic perturbations have increased PHB accumulation in Synechocystis and serve as markers for engineering increased polymer production in higher photosynthetic organisms. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 |
| doi_str_mv | 10.1002/btpr.228 |
| format | Article |
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J. ; Jin, Yong-Su ; Espinoza, Freddy A. ; Stephanopoulos, Gregory</creator><creatorcontrib>Tyo, Keith E. J. ; Jin, Yong-Su ; Espinoza, Freddy A. ; Stephanopoulos, Gregory</creatorcontrib><description>Inverse metabolic engineering (IME) is a combinatorial approach for identifying genotypes associated with a particular phenotype of interest. In this study, gene disruptions that increase the biosynthesis of poly‐3‐hydroxybutyrate (PHB) in the photosynthetic bacterium Synechocystis PCC6803 were identified. A Synechocystis mutant library was constructed by homologous recombination between the Synechocystis genome and a mutagenized genomic plasmid library generated through transposon insertion. Using a fluorescence‐activated cell sorting‐based high throughput screen, high PHB accumulating mutants from the library grown in different nutrient conditions were isolated and characterized. While several mutants isolated from the screen had increased PHB accumulation, transposon insertions in only two ORFs could be linked to increased PHB production. Disruptions of sll0461, coding for gamma‐glutamyl phosphate reductase (proA), and sll0565, a hypothetical protein, resulted in increased accumulation in standard growth media and acetate supplemented media. These genetic perturbations have increased PHB accumulation in Synechocystis and serve as markers for engineering increased polymer production in higher photosynthetic organisms. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009</description><identifier>ISSN: 8756-7938</identifier><identifier>ISSN: 1520-6033</identifier><identifier>EISSN: 1520-6033</identifier><identifier>DOI: 10.1002/btpr.228</identifier><identifier>PMID: 19606467</identifier><identifier>CODEN: BIPRET</identifier><language>eng</language><publisher>Hoboken: Wiley Subscription Services, Inc., A Wiley Company</publisher><subject>Aldehyde Oxidoreductases - genetics ; Aldehyde Oxidoreductases - metabolism ; Bacterial Proteins - genetics ; Bacterial Proteins - metabolism ; Biological and medical sciences ; Biotechnology ; Cloning, Molecular - methods ; Combinatorial Chemistry Techniques - methods ; Culture Media - chemistry ; Culture Media - metabolism ; DNA Transposable Elements - genetics ; Flow Cytometry ; fluorescence-activated cell sorting (FACS) ; Fundamental and applied biological sciences. 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J.</creatorcontrib><creatorcontrib>Jin, Yong-Su</creatorcontrib><creatorcontrib>Espinoza, Freddy A.</creatorcontrib><creatorcontrib>Stephanopoulos, Gregory</creatorcontrib><title>Identification of gene disruptions for increased poly-3-hydroxybutyrate accumulation in Synechocystis PCC 6803</title><title>Biotechnology progress</title><addtitle>Biotechnol Progress</addtitle><description>Inverse metabolic engineering (IME) is a combinatorial approach for identifying genotypes associated with a particular phenotype of interest. In this study, gene disruptions that increase the biosynthesis of poly‐3‐hydroxybutyrate (PHB) in the photosynthetic bacterium Synechocystis PCC6803 were identified. A Synechocystis mutant library was constructed by homologous recombination between the Synechocystis genome and a mutagenized genomic plasmid library generated through transposon insertion. Using a fluorescence‐activated cell sorting‐based high throughput screen, high PHB accumulating mutants from the library grown in different nutrient conditions were isolated and characterized. While several mutants isolated from the screen had increased PHB accumulation, transposon insertions in only two ORFs could be linked to increased PHB production. Disruptions of sll0461, coding for gamma‐glutamyl phosphate reductase (proA), and sll0565, a hypothetical protein, resulted in increased accumulation in standard growth media and acetate supplemented media. These genetic perturbations have increased PHB accumulation in Synechocystis and serve as markers for engineering increased polymer production in higher photosynthetic organisms. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009</description><subject>Aldehyde Oxidoreductases - genetics</subject><subject>Aldehyde Oxidoreductases - metabolism</subject><subject>Bacterial Proteins - genetics</subject><subject>Bacterial Proteins - metabolism</subject><subject>Biological and medical sciences</subject><subject>Biotechnology</subject><subject>Cloning, Molecular - methods</subject><subject>Combinatorial Chemistry Techniques - methods</subject><subject>Culture Media - chemistry</subject><subject>Culture Media - metabolism</subject><subject>DNA Transposable Elements - genetics</subject><subject>Flow Cytometry</subject><subject>fluorescence-activated cell sorting (FACS)</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Gene Library</subject><subject>Hydroxybutyrates - metabolism</subject><subject>inverse metabolic engineering</subject><subject>Mutation</subject><subject>Plasmids - genetics</subject><subject>Polyesters - metabolism</subject><subject>polyhydroxyalkanoate</subject><subject>Synechocystis</subject><subject>Synechocystis - enzymology</subject><subject>Synechocystis - genetics</subject><subject>Synechocystis - metabolism</subject><subject>Systems Biology - methods</subject><subject>transposon mutagenesis</subject><issn>8756-7938</issn><issn>1520-6033</issn><issn>1520-6033</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2009</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqF0U2L1DAcBvAgijuugp9AchG9dM1L89KjjjouLDq7rugtpMm_brTT1KTF7be3Q8t6Ek-B8ON5SB6EnlJyRglhr-qhT2eM6XtoQwUjhSSc30cbrYQsVMX1CXqU8w9CiCaSPUQntJJEllJtUHfuoRtCE5wdQuxwbPB36AD7kNPYH68ybmLCoXMJbAaP-9hOBS9uJp_i7VSPw5TsANg6Nx7GdkkJHf48deBuopvyEDLeb7dYasIfoweNbTM8Wc9T9OX9u-vth-Li0-58-_qicIJqXTBW11CBFY6X4GpX0ZJ6sLQsvaCSACNKQa2ACMp42ZSU1b4G75xUwoJv-Cl6seT2Kf4aIQ_mELKDtrUdxDEbrTUlFeH6_1JWQomKqVm-XKRLMecEjelTONg0GUrMcQZznMHMM8z02Ro61gfwf-H67zN4vgKbnW2bZDsX8p1j8wOFJtXsisX9Di1M_yw0b673V0vx6kMe4PbO2_TTzK1KmK8fd2Z3ebXnb-U3c8n_AHpCr50</recordid><startdate>200909</startdate><enddate>200909</enddate><creator>Tyo, Keith E. 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Psychology</topic><topic>Gene Library</topic><topic>Hydroxybutyrates - metabolism</topic><topic>inverse metabolic engineering</topic><topic>Mutation</topic><topic>Plasmids - genetics</topic><topic>Polyesters - metabolism</topic><topic>polyhydroxyalkanoate</topic><topic>Synechocystis</topic><topic>Synechocystis - enzymology</topic><topic>Synechocystis - genetics</topic><topic>Synechocystis - metabolism</topic><topic>Systems Biology - methods</topic><topic>transposon mutagenesis</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Tyo, Keith E. 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J.</au><au>Jin, Yong-Su</au><au>Espinoza, Freddy A.</au><au>Stephanopoulos, Gregory</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Identification of gene disruptions for increased poly-3-hydroxybutyrate accumulation in Synechocystis PCC 6803</atitle><jtitle>Biotechnology progress</jtitle><addtitle>Biotechnol Progress</addtitle><date>2009-09</date><risdate>2009</risdate><volume>25</volume><issue>5</issue><spage>1236</spage><epage>1243</epage><pages>1236-1243</pages><issn>8756-7938</issn><issn>1520-6033</issn><eissn>1520-6033</eissn><coden>BIPRET</coden><abstract>Inverse metabolic engineering (IME) is a combinatorial approach for identifying genotypes associated with a particular phenotype of interest. In this study, gene disruptions that increase the biosynthesis of poly‐3‐hydroxybutyrate (PHB) in the photosynthetic bacterium Synechocystis PCC6803 were identified. A Synechocystis mutant library was constructed by homologous recombination between the Synechocystis genome and a mutagenized genomic plasmid library generated through transposon insertion. Using a fluorescence‐activated cell sorting‐based high throughput screen, high PHB accumulating mutants from the library grown in different nutrient conditions were isolated and characterized. While several mutants isolated from the screen had increased PHB accumulation, transposon insertions in only two ORFs could be linked to increased PHB production. Disruptions of sll0461, coding for gamma‐glutamyl phosphate reductase (proA), and sll0565, a hypothetical protein, resulted in increased accumulation in standard growth media and acetate supplemented media. These genetic perturbations have increased PHB accumulation in Synechocystis and serve as markers for engineering increased polymer production in higher photosynthetic organisms. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009</abstract><cop>Hoboken</cop><pub>Wiley Subscription Services, Inc., A Wiley Company</pub><pmid>19606467</pmid><doi>10.1002/btpr.228</doi><tpages>8</tpages></addata></record> |
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| ispartof | Biotechnology progress, 2009-09, Vol.25 (5), p.1236-1243 |
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| source | MEDLINE; Wiley Online Library Journals Frontfile Complete |
| subjects | Aldehyde Oxidoreductases - genetics Aldehyde Oxidoreductases - metabolism Bacterial Proteins - genetics Bacterial Proteins - metabolism Biological and medical sciences Biotechnology Cloning, Molecular - methods Combinatorial Chemistry Techniques - methods Culture Media - chemistry Culture Media - metabolism DNA Transposable Elements - genetics Flow Cytometry fluorescence-activated cell sorting (FACS) Fundamental and applied biological sciences. Psychology Gene Library Hydroxybutyrates - metabolism inverse metabolic engineering Mutation Plasmids - genetics Polyesters - metabolism polyhydroxyalkanoate Synechocystis Synechocystis - enzymology Synechocystis - genetics Synechocystis - metabolism Systems Biology - methods transposon mutagenesis |
| title | Identification of gene disruptions for increased poly-3-hydroxybutyrate accumulation in Synechocystis PCC 6803 |
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