Identification of gene disruptions for increased poly-3-hydroxybutyrate accumulation in synechocystis PCC 6803

Keith E J Tyo, Freddy A. Espinoza, Gregory Stephanopoulos*, Yong Su Jin

*Corresponding author for this work

Research output: Contribution to journalArticle

32 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)1236-1243
Number of pages8
JournalBiotechnology Progress
Volume25
Issue number5
DOIs
StatePublished - Sep 1 2009

Keywords

  • Fluorescence-activated cell sorting (FACS)
  • Inverse metabolic engineering
  • Polyhydroxyalkanoate
  • Synechocystis
  • Transposon mutagenesis

ASJC Scopus subject areas

  • Biotechnology

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