Maximizing PHB content in Synechocystis sp . PCC 6803: a new metabolic engineering strategy based on the regulator PirC
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Microbial Cell Factories Open Access
RESEARCH
Maximizing PHB content in Synechocystis sp. PCC 6803: a new metabolic engineering strategy based on the regulator PirC Moritz Koch1 , Jonas Bruckmoser2, Jörg Scholl1, Waldemar Hauf1, Bernhard Rieger2 and Karl Forchhammer1*
Abstract Background: PHB (poly-hydroxy-butyrate) represents a promising bioplastic alternative with good biodegradation properties. Furthermore, PHB can be produced in a completely carbon–neutral fashion in the natural producer cyanobacterium Synechocystis sp. PCC 6803. This strain has been used as model system in past attempts to boost the intracellular production of PHB above ~ 15% per cell-dry-weight (CDW). Results: We have created a new strain that lacks the regulatory protein PirC (product of sll0944), which exhibits a higher activity of the phosphoglycerate mutase resulting in increased PHB pools under nutrient limiting conditions. To further improve the intracellular PHB content, two genes involved in PHB metabolism, phaA and phaB, from the known producer strain Cupriavidus necator, were introduced under the control of the strong promotor PpsbA2. The resulting strain, termed PPT1 (ΔpirC-REphaAB), produced high amounts of PHB under continuous light as well under a day-night regime. When grown in nitrogen and phosphorus depleted medium, the cells produced up to 63% per CDW. Upon the addition of acetate, the content was further increased to 81% per CDW. The produced polymer consists of pure PHB, which is highly isotactic. Conclusion: The amounts of PHB achieved with PPT1 are the highest ever reported in any known cyanobacterium and demonstrate the potential of cyanobacteria for a sustainable, industrial production of PHB. Keywords: Cyanobacteria, PHB, Metabolic engineering, Synechocystis 6803, Biopolymers, Sustainable Introduction The global contamination with non-degradable plastic is a huge environmental burden of our time [15, 29]. While bioplastics have been suggested as potential solution, they still represent only a very small fraction of the plastics overall used [11]. Furthermore, many of these bioplastics have unsatisfying biodegradation properties. The most common bioplastic, PLA (polylactic-acid), is barely degraded in marine environments [34]. This has led to an increasing interest into *Correspondence: karl.forchhammer@uni‑tuebingen.de 1 Interfaculty Institute of Microbiology and Infection Medicine Tübingen, Eberhard-Karls-Universität Tübingen, Tübingen, Germany Full list of author information is available at the end of the article
another class of bioplastics with improved degradation properties: poly-hydroxy-alkanoates (PHAs). The most common variant of this chemical class is poly-hydroxybutyrate (PHB) which is produced by various microorganisms. Currently, PHB is produced by fermentation using heterotrophic bacteria, such as Cupriavidus necator or Escherichia coli [6]. However, these production processes require crop-derived organic carbon sources for growth and production and pose a threat to human food-supply. An alternat
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