Genetic and ultrastructural analysis of different mutants of Pseudomonas putida affected in the poly‐3‐hydroxy‐n‐alkanoate gene cluster

Abstract

Functional analyses of the different proteins involved in the synthesis and accumulation of polyhydroxyalkanoates (PHAs) in P. putida U were performed using a mutant in which the pha locus had been deleted (PpUDeltapha). These studies showed that: (i) Pha enzymes cannot be replaced by other proteins in this bacterium, (ii) the transformation of PpDeltapha with a plasmid containing the locus pha fully restores the synthesis of bioplastics, (iii) the transformation of PpDeltapha with a plasmid harbouring the gene encoding the polymerase PhaC1 (pMCphaC1) permits the synthesis of polyesters (even in absence of phaC2ZDFI); however, in this strain (PpUDeltapha-pMCphaC1) the number of PHAs granules was higher than in the wild type, (iv) the expression of phaF in PpUDeltapha-pMCphaC1 restores the original phenotype, showing that PhaF is involved in the coalescence of the PHAs granules. Furthermore, the deletion of the phaDFI genes in P. putida U considerably decreases (> 70%) the biosynthesis of PHAs consisting of hydroxyalkanoates with aliphatic constituents, and completely prevents the synthesis of those ones containing aromatic monomers. Additional experiments revealed that the deletion of phaD in P. putida U strongly reduces the synthesis of PHA, this effect being restored by PhaF. Moreover, the overexpression of phaF in P. putida U, or in its DeltafadBA mutant, led to the collection of PHA over-producer strains.

New insights in the formation of polyhydroxyalkanoate granules (carbonosomes) and novel functions of poly(3‐hydroxybutyrate)

“…Oligomer hydrolase ACT, (DEL), WB (partially soluble) (Kobayashi et al, 2003;2005;Uchino et al, 2008) phaZc* (phaY2) Oligomer hydrolase ACT, (DEL), WB (mostly soluble) (Kobayashi et al, 2003;2005;Uchino et al, 2008) Pseudomonas putida mcl-PHA phaC1 PHA synthase (ACT), PROT Stuart et al, 1996;Ren et al, 2009a) (Pseudomonas oleovorans) strains GP01, KT2442 phaC2 PHA synthase (not expressed in wt) (Ren et al, 2005;2009b) phaZ PHB depolymerase ACT, WB, OE/DEL, PROT de Eugenio et al, 2007;Ren et al, 2009a) phaI Phasin PROT (Prieto et al, 1999;Klinke et al, 2000;Moldes et al, 2004;Sandoval et al, 2007;Ren et al, 2009b) phaF Granule segregation factor, phasin (FM), DEL, PROT (Prieto et al, 1999;Klinke et al, 2000;Moldes et al, 2004;Sandoval et al, 2007;Ren et al, 2009b) (Prieto et al, 1999;Klinke et al, 2000;Galán et al, 2011) phaD* Transcriptional regulator…”

Identification of the Haloarchaeal Phasin (PhaP) That Functions in Polyhydroxyalkanoate Accumulation and Granule Formation in Haloferax mediterranei

“…In Pseudomonas oleovorans, two independent promoters for the major phasin gene phaF have been identified (30,35). As the protein level of PhaP is much higher than that of GAP12, as detected by 2-DE (Fig.…”

“…During the investigation of the organization of the phaP genes and their neighbor genes in bacteria, most of the phaP genes were found to be carried in monocistronic operons (23,40). In the Pseudomonas genus, although the two phasin genes phaI and phaF are in an operon, the gene phaF still possesses its own promoter nearby (30,35). In the known regulation models for phasins, all phasin genes harbor their own promoter, which is regulated by PhaR.…”

Conversion of Biomass into Bioplastics and Their Potential Environmental Impacts

“…There were a couple of reports on PHAs with unusual properties, such as adding functional groups side groups to the PHA monomer mediated by low specific PHA synthases (Hiraishi et al, 2006;Luengo et al, 2003;Sandoval, et al, 2007;Tsuge et al, 2005), PHA monomer of high molecular weight by mutated synthases (Zheng et al, 2006), and funtionalized PHAs containing C-C double bonds when fed unsaturated fatty acids (methanol) (Hofer, et al, 2010). One of the key issues hindering the large scale biopolymer production is the cost of sugars and fatty acids as feed stocks.…”

“…Several refactored microbes have been constructed to produce PHAs (Aldor and Keasling, 2003;Garcia, et al, 2004;Hofer et al, 2010;Lee, et al, 1999;Li et al, 2007;Park et al, 2002;Qiu et al, 2006;Sandoval et al, 2007;Zhang et al, 2009). One commonly used engineering strain is E. coli.…”