The turnover of medium‐chain‐length polyhydroxyalkanoates in Pseudomonas putida KT2442 and the fundamental role of PhaZ depolymerase for the metabolic balance

Abstract

Polyhydroxyalkanoates (PHAs) are biodegradable polymers produced by a wide range of bacteria, including Pseudomonads. These polymers are accumulated in the cytoplasm as carbon and energy storage materials when culture conditions are unbalanced and hence, they have been classically considered to act as sinks for carbon and reducing equivalents when nutrients are limited. Bacteria facing carbon excess and nutrient limitation store the extra carbon as PHAs through the PHA polymerase (PhaC). Thereafter, under starvation conditions, PHA depolymerase (PhaZ) degrades PHA and releases R-hydroxyalkanoic acids, which can be used as carbon and energy sources. To study the influence of a deficient PHA metabolism in the growth of Pseudomonas putida KT2442 we have constructed two mutant strains defective in PHA polymerase (phaC1)- and PHA depolymerase (phaZ)-coding genes respectively. By using these mutants we have demonstrated that PHAs play a fundamental role in balancing the stored carbon/biomass/number of cells as function of carbon availability, suggesting that PHA metabolism allows P. putida to adapt the carbon flux of hydroxyacyl-CoAs to cellular demand. Furthermore, we have established that the coordination of PHA synthesis and mobilization pathways configures a functional PHA turnover cycle in P. putida KT2442. Finally, a new strain able to secrete enantiomerically pure R-hydroxyalkanoic acids to the culture medium during cell growth has been engineering by redirecting the PHA cycle to biopolymer hydrolysis.

Medium-chain-length polyhydroxyalkanoates synthesis by Pseudomonas putida KT2440 relA/spoT mutant: bioprocess characterization and transcriptome analysis

“…It could be suggested that both mutants do not effectively activate processes leading to recovery of energy from PHAs. The number of transcripts of phaC1, phaZ, phaC2 and phaD differed significantly from those of phaF and phaI , which indicates that these operons are differentially regulated, as in de Eugenio et al (2010). According to previous studies (Klinke et al 2000; de Eugenio et al 2010), the phaD gene acts as an activator of the phaC1 and phaI promoters.…”

Polyester hydrolytic and synthetic activity catalyzed by the medium-chain-length poly(3-hydroxyalkanoate) depolymerase from Streptomyces venezuelae SO1

“…Interestingly, more than 150 3-HAs have been identified as constituents of these microbial polymers (most of them summarized by Steinbü-chel and Valentin 1995). Additionally, all HAs are enantiomerically pure and in R-configuration (Ruth et al 2007;De Eugenio et al 2010).…”

High Polyhydroxybutyrate Production in Pseudomonas extremaustralis Is Associated with Differential Expression of Horizontally Acquired and Core Genome Polyhydroxyalkanoate Synthase Genes

“…This cluster encode two class II synthase genes ( phaC1 and phaC2 ) separated by a gene encoding a PHA depolymerase ( phaZ ); a transcriptional activator ( phaD ) and two genes ( phaFI ) encoding phasin-like proteins [2]. In most Pseudomonas, β-oxidation and fatty acid de novo synthesis are used to convert fatty acid or carbohydrate intermediates, respectively into (R)-3-hydroxyacyl-CoAs, which are used as substrates by the Class II PHA synthases [3].…”