Tight coupling of polymerization and depolymerization of polyhydroxyalkanoates ensures efficient management of carbon resources in Pseudomonas putida

Abstract

SummaryEnvironmental microbes oscillate between feast and famine and need to carefully manage utilization, storage and conversion of reserve products to exploitable sources of carbon and energy. Polyhydroxyalkanoates (PHAs) are storage polymers that serve bacteria as sources of food materials under physiological conditions of carbon demand. In order to obtain insights into the role of PHA depolymerase (PhaZ) and its relationship to a PHA polymerase (PhaC2) in the carbon management activity of Pseudomonas putida strain U, we created a polymerase hyperexpression strain and a depolymerase knockout mutant of this strain, and examined their synthesis of PHA and expression of their PHA genes. This study revealed that hyperexpression of PhaC2 led to the accumulation of higher amounts of PHA (44%wt) than in the wild-type strain (24%wt) after 24 h of cultivation, which then returned to wild-type levels by 48 h, as a result of elevated depolymerization. The phaZ mutant, however, accumulated higher levels of PHA than the parental strain (62%wt), which were maintained for at least 96 h. Transcriptional analysis of the pha cluster by RT-PCR revealed that PHA operon proteins, including depolymerase, are expressed from the beginning of the growth phase. Hyperexpression of the PhaC2 polymerase was accompanied by an increase in the expression of the PhaZ depolymerase and a decrease in expression of another PHA polymerase, PhaC1. This suggests tight regulatory coupling of PHA polymerase and depolymerase activities that act in synergy, and in concert with other PHA proteins, to provide dynamic PHA granule synthesis and remodelling that rapidly and sensitively respond to changes in availability of carbon and the physiological-metabolic needs of the cell, to ensure optimal carbon resource management.

The loss of function of PhaC1 is a survival mechanism that counteracts the stress caused by the overproduction of poly‐3‐hydroxyalkanoates in Pseudomonas putidaΔfadBA

“…The essential role played by PhaC1 in PHA synthesis agrees with the data reported by Arias and colleagues () showing that maximal PHA accumulation correlates with a rapid and significant increase of phaC1 transcription. Sandoval and colleagues () reported that the expression of phaC1 in a mutant carrying a deletion of the whole cluster ( Pp UΔ pha ) was sufficient to restore PHA synthesis.…”

A holistic view of polyhydroxyalkanoate metabolism in Pseudomonas putida

“…The peripheral supplier routes and regulatory networks ensure the funnelling of monomeric precursors towards the polymer. PHA synthesis and degradation are linked to the metabolic network thanks to a continuous cycle in which PHA synthase and depolymerase are simultaneously active, thus ensuring PHA turnover (Ren et al ., ; de Eugenio et al ., ; Arias et al ., ). Under PHA‐producing conditions, PhaZ KT depolymerase continuously releases 3‐hydroxy fatty acids from PHA granules.…”

Features of pseudomonads growing at low temperatures: another facet of their versatility

“…In support of this view, the growth of P. extremaustralis under microaerobic conditions rescued the growth defect of the mutant strain unable to produce PHAs at low temperature (Tribelli and López, ). PHA metabolism is a dynamic process, with a continuous cycle of synthesis and degradation that helps adapt the carbon flow to the transient demand for metabolic intermediates (Escapa et al ., ; Arias et al ., ). Its importance would seem to be greater at low temperatures.…”