A Patatin-Like Protein Associated with the Polyhydroxyalkanoate (PHA) Granules of Haloferax mediterranei Acts as an Efficient Depolymerase in the Degradation of Native PHA

Abstract

cThe key enzymes and pathways involved in polyhydroxyalkanoate (PHA) biosynthesis in haloarchaea have been identified in recent years, but the haloarchaeal enzymes for PHA degradation remain unknown. In this study, a patatin-like PHA depolymerase, PhaZh1, was determined to be located on the PHA granules in the haloarchaeon Haloferax mediterranei. PhaZh1 hydrolyzed the native PHA (nPHA) [including native polyhydroxybutyrate (nPHB) and native poly(3-hydroxybutyrateco-3-hydroxyvalerate) (nPHBV) in this study] granules in vitro with 3-hydroxybutyrate (3HB) monomer as the primary product. The site-directed mutagenesis of PhaZh1 indicated that Gly 16 , Ser 47 (in a classical lipase box, G-X-S 47 -X-G), and Asp 195 of this depolymerase were essential for its activity in nPHA granule hydrolysis. Notably, phaZh1 and bdhA (encoding putative 3HB dehydrogenase) form a gene cluster (HFX_6463 to _6464) in H. mediterranei. The 3HB monomer generated from nPHA degradation by PhaZh1 could be further converted into acetoacetate by BdhA, indicating that PhaZh1-BdhA may constitute the first part of a PHA degradation pathway in vivo. Interestingly, although PhaZh1 showed efficient activity and was most likely the key enzyme in nPHA granule hydrolysis in vitro, the knockout of phaZh1 had no significant effect on the intracellular PHA mobilization, implying the existence of an alternative PHA mobilization pathway(s) that functions effectively within the cells of H. mediterranei. Therefore, identification of this patatin-like depolymerase of haloarchaea may provide a new strategy for producing the high-value-added chiral compound (R)-3HB and may also shed light on the PHA mobilization in haloarchaea. P olyhydroxyalkanoate (PHA) is accumulated in the form of granules and serves as storage compound of carbon and energy in bacteria (1) and archaea (2) during growth in the presence of excess carbon sources. Several proteins, which are known as PHA granule-associated proteins (PGAPs), are embedded on or attached to the PHA granules. These include PHA synthases, phasins, regulatory proteins, and depolymerases (3). A PHA-accumulating host may utilize the accumulated PHA for growth and survival under conditions of carbon starvation (4). PHA depolymerase (PhaZ) is the key enzyme that functions in PHA mobilization.In bacteria, PHA depolymerases are grouped into two classes: intracellular (catalyzing the degradation of endogenous PHA) (iPhaZ) and extracellular (catalyzing the degradation of exogenous PHA) (ePhaZ) PHA depolymerases (5). The extracellular PHA depolymerase degradation process is well known, but the mechanism underlying the metabolic pathway and regulation of PHA degradation in vivo remains poorly understood (5). Native polyhydroxybutyrate (nPHB) is degraded by iPhaZs in vitro to 3-hydroxybutyrate (3HB) (6, 7) or 3-hydroxybutyryl-coenzyme A (3HB-CoA) in the presence of CoA (8, 9). The PHB degradation product, 3HB-CoA, is the precursor of PHB synthesis; hence, the simultaneous synthesis and mobilization of PHB (10, 11) (8). Notably, th...

Production of Polyhydroxyalkanoate (PHA) Biopolyesters by Extremophiles?

“…mediteranneimediated PHA production has been further optimized by researchers all around the world. Whereas some of these groups focused on the application of inexpensive carbon feedstocks to safe substrate costs, such as whey permeate from dairy industry [12,13,48], rice-based ethanol stillage [49], extruded rice bran [50], enzymatically extruded starch [51], crude glycerol phase [28], olive mill waste water [52], vinasse [53], etc., others clarified the enzymatic and genetic background of PHA-synthesis [54][55][56][57] and in vitro degradation [58] by this organisms. Mathematic models of PHA-production [59], and kinetic studies of PHA and by-product synthesis and degradation [60] were reported for Hfx.…”

Enoyl-CoA hydratase mediates polyhydroxyalkanoate mobilization in Haloferax mediterranei

“…Comparing to the R-ECH research in bacteria, the characterization of R-ECH in archaea was barely known until recently. Previously, six PHA-granule-associated proteins (PGAPs) 15 16 were identified in the haloarchaeon Haloferax mediterranei , a model strain for PHA metabolism research in archaea 17 18 19 . The six PGAPs include putative R-ECH (HFX_5217), phasin PhaP 15 , regulatory protein PhaR 20 , PHA synthase subunits PhaE and PhaC 21 , and PHA depolymerase PhaZh1 16 .…”

Current developments on polyhydroxyalkanoates synthesis by using halophiles as a promising cell factory

“…The intracellular PHBV gradually degrades when the PHA-rich cells of Hfx. mediterranei are transferred into a fresh medium without carbon source [121]. Strikingly, in addition to the five PHA granule associated proteins, two new proteins were observed on the PHA granules in this strain.…”

“…Interestingly, the expression level of phaRP was enhanced in the presence of PHA as the activity of the phaRP promoter decreased by twofolds in the ΔphaEC and ΔphaPEC mutants of Hfx. mediterranei [121]. However, knocking out of the phaR gene increased the activity of the phaRP promoter by fourfolds in the ΔphaRPEC mutant, indicating that PhaR is a negative regulator of the phaRP operon [118].…”

“…mediterranei. The other protein BdhA (3HB dehydrogenase) was encoded by the gene located upstream of phaZh1 [121]. BdhA converted 3HB monomers generated by PhaZ1 from natural PHA granules into acetoacetate.…”