Biochemical characterization of a new type of intracellular PHB depolymerase from Rhodospirillum rubrum with high hydrolytic activity on native PHB granules

ABSTRACTThe obligate predatorBdellovibrio bacteriovorusHD100 shows a large set of proteases and other hydrolases as part of its hydrolytic arsenal needed for its predatory life cycle. We present genetic and biochemical evidence that open reading frame (ORF) Bd3709 ofB. bacteriovorusHD100 encodes a novel medium-chain-length polyhydroxyalkanoate (mcl-PHA) depolymerase (PhaZBd). The primary structure of PhaZBdsuggests that this enzyme belongs to the α/β-hydrolase fold family and has a typical serine hydrolase catalytic triad (serine-histidine-aspartic acid) in agreement with other PHA depolymerases and lipases. PhaZBdhas been extracellularly produced using different hypersecretor Tol-pal mutants ofEscherichia coliandPseudomonas putidaas recombinant hosts. The recombinant PhaZBdhas been characterized, and its biochemical properties have been compared to those of other PHA depolymerases. The enzyme behaves as a serine hydrolase that is inhibited by phenylmethylsulfonyl fluoride. It is also affected by the reducing agent dithiothreitol and nonionic detergents like Tween 80. PhaZBdis an endoexohydrolase that cleaves both large and small PHA molecules, producing mainly dimers but also monomers and trimers. The enzyme specifically degrades mcl-PHA and is inactive toward short-chain-length polyhydroxyalkanoates (scl-PHA) like polyhydroxybutyrate (PHB). These studies shed light on the potentiality of these predators as sources of new biocatalysts, such as an mcl-PHA depolymerase, for the production of enantiopure hydroxyalkanoic acids and oligomers as building blocks for the synthesis of biobased polymers.

Section: Discussionmentioning

confidence: 89%

“…The depolymerases have been classified as intra-or extracellular enzymes, with the exception of the PHB depolymerase from Rhodospirillum rubrum (PhaZ1 Rru ), which is located in the periplasm (25,59). The physiological function of R. rubrum PHB depolymerase remains to be elucidated, since PHB granules are located intracellularly.…”

Section: Discussionmentioning

confidence: 99%

Identification and Biochemical Evidence of a Medium-Chain-Length Polyhydroxyalkanoate Depolymerase in the Bdellovibrio bacteriovorus Predatory Hydrolytic Arsenal

Martínez

Peña

García-Hidalgo

et al. 2012

“…Subcellular localization of PhaZd1 and PhaZd2. If the physiological function of PhaZd1 and PhaZd2 is the depolymerization of accumulated PHB, the enzymes should be able to specifically bind to PHB in vivo, as has been previously described for other iPHB depolymerases (22,44). Attachment to and detachment from PHB granules of proteins with a PHB depolymerase function could be a way to regulate the accumulation and mobilization of accumulated PHB.…”

Section: Resultsmentioning

confidence: 99%

To Be or Not To Be a Poly(3-Hydroxybutyrate) (PHB) Depolymerase: PhaZd1 (PhaZ6) and PhaZd2 (PhaZ7) of Ralstonia eutropha, Highly Active PHB Depolymerases with No Detectable Role in Mobilization of Accumulated PHB

Sznajder

Jendrossek

2014

Self Cite

The putative physiological functions of two related intracellular poly(3-hydroxybutyrate) (PHB) depolymerases, PhaZd1 and PhaZd2, of Ralstonia eutropha H16 were investigated. Purified PhaZd1 and PhaZd2 were active with native PHB granules in vitro. Partial removal of the proteinaceous surface layer of native PHB granules by trypsin treatment or the use of PHB granules isolated from ⌬phaP1 or ⌬phaP1-phaP5 mutant strains resulted in increased specific PHB depolymerase activity, especially for PhaZd2. Constitutive expression of PhaZd1 or PhaZd2 reduced or even prevented the accumulation of PHB under PHB-permissive conditions in vivo. Expression of translational fusions of enhanced yellow fluorescent protein (EYFP) with PhaZd1 and PhaZd2 in which the active-site serines (S190 and Ser193) were replaced with alanine resulted in the colocalization of only PhaZd1 fusions with PHB granules. C-terminal fusions of inactive PhaZd2(S193A) with EYFP revealed the presence of spindlelike structures, and no colocalization with PHB granules was observed. Chromosomal deletion of phaZd1, phaZd2, or both depolymerase genes had no significant effect on PHB accumulation and mobilization during growth in nutrient broth (NB) or NBgluconate medium. Moreover, neither proteome analysis of purified native PHB granules nor lacZ fusion studies gave any indication that PhaZd1 or PhaZd2 was detectably present in the PHB granule fraction or expressed at all during growth on NBgluconate medium. In conclusion, PhaZd1 and PhaZd2 are two PHB depolymerases with a high capacity to degrade PHB when artificially expressed but are apparently not involved in PHB mobilization in the wild type. The true in vivo functions of PhaZd1 and PhaZd2 remain obscure. Ralstonia eutropha H16 is a chemolithoautotrophic betaproteobacterium that has become famous because of its ability to accumulate large amounts of poly(3-hydroxybutyrate) (PHB). R. eutropha is used for the commercial production of bioplastics (polyhydroxyalkanoates [PHAs] such as PHB and copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate) and is considered a model organism for PHA research (1-5). Investigation of the composition of the surface layer of PHB granules of R. eutropha H16 revealed an astonishingly high number of polypeptides that are predicted or have already been shown to be specifically attached to the PHB granule surface. These proteins include enzymes involved in biosynthesis (PHB synthase PhaC1) (6), in granule structure integrity (phasins PhaP1-PhaP7), in PHB mobilization (PhaZa1 to PhaZa5, PhaZb, PhaZc, PhaZd), and in other functions (PhaR, PhaM). For reviews, see references 1, 5, 7, and 8. Other PHA-accumulating bacteria and Archaea are also known to have proteins specifically attached to the PHA surface (9-15). For an overview and more references, see Table 2 in reference 5. Unfortunately, designation of PHB granule-associated proteins in R. eutropha is not uniformly used in literature: for alternative designations of PHB depolymerases and PHB oligomer hydrolases in R. eutropha, s...

“…PhaZ1 was shown to be located in the cell's periplasm and is unlikely physically involved in in vivo polyhydroxybutyrate (PHB) degradation (20). In vitro studies on the gene product of phaZ3 showed that this PHA depolymerase was specific for amorphous PHA SCL but was predicted to be inactive at cytoplasm-typical concentrations of divalent cations, such as Mg 2ϩ (32). In contrast, PhaZ2 shows significant homologies to the well-characterized intracellular PHA SCL depolymerase of Ralstonia eutropha (PhaZ1) and was therefore predicted to be responsible for intracellular PHA degradation in R. rubrum (20,32,33).…”

Section: Resultsmentioning

confidence: 99%

“…In vitro studies on the gene product of phaZ3 showed that this PHA depolymerase was specific for amorphous PHA SCL but was predicted to be inactive at cytoplasm-typical concentrations of divalent cations, such as Mg 2ϩ (32). In contrast, PhaZ2 shows significant homologies to the well-characterized intracellular PHA SCL depolymerase of Ralstonia eutropha (PhaZ1) and was therefore predicted to be responsible for intracellular PHA degradation in R. rubrum (20,32,33). Thus, phaZ2 (locus tag Rru_A3356) was chosen for (i) deletion in R. rubrum ⌬phaC1 ⌬phaC2, as well as (ii) site-directed mutagenesis leading to an amino acid exchange in the putative active site of PhaZ2.…”

Section: Resultsmentioning

confidence: 99%

Synthesis Gas (Syngas)-Derived Medium-Chain-Length Polyhydroxyalkanoate Synthesis in Engineered Rhodospirillum rubrum

Heinrich

Raberg

Fricke

et al. 2016

The purple nonsulfur alphaproteobacterium Rhodospirillum rubrum S1 was genetically engineered to synthesize a heteropolymer of mainly 3-hydroxydecanoic acid and 3-hydroxyoctanoic acid [P(3HD-co-3HO)] from CO-and CO 2 -containing artificial synthesis gas (syngas). For this, genes from Pseudomonas putida KT2440 coding for a 3-hydroxyacyl acyl carrier protein (ACP) thioesterase (phaG), a medium-chain-length (MCL) fatty acid coenzyme A (CoA) ligase (PP_0763), and an MCL polyhydroxyalkanoate (PHA) synthase (phaC1) were cloned and expressed under the control of the CO-inducible promoter P cooF from R. rubrum S1 in a PHA-negative mutant of R. rubrum. P(3HD-co-3HO) was accumulated to up to 7.1% (wt/wt) of the cell dry weight by a recombinant mutant strain utilizing exclusively the provided gaseous feedstock syngas. In addition to an increased synthesis of these medium-chain-length PHAs (PHA MCL ), enhanced gene expression through the P cooF promoter also led to an increased molar fraction of 3HO in the synthesized copolymer compared with the P lac promoter, which regulated expression on the original vector. The recombinant strains were able to partially degrade the polymer, and the deletion of phaZ2, which codes for a PHA depolymerase most likely involved in intracellular PHA degradation, did not reduce mobilization of the accumulated polymer significantly. However, an amino acid exchange in the active site of PhaZ2 led to a slight increase in PHA MCL accumulation. The accumulated polymer was isolated; it exhibited a molecular mass of 124.3 kDa and a melting point of 49.6°C. With the metabolically engineered strains presented in this proof-of-principle study, we demonstrated the synthesis of elastomeric second-generation biopolymers from renewable feedstocks not competing with human nutrition. IMPORTANCEPolyhydroxyalkanoates (PHAs) are natural biodegradable polymers (biopolymers) showing properties similar to those of commonly produced petroleum-based nondegradable polymers. The utilization of cheap substrates for the microbial production of PHAs is crucial to lower production costs. Feedstock not competing with human nutrition is highly favorable. Syngas, a mixture of carbon monoxide, carbon dioxide, and hydrogen, can be obtained by pyrolysis of organic waste and can be utilized for PHA synthesis by several kinds of bacteria. Up to now, the biosynthesis of PHAs from syngas has been limited to short-chain-length PHAs, which results in a stiff and brittle material. In this study, the syngas-utilizing bacterium Rhodospirillum rubrum was genetically modified to synthesize a polymer which consisted of medium-chain-length constituents, resulting in a rubber-like material. This study reports the establishment of a microbial synthesis of these so-called medium-chain-length PHAs from syngas and therefore potentially extends the applications of syngas-derived PHAs. Biodegradable and biocompatible polyhydroxyalkanoates (PHAs) are naturally occurring polyesters, which are synthesized by various bacteria. Depending on their...