Characterization of polyhydroxyalkanoate synthases from Halomonas sp. O-1 and Halomonas elongata DSM2581: Site-directed mutagenesis and recombinant expression

Ilham, Mulyana; Nakanomori, Satoko; Kihara, Takahiro; Hokamura, Ayaka; Matsusaki, Hiromi; Tsuge, Takeharu; Mizuno, Kouhei

doi:10.1016/j.polymdegradstab.2014.04.024

Cited by 27 publications

(35 citation statements)

References 34 publications

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“…Lipase box-like sequences are highly conserved domains which have been identified as active sites of the enzymes [31][32][33] and play a crucial role in elongation of the polymer [34] in several PHA-producing species. These domains expose similarities with those of lipase, but the difference is in the replacement of the essential active site of lipase, a serine, by a cysteine in the lipase box-like domain of PHA synthase [35], leading to renaming these sequences as PhaC box consensus sequences [34].…”

Section: Discussionmentioning

confidence: 99%

“…In this pattern, similar to lipase, Cysteine (Cys or C) represents the catalytic amino acid and is involved in a catalytic triad (C-H-D) participating, supposedly, in the elongation step of the PHA polymer [34]. The most common described pattern is Glycine-X-Cysteine-X-Glycine (G-X-C-X-G), including Glycine-Tyrosine-Cysteine-Methionine-Glycine sequence (G-Y-C-M-G) detected in Bacillus cereus (ATCC 14579) or Haloferax mediterranei (ATCC 33500), Glycine-Tyrosine-Cysteine-Leucine-Glycine sequence (G-Y-C-L-G) found in Cupriavidus metallidurans strain CH34 or Halomonas boliviensis LC1 (DSM15516), or Glycine-Alanine-Cysteine-Serine-Glycine sequence (G-A-C-S-G) in Cupriavidus necator strain N-1 or Pseudomonas fulva strain 12-X [31,[34][35][36]. However, variations in amino acid composition have also been described in various bacterial species like H. elongata (DSM 2581) or Halomonas sp.…”

Section: Discussionmentioning

confidence: 99%

“…However, variations in amino acid composition have also been described in various bacterial species like H. elongata (DSM 2581) or Halomonas sp. KM-1 for which sequences have been described (Figure 3) [31].…”

Section: Discussionmentioning

confidence: 99%

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PHA Production and PHA Synthases of the Halophilic Bacterium Halomonas sp. SF2003

et al. 2020

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Among the different tools which can be studied and managed to tailor-make polyhydroxyalkanoates (PHAs) and enhance their production, bacterial strain and carbon substrates are essential. The assimilation of carbon sources is dependent on bacterial strain’s metabolism and consequently cannot be dissociated. Both must wisely be studied and well selected to ensure the highest production yield of PHAs. Halomonas sp. SF2003 is a marine bacterium already identified as a PHA-producing strain and especially of poly-3-hydroxybutyrate (P-3HB) and poly-3-hydroxybutyrate-co-3-hydroxyvalerate (P-3HB-co-3HV). Previous studies have identified different genes potentially involved in PHA production by Halomonas sp. SF2003, including two phaC genes with atypical characteristics, phaC1 and phaC2. At the same time, an interesting adaptability of the strain in front of various growth conditions was highlighted, making it a good candidate for biotechnological applications. To continue the characterization of Halomonas sp. SF2003, the screening of carbon substrates exploitable for PHA production was performed as well as production tests. Additionally, the functionality of both PHA synthases PhaC1 and PhaC2 was investigated, with an in silico study and the production of transformant strains, in order to confirm and to understand the role of each one on PHA production. The results of this study confirm the adaptability of the strain and its ability to exploit various carbon substrates, in pure or mixed form, for PHA production. Individual expression of PhaC1 and PhaC2 synthases in a non-PHA-producing strain, Cupriavidus necator H16 PHB¯4 (DSM 541), allows obtaining PHA production, demonstrating at the same time, functionality and differences between both PHA synthases. All the results of this study confirm the biotechnological interest in Halomonas sp. SF2003.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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PHA Production and PHA Synthases of the Halophilic Bacterium Halomonas sp. SF2003

et al. 2020

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“…R5-57 carries three genes annotated as polyhydroxyalkanoate synthases (PHA Cs); the enzymes responsible for carrying out the final polymerization step in PHA biosynthesis [28]. The product of phaC HALO1802 has high homology with PHA C1sequences of H. boliviensis (91 %) and H. campaniensis (86 %) and with enzymes from Halomonas spp.O-1 (86 %) and H. elongata (77 %) which have recently been heterologously produced and characterized [29]. The putative PHA C (HALO2716) of Halomonas sp.…”

Section: Insights From the Genome Sequencementioning

confidence: 99%

Complete genome sequence of Halomonas sp. R5-57

Williamson

Santi

Altermark

et al. 2016

Stand in Genomic Sci

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The marine Arctic isolate Halomonas sp. R5-57 was sequenced as part of a bioprospecting project which aims to discover novel enzymes and organisms from low-temperature environments, with potential uses in biotechnological applications. Phenotypically, Halomonas sp. R5-57 exhibits high salt tolerance over a wide range of temperatures and has extra-cellular hydrolytic activities with several substrates, indicating it secretes enzymes which may function in high salinity conditions. Genome sequencing identified the genes involved in the biosynthesis of the osmoprotectant ectoine, which has applications in food processing and pharmacy, as well as those involved in production of polyhydroxyalkanoates, which can serve as precursors to bioplastics. The percentage identity of these biosynthetic genes from Halomonas sp. R5-57 and current production strains varies between 99 % for some to 69 % for others, thus it is plausible that R5-57 may have a different production capacity to currently used strains, or that in the case of PHAs, the properties of the final product may vary. Here we present the finished genome sequence (LN813019) of Halomonas sp. R5-57 which will facilitate exploitation of this bacterium; either as a whole-cell production host, or by recombinant expression of its individual enzymes.Electronic supplementary materialThe online version of this article (doi:10.1186/s40793-016-0192-4) contains supplementary material, which is available to authorized users.

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“…. It was recently demonstrated that PhaCs from Halomonas strains have a similar sequence (S‐X‐C‐X‐G) at the active site, where the first Gly in the lipase box‐like sequence is replaced with Ser . PhaCs from Paracoccus denitrificans and several other strains (e.g., Rhodobacter sphaeroides and Rhodobacter capsulatus ) have been proposed to have an alternate active site sequence (G‐X‐C‐X‐A) in which the second Gly is replaced with Ala .…”

mentioning

confidence: 99%

Expanded amino acid sequence of the PhaC box in the active center of polyhydroxyalkanoate synthases

et al. 2019

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Polyhydroxyalkanoate (PHA) synthases catalyze the polymerization reaction of the acyl moiety of hydroxyacyl‐coenzyme A into polyester. The catalytic subunit PhaC of PHA synthase has the PhaC box sequence at the active site that is typically described as G‐X‐C‐X‐G‐G (X is an arbitrary amino acid), and cysteine is an active center. In this study, an amino acid replacement was introduced into the PhaC box of the PHA synthase derived from Ralstonia eutropha (PhaCRe) to investigate the importance of highly conserved residues in polymerizing activity. Point mutagenesis revealed that PhaCRe mutants with the expanded PhaC box sequence ([GAST]‐X‐C‐X‐[GASV]‐[GA]) are functional PHA synthases. These findings highlight the low mutational robustness of the last glycine residue in the PhaC box as well as that of the active center cysteine.

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Characterization of polyhydroxyalkanoate synthases from Halomonas sp. O-1 and Halomonas elongata DSM2581: Site-directed mutagenesis and recombinant expression

Cited by 27 publications

References 34 publications

PHA Production and PHA Synthases of the Halophilic Bacterium Halomonas sp. SF2003

PHA Production and PHA Synthases of the Halophilic Bacterium Halomonas sp. SF2003

Complete genome sequence of Halomonas sp. R5-57

Expanded amino acid sequence of the PhaC box in the active center of polyhydroxyalkanoate synthases

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