Comparison of four phaC genes from Haloferax mediterranei and their function in different PHBV copolymer biosyntheses in Haloarcula hispanica.

State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No,1 West Beichen Road, Chaoyang District, Beijing 100101, China. .
Saline Systems (Impact Factor: 1.18). 01/2010; 6:9. DOI: 10.1186/1746-1448-6-9
Source: PubMed

ABSTRACT The halophilic archaeon Haloferax mediterranei is able to accumulate large amounts of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with high molar fraction of 3-hydroxyvalerate (3HV) from unrelated carbon sources. A Polyhydroxyalkanoate (PHA) synthase composed of two subunits, PhaCHme and PhaEHme, has been identified in this strain, and shown to account for the PHBV biosynthesis.
With the aid of the genome sequence of Hfx. mediterranei CGMCC 1.2087, three additional phaC genes (designated phaC1, phaC2, and phaC3) were identified, which encoded putative PhaCs. Like PhaCHme (54.8 kDa), PhaC1 (49.7 kDa) and PhaC3 (62.5 kDa) possessed the conserved motifs of type III PHA synthase, which was not observed in PhaC2 (40.4 kDa). Furthermore, the longer C terminus found in the other three PhaCs was also absent in PhaC2. Reverse transcription PCR (RT-PCR) revealed that, among the four genes, only phaCHme was transcribed under PHA-accumulating conditions in the wild-type strain. However, heterologous coexpression of phaEHme with each phaC gene in Haloarcula hispanica PHB-1 showed that all PhaCs, except PhaC2, could lead to PHBV accumulation with various 3HV fractions. The three kinds of copolymers were characterized using gel-permeation chromatography (GPC), differential scanning calorimetry (DSC), and thermogravimetric analysis (TGA). Their thermal properties changed with the variations in monomer composition as well as the different molecular weights (Mw), thus might meet various application requirements.
We discover three cryptic phaC genes in Hfx. mediterranei, and demonstrate that genetic engineering of these newly identified phaC genes has biotechnological potential for PHBV production with tailor-made material properties.

  • [Show abstract] [Hide abstract]
    ABSTRACT: Utilization of by-products from oilseed-based biodiesel production (crude glycerol, rapeseed meal hydrolysates) for microbial polyhydroxyalkanoate (PHA) production could lead to the replacement of expensive carbon sources, nutrient supplements and precursors for co-polymer production. Batch fermentations in shake flasks with varying amounts of free amino nitrogen led to the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)) with a 2.8-8% 3HV content. Fed-batch fermentations in shake flasks led to the production of 10.9g/L P(3HB-co-3HV) and a 55.6% P(3HB-co-3HV) content. NaCl concentrations between 2 and 6g/L gradually became inhibitory to bacterial growth and PHA formation, whereas in the case of K(2)SO(4), the inhibitory effect was observed only at concentrations higher than 20g/L. Differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and nuclear magnetic resonance ((13)C NMR) demonstrated that the incorporation of 3HV into the obtained P(3HB-co-3HV) lowered glass transition temperature, crystallinity and melting point as compared to polyhydroxybutyrate. Integrating PHA production in existing oilseed-based biodiesel plants could enhance the viability and sustainability of this first generation biorefinery.
    Bioresource Technology 11/2012; 130C:16-22. · 5.04 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Haloarcula hispanica has been widely used in haloarchaeal studies, particularly in the isolation of haloviruses. The genome of strain N601, a laboratory derivative of the type strain ATCC 33960, was sequenced. Several potentially significant differences from the published sequence of the type strain (CGMCC 1.2049 = ATCC 33960) were observed.
    Genome announcements. 03/2014; 2(2).
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Haloferax mediterranei is able to accumulate the bioplastic poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) with more than 10 mol% 3-hydroxyvalerate (3HV), from unrelated carbon sources. However, the pathways that produce propionyl-CoA, an important precursor of 3HV monomer, have not yet been determined. Bioinformatic analysis of H. mediterranei genome indicated that this strain uses multiple pathways for propionyl-CoA biosynthesis, including the citramalate/2-oxobutyrate pathway, aspartate/2-oxobutyrate pathway, methylmalonyl-CoA pathway, and a novel 3-hydroxypropionate pathway. Co-feeding of pathway intermediates and inactivating pathway-specific genes supported that these four pathways were indeed involved in the biosynthesis of 3HV monomer. The novel 3-hydroxypropionate pathway that couples CO(2) assimilation with PHBV biosynthesis was further confirmed by analysis of (13)C positional enrichment in 3HV. Notably, (13)C metabolic flux analysis showed that the citramalate/2-oxobutyrate pathway (53.0% flux) and the 3-hydroxypropionate pathway (30.6% flux) were the two main generators of propionyl-CoA from glucose. Additionally, genetic perturbation on the transcriptome of the ΔphaEC mutant (deficient in PHBV accumulation) revealed that a considerable number of genes in the four propionyl-CoA synthetic pathways were significantly down-regulated. Taken together, this study for the first time determined four propionyl-CoA supplying pathways for PHBV production in haloarchaea, particularly including a new 3-hydroxypropionate pathway. These results would provide novel strategies for the production of PHBV with controllable 3HV molar fraction.
    Applied and Environmental Microbiology 02/2013; · 3.95 Impact Factor

Full-text (2 Sources)

Available from
Nov 25, 2014