Biosynthesis Of PHBV With Haloarchaeon Halogranum Amylolyticum TNN58

Polyhydroxyalkanoates(PHAs) is a class of natural biopolyesters accumulated intracellularly by many microorganisms. These polymers have attracted particular attention as green plastic in biomedical and industrial applications due to their good biodegradability and biocompatibility. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)(PHBV) is one of the most common members of PHAs. Haloarchaea is an important group of PHA(polyhydroxyalkanoate)-accumulating organisms.It was discovered that Halogranum amylolyticum TNN58 could efficiently accumulate poly(3-hydroxybutyrate-co-3-hydroxyvalerate)(PHBV) with a high 3-hydroxyvalerate(3HV) fraction using glucose as carbon source. Briefly, transmission electron microscopy(TEM) analysis revealed the presence of a large number of PHA granules in the cells. Gas chromatography-mass spectrometry(GC-MS) and 1H nuclear magnetic resonance(NMR) analyses showed that PHAs synthesized from glucose was PHBV. Moreover, the 3HV content reached 20~ mol%, which is the highest 3HV fraction from unrelated carbon courses.Fermentation experiments suggested that nitrogen-limited MG medium was better than nutrient-rich NOMG and AS168 medium for PHBV production. Additionally, glucose was the most suitable carbon source among the tested carbon sources. Interestingly, PHBV accumulation was almost paralleled by cell growth and glucose consumption. By applying the fed-batch process in fermentor, the cell dry weight was 29 g/L and the PHBV production reach to 14 g/L.The classical PHA synthase genes were successfully cloned via consensus-degenerate hybrid oligonucleotide primers(CODEHOPs) and high-efficiency thermal asymmetric interlaced(hiTAIL) PCR methods. Then the phaEC genes were identified to encode the two submit of PHA synthase, PhaE and PhaC. They were proved to be expressed by RT-PCR and Western blot analysis when transformed into Haloarcula hispanica PHB-1, a mutant of PHA synthase. H. hispanica PHB-1 completely lost the ability of PHB accumulation Complementation with phaEHm and phaCHm genes of H.amylolyticum TNN58, restored this mutantthe capability of PHB accumulation. These results indicated that the phaEC genes were required for biosynthesis of PHB, and might encode an active PHA synthase.PHA can be accumulated in many microorganisms. But, there is no report comparing the properties of PHBV from different groups of producers, e.g. bacteria and haloarchaea. In this study, two types of PHBV copolymers were synthesized in Halogranum amylolyticum and Ralstonia eutropha, respectively, by feeding different carbon sources. They possessed a similar concentration of 3HV monomers(21 mol%) and were named PHBV-HA(produced by H.amylolyticum) and PHBV-RE(produced by R. eutropha) based on their source. Interestingly, they exhibited different behaviors especially in thermal stability, melting temperature, crystallinity percentage, and mechanical properties. Furthermore, the films of PHBV-HA and PHBV-RE possessed different surface properties, such as surface roughness, wettability, and surface free energy. The value of hemolysis on the PHBV-HA film was lower in comparison with the PHBV-RE film, although the values were both within the limit of 5% permissible for biomaterials. Notably, few inactivated platelets adhered to the surface of the PHBV-HA film, whereas numerous activated platelets were seen on film PHBV-RE. These results indicated that PHBV-HA was a better potential component of blood-contact biomaterials than PHBV-RE. Our study clearly revealed that the properties of PHAs are source-dependent and haloarchaeal species provide a new opportunity for the production of desired PHAs.In order to get a systematic knowledge about the PHBV synthesis in H.amylolyticum TNN58, this study used the shot-gun sequencing technology to sequence the genome of H.amylolyticum TNN58. Then, the Velvet software was used to stitch and got 32 scaffolds. The whole genome is about 4.71 M, the sequencing depth was 218 X. According to the analysis of genome sequence, the PHA synthesis related genes were identified and the carbon metabolic pathways were predicted in this strain. These results will help to reveal the pathways of PHBV synthesis and provide genome sequence for the deep researches in H.amylolyticum TNN58.