title: Synthesis of high 4-hydroxybutyrate copolymer by Cupriavidus sp. transformants using one-stage cultivation and mixed precursor substrates strategy.
abstract: Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] copolymer is noted for its high biocompatibility, which makes it an excellent candidate for biopharmaceutical applications. The wild-type Cupriavidus sp. USMAA1020 strain is able to synthesize P(3HB-co-4HB) copolymers with different 4HB monomer compositions (up to 70mol%) in shaken flask cultures. Combinations of 4HB carbon precursors consisting of 1,6-hexanediol and γ-butyrolactone were applied for the production of P(3HB-co-4HB) with different 4HB molar fraction. A sharp increase in 4HB monomer composition was attained by introducing additional copies of PHA synthase gene (phaC), responsible for P(3HB-co-4HB) polymerization. The phaC of Cupriavidus sp. USMAA1020 and Cupriavidus sp. USMAA2-4 were cloned and heterologously introduced into host, wild-type Cupriavidus sp. USMAA1020. The gene dosage treatment resulted in the accumulation of 93mol% 4HB by the transformant strains when grown in similar conditions as the wild-type USMAA1020. The PHA synthase activities for both transformants were almost two-fold higher than the wild-type. The ability of the transformants to produce copolymers with high 4HB monomer composition was also tested in large scale production system using 5L and 30L bioreactors with a constant oxygen mass transfer rate. The 4HB monomer composition could be maintained at a range of 83-89mol%. The mechanical and thermal properties of copolymers improved with increasing 4HB monomer composition. The copolymers produced could be tailored for specific biopharmaceutical applications based on their properties.
authors.affiliation: School of Marine and Environmental Sciences, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu, Malaysia; Malaysian Institute of Pharmaceuticals and Nutraceuticals, NIBM MOSTI, Malaysia.
authors.forename: Ishak Muhammad
authors.initials: IM
authors.lastname: Syafiq
authors.affiliation: Malaysian Institute of Pharmaceuticals and Nutraceuticals, NIBM MOSTI, Malaysia.
authors.forename: Kai-Hee
authors.initials: KH
authors.lastname: Huong
authors.affiliation: School of Biological Sciences, Universiti Sains Malaysia, 11800 Pulau Pinang, Malaysia.
authors.forename: K
authors.initials: K
authors.lastname: Shantini
authors.affiliation: Malaysian Institute of Pharmaceuticals and Nutraceuticals, NIBM MOSTI, Malaysia; School of Fundamental Science, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu, Malaysia.
authors.forename: S
authors.initials: S
authors.lastname: Vigneswari
authors.affiliation: School of Biological Sciences, Universiti Sains Malaysia, 11800 Pulau Pinang, Malaysia.
authors.forename: Nursolehah Abd
authors.initials: NA
authors.lastname: Aziz
authors.affiliation: Malaysian Institute of Pharmaceuticals and Nutraceuticals, NIBM MOSTI, Malaysia; School of Biological Sciences, Universiti Sains Malaysia, 11800 Pulau Pinang, Malaysia; Centre for Chemical Biology, Universiti Sains Malaysia, 11900 Bayan Lepas, Pulau Pinang, Malaysia.
authors.forename: Al-Ashraf Abdullah
authors.initials: AA
authors.lastname: Amirul
authors.affiliation: School of Marine and Environmental Sciences, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu, Malaysia; Malaysian Institute of Pharmaceuticals and Nutraceuticals, NIBM MOSTI, Malaysia; Institute of Marine Biotechnology, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu, Malaysia. Electronic address: kesaven@umt.edu.my.
authors.forename: Kesaven
authors.initials: K
authors.lastname: Bhubalan
mesh_terms: D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D019149:Bioreactors / Q000382:microbiology; D001709:Biotechnology; D049921:Cupriavidus / Q000235:genetics / Q000378:metabolism*; D018628:Gene Dosage; D005798:Genes, Bacterial; D006885:Hydroxybutyrates / Q000737:chemistry / Q000378:metabolism*; D007218:Industrial Microbiology; D011091:Polyesters / Q000737:chemistry / Q000378:metabolism*; D014170:Transformation, Genetic; D000094122:Polyhydroxybutyrates
chemical_list: D000217:Acyltransferases; D001426:Bacterial Proteins; D006885:Hydroxybutyrates; D011091:Polyesters; C068056:poly(3-hydroxybutyrate-co-4-hydroxybutyrate); D000094122:Polyhydroxybutyrates; C077225:poly(3-hydroxyalkanoic acid) synthase
keywords: Cupriavidus sp. USMAA1020; Gene dosage; PHA synthase; Poly(3-hydroxybutyrate-co-4-hydroxybutyrate); Polyhydroxyalkanoate