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 70 mol%) in shaken flask cultures. Combinations of 4HB carbon precursors consisting of 1,6-hexanediol and gamma-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 93 mol% 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 5 L and 30 L bioreactors with a constant oxygen mass transfer rate. The 4HB monomer composition could be maintained at a range of 83-89 mol%. 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. (C) 2016 Elsevier Inc. All rights reserved.