Enhanced 4-hydroxybutyrate incorporation into the PHA terpolymer of Haloferax mediterranei by heterologous expression of 4-hydroxybutyrate-CoA transferases/synthetases

The polyhydroxyalkanoate terpolymer, P[(3HB)- co -(3HV)- co -(4HB)], is a promising plastic alternative for specialized applications, notably in medical and pharmaceutical sectors. Haloferax mediterranei (Hfx), an extreme halophile archaeon, is a P[(3HB)- co -(3HV)- co -(4HB)] terpolymer production host, however the native molar proportion of 4HB incorporated into the terpolymer is low. To improve incorporation, four 4-hydroxybutyrate-CoA transferases/synthetases from Clostridum kluyveri (OrfZ), Clostridium aminobutyricum (AbfT), Nitrosopumilis maritimus ( Nm CAT), and Cupriavidus necator N-1 ( Cn CAT), were heterologously expressed in H. mediterranei , and evaluated for their ability to supply 4HB-CoA for PHA terpolymer production. Growth, PHA synthesis, and polymer composition were evaluated for the four heterologous strains in shake-flask, with Hfx_ Nm CAT demonstrating superior growth, terpolymer titre and 4HB molar ratio. Co-feeding with γ-butyrolactone was optimised, and Hfx_ Nm CAT was further evaluated under fed-batch fermentation where a maximum PHA titre of 0.7 g/L, containing 52 mol% 4HB, was achieved. This is an order of magnitude improvement in 4HB terpolymer incorporation by H. mediterranei . • Heterologous 4HB-CoA transferase/synthetase expression in H. mediterranei . • PHA terpolymer production from glucose and γ-butyrolactone co-feed. • P[(3HB)- co -(5 mol% 3HV)- co -(52 mol% 4HB)] production in fed-batch fermentation.