Abstract

Poly(3-hydroxybutyrate-co-3-hydroxypropionate), also known as P(3HB-co-3HP), is a microbially produced biopolymer proposed as a sustainable alternative to petroleum-based plastics. However, most biosynthetic pathways for its production rely on costly precursor or cofactor supplementation, limiting their industrial applicability. In this study, Cupriavidus necator H16 was engineered to produce P(3HB-co-3HP) directly from central metabolism by introducing a heterologous beta-alanine pathway for 3HP biosynthesis. Two strategies were employed to modulate copolymer composition: (i) a constitutive pathway modulated by nitrogen availability, and (ii) an arabinose-inducible system regulating the synthesis of the pathway intermediate beta-alanine. Both approaches enabled the production of copolymers with adjustable 3HP content without the need for external precursors or cofactors. The N-based strategy achieved a maximum 3HP content of 66.5 mol% with a PHA titer of 1.1 g/L, while the inducible system allowed finer monomer control and reached up to 51.7 mol% 3HP with a PHA titer of 2.5 g/L. Additionally, copolymer production was demonstrated using crude glycerol as the sole carbon source, where the 3HP incorporation was tunable from 6.9 to 37.7 mol%, depending on the arabinose concentration, and the PHA titer ranged from 1.6 to 2.0 g/L. This study highlights the potential of the beta-alanine pathway for sustainable 3HP-containing copolymers biosynthesis and demonstrates the feasibility of using crude glycerol, to support cost-effective bioplastic production.