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To develop a microbial strain producing poly(3-hydroxybutyrate) [P(3HB)], in the absence of antibiotic supplementation (normally required to stabilize a recombinant plasmid), by constructing a recombinant Escherichia coli strain with phaCAB and vgb integrated into the chromosome.The polyhydroxyalkanoate (PHA) synthesis operon (phaCAB) and the bacterial haemoglobin gene (vgb) were integrated downstream of nlpB (novel lipoprotein B) in E. coli K12, via homologous recombination, to form a recombinant strain, termed YH100. VHb encoded by the vgb gene was successfully expressed in YH100, as confirmed by Western blotting. P(3HB) synthesis by the YH100 strain grown in the absence of antibiotic was analysed by transmission electron microscopy. The yield of P(3HB) is 208 mg g−1. The thermal stability of P(3HB) produced from YH100 was similar to that of commercial P(3HB). Further, the polydispersity index (PDI) of the P(3HB) polymer derived from YH100 was 1·37, indicating that polymer uniformity was greater than that of commercial P(3HB), which had a PDI of 1·47.We successfully constructed a recombinant E. coli strain expressing exogenous genes, specifically phaCAB from Cupriavidus necator and vgb from Vitreoscilla stercoraria, integrated into the downstream of chromosomal dapA-nlpB locus. P(3HB) was stably produced by this strain, without any need for antibiotic supplementation to stabilize a recombinant plasmid at least for 48h.We report a genetic locus, the downstream of the nlpB locus in E. coli, in which the transcription of the exogenous genes is driven by the dapA-nlpB promoter without the need for the addition of inducer and antibiotic.