Chromosomal integration and expression of heterologous gene(s) are favored in industrial biotechnology due to the inheriting expression stability. Yet, chromosomal expression is commonly weaker than plasmid one. The effect on gene expression level at 13 chromosomal locations in Escherichia coli was investigated using the polyhydroxybutyrate (PHB) synthesis pathway encoded by a phaCAB operon as a reporter. When 11 copies of phaCAB were randomly integrated into 11 of the 13 chromosomal locations, respectively, 5.2 wt% of PHB was produced. PHB (34.1 wt%) was accumulated by a recombinant E. coli inserted chromosomally with 50 copies of phaCAB in the active asnB site using a Cre-loxP recombination method. This PHB accumulation level was equivalent to a medium-copy-number plasmid expression system, suggesting the importance of chromosomal gene copy number for PHB production by E. coli. This result was used to manipulate a Halomonas strain. One copy of genes scpAB encoding methylmalonyl-CoA mutase and methylmalonyl-CoA decarboxylase was inserted into the strongest expression site porin in the chromosome of the 2-methylcitrate synthase (prpC) deleted mutant Halomonas TD08, leading to the synthesis of poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) from glucose as the sole carbon source. The chromosome-engineered strain produced PHBV consisting of 5-12 mol% 3-hydroxyvalerate (3HV) stably compared with unstable fluctuation of 7-25 mol% 3HV by a medium-copy-number plasmid system. These results demonstrated that chromosome engineering based on active transcriptional site and gene copy number is more feasible for polyhydroxyalkanoate (PHA) synthesis in Halomonas TD08 compared with in E. coli.