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Cupriavidus necator  strain A-04 has shown 16S rRNA gene identity to the well-known industrial strain  C. necator  H16. Nevertheless, the cell characteristics and polyhydroxyalkanoate (PHA) production ability of  C. necator  strain A-04 were different from those of  C. necator  H16. This study aimed to express PHA biosynthesis genes of  C. necator  strain A-04 in  Escherichia coli  via an arabinose-inducible expression system. In this study, the PHA biosynthesis operon of  C. necator  strain A-04, consisting of three genes encoding acetyl-CoA acetyltransferase ( phaA  A–04 , 1182 bp, 40.6 kDa), acetoacetyl-CoA reductase ( phaB  A–04 , 741 bp, 26.4 kDa) and PHB synthase Class I ( phaC  A–04 , 1770 bp), was identified. Sequence analysis of the  phaA  A–04 ,  phaB  A–04 , and phaC A–04  genes revealed that  phaC  A–04  was 99% similar to  phaC  H16  from  C. necator  H16. The difference in amino acid residue situated at position 122 of  phaC  A–04  was proline, whereas that of  C. necator  H16 was leucine. The intact  phaCAB  A–04  operon was cloned into the arabinose-inducible araBAD promoter and transformed into  E. coli  strains Top 10, JM109 and XL-1 blue. The results showed that optimal conditions obtained from shaken flask experiments yielded 6.1 ± 1.1 g/L cell dry mass (CDM), a PHB content of 93.3 ± 0.9% (w/w) and a productivity of 0.24 g/(L⋅h), whereas the wild-type  C. necator  strain A-04 accumulated 78% (w/w) PHB with a productivity of 0.09 g/(L⋅h). Finally, for the scaled-up studies, fed-batch cultivations by pH-stat control in a 5-L fermenter of  E. coli  strains XL1-Blue harboring pBAD/Thio-TOPO- phaCAB  A–04  and pColdTF- phaCAB  A–04  in MR or LB medium, leading to a PHB production of 31.4 ± 0.9 g/L at 54 h with a PHB content of 83.0 ± 3.8% (w/w), a CDM of 37.8 ± 1.2 g/L, a Y  P/S   value of 0.39 g PHB/g glucose and a productivity of 0.6 g PHB/(L⋅h) using pColdTF- phaCAB  A–04  in MR medium. In addition, PHB production was 29.0 ± 1.1 g/L with 60.2 ± 2.3% PHB content in the CDM of 53.1 ± 1.0 g/L, a Y  P/S   value of 0.21 g PHB/g glucose and a productivity of 0.4 g PHB/(L⋅h) using pBAD/Thio-TOPO- phaCAB  A–04  in LB medium. Thus, a relatively high PHB concentration and productivity were achieved, which demonstrated the possibility of industrial production of PHB.

frontiers in bioengineering and biotechnology

Polyhydroxybutyrate (PHB) Production Using an Arabinose-Inducible Expression System in Comparison With Cold Shock Inducible Expression System in Escherichia coli