AUTHOR=Napathorn Suchada Chanprateep , Visetkoop Sirirat , Pinyakong Onruthai , Okano Kenji , Honda Kohsuke 

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

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=9

YEAR=2021

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2021.661096

DOI=10.3389/fbioe.2021.661096

ISSN=2296-4185

ABSTRACT=<p><italic>Cupriavidus necator</italic> strain A-04 has shown 16S rRNA gene identity to the well-known industrial strain <italic>C. necator</italic> H16. Nevertheless, the cell characteristics and polyhydroxyalkanoate (PHA) production ability of <italic>C. necator</italic> strain A-04 were different from those of <italic>C. necator</italic> H16. This study aimed to express PHA biosynthesis genes of <italic>C. necator</italic> strain A-04 in <italic>Escherichia coli</italic> via an arabinose-inducible expression system. In this study, the PHA biosynthesis operon of <italic>C. necator</italic> strain A-04, consisting of three genes encoding acetyl-CoA acetyltransferase (<italic>phaA</italic><sub>A–04</sub>, 1182 bp, 40.6 kDa), acetoacetyl-CoA reductase (<italic>phaB</italic><sub>A–04</sub>, 741 bp, 26.4 kDa) and PHB synthase Class I (<italic>phaC</italic><sub>A–04</sub>, 1770 bp), was identified. Sequence analysis of the <italic>phaA</italic><sub>A–04</sub>, <italic>phaB</italic><sub>A–04</sub>, and phaC<sub>A–04</sub> genes revealed that <italic>phaC</italic><sub>A–04</sub> was 99% similar to <italic>phaC</italic><sub>H16</sub> from <italic>C. necator</italic> H16. The difference in amino acid residue situated at position 122 of <italic>phaC</italic><sub>A–04</sub> was proline, whereas that of <italic>C. necator</italic> H16 was leucine. The intact <italic>phaCAB</italic><sub>A–04</sub> operon was cloned into the arabinose-inducible araBAD promoter and transformed into <italic>E. coli</italic> 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 <italic>C. necator</italic> 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 <italic>E. coli</italic> strains XL1-Blue harboring pBAD/Thio-TOPO-<italic>phaCAB</italic><sub>A–04</sub> and pColdTF-<italic>phaCAB</italic><sub>A–04</sub> 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<sub><italic>P/S</italic></sub> value of 0.39 g PHB/g glucose and a productivity of 0.6 g PHB/(L⋅h) using pColdTF-<italic>phaCAB</italic><sub>A–04</sub> 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<sub><italic>P/S</italic></sub> value of 0.21 g PHB/g glucose and a productivity of 0.4 g PHB/(L⋅h) using pBAD/Thio-TOPO-<italic>phaCAB</italic><sub>A–04</sub> in LB medium. Thus, a relatively high PHB concentration and productivity were achieved, which demonstrated the possibility of industrial production of PHB.</p>