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

Abstract

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 (phaAA–04, 1182 bp, 40.6 kDa), acetoacetyl-CoA reductase (phaBA–04, 741 bp, 26.4 kDa) and PHB synthase Class I (phaCA–04, 1770 bp), was identified. Sequence analysis of the phaAA–04, phaBA–04, and phaCA–04 genes revealed that phaCA–04 was 99% similar to phaCH16 from C. necator H16. The difference in amino acid residue situated at position 122 of phaCA–04 was proline, whereas that of C. necator H16 was leucine. The intact phaCABA–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-phaCABA–04 and pColdTF-phaCABA–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 YP/S value of 0.39 g PHB/g glucose and a productivity of 0.6 g PHB/(L⋅h) using pColdTF-phaCABA–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 YP/S value of 0.21 g PHB/g glucose and a productivity of 0.4 g PHB/(L⋅h) using pBAD/Thio-TOPO-phaCABA–04 in LB medium. Thus, a relatively high PHB concentration and productivity were achieved, which demonstrated the possibility of industrial production of PHB.

Characterization of Polyhydroxybutyrate, PHB, Synthesized by Newly Isolated Haloarchaea Halolamina spp.

“…One of the solutions to the problem of petroleum plastics is to replace them with environmentally friendly, renewable, economically profitable, and biodegradable plastics. Bioplastics are biopolymers made up of a variety of compounds, with most of them being renewable, such as cellulose, starch, or bioethanol [ 2 ]. They are produced and accumulated by several microorganisms, such as bacteria, cyanobacteria, and archaea, as intracellular granules and serve as carbon storage for energy [ 3 ].…”

Strategies for Poly(3-hydroxybutyrate) Production Using a Cold-Shock Promoter in Escherichia coli

“…To verify that the cold-shock cspA promoter works together with the TF chaperone to improve the solubility of PhaC A–04 , the hydrophilic GST tag was fused to the N-terminus of PhaC A–04 (pGEX-6P-1- phaCAB A–04 ), and the effect of the GST tag at 37°C on the polymerization reaction of phaC A–04 based on the amount of PHB production was investigated. In addition, pBAD/Thio-TOPO- phaCAB A–04 , encoding a hydrophilic N-terminal thioredoxin fusion protein and C-terminal 6His-fusion protein induced by arabinose, was also used for comparison ( Napathorn et al, 2021 ). The control strain, harboring pUC19-nativeP- phaCAB A–04 , was under the control of the native promoter derived from C. necator strain A-04, and no induction agent was required under the same conditions.…”

“…The difference was in the amino acid residue situated at position 122, which in phaC A-04 was proline but in C. necator H16 was leucine. The total amino acid sequences of phaA A-04 and phaB A-04 were 100% matched with those of C. necator H16 (Napathorn et al, 2021). Notably, C. necator strain A-04 prefers fructose over glucose as a carbon source, accumulating PHB at 78% of the CDM under a C/N ratio of 200, whereas it could incorporate a high mole fraction of monomeric 4-hydroxybutyrate monomeric into the poly(3-hydroxybutyrateco-4-hydroxybutyrate) [P(3HB-co-4HB)] copolymer under a C/N ratio of 20 (Chanprateep et al, 2010), as well as the poly(3hydroxybutyrate-co-3-hydroxyvaterate-co-4-hydroxybutyrate) [P(3HB-co-3HV-co-4HB)] terpolymer (Chanprateep and Kulpreecha, 2006).…”

“…amount of PHB production was investigated. In addition, pBAD/Thio-TOPO-phaCAB A-04 , encoding a hydrophilic N-terminal thioredoxin fusion protein and C-terminal 6His-fusion protein induced by arabinose, was also used for comparison (Napathorn et al, 2021). The control strain, harboring pUC19-nativeP-phaCAB A-04 , was under the control of the native promoter derived from C. necator strain A-04, and no induction agent was required under the same conditions.…”

“…Another benefit of using the pCold system is that the induction was carried out at cold temperatures, which has been shown to significantly improve protein folding by decreasing the rate of transcription and translation, thus providing more time for the protein to be folded (Agashe et al, 2004;Baneyx and Mujacic, 2004). Compared with previous reports, the PHA biosynthesis operon of C. necator strain A-04 was also cloned into pGEX-6P-1 [tac promoter, isopropyl-β-D-thiogalactopyranoside (IPTG)-inducible vector, N-terminal GST fusion protein], pBAD/Thio-TOPO (araBAD promoter, arabinose-inducible vector, N-terminal thioredoxin fusion protein and C-terminal 6His-fusion protein) (Napathorn et al, 2021) and pUC19 (control strain, phaCAB A-04 biosynthesis genes with native promoter of phaC A-04 ) and transformed into E. coli JM109 (Table 1). Next, to optimize phaC A-04 overexpression in shake flask cultivation, three induction methods were tested and compared with conventional induction method (Figure 1, details are described in the section "Materials ) and E. coli JM109 (pColdTF-phaCAB A-04 ).…”