Dataset Information

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

ABSTRACT: The present study attempted to increase poly(3-hydroxybutyrate) (PHB) production by improving expression of PHB biosynthesis operon derived from Cupriavidus necator strain A-04 using various types of promoters. The intact PHB biosynthesis operon of C. necator A-04, an alkaline tolerant strain isolated in Thailand with a high degree of 16S rRNA sequence similarity with C. necator H16, was subcloned into pGEX-6P-1, pColdI, pColdTF, pBAD/Thio-TOPO, and pUC19 (native promoter) and transformed into Escherichia coli JM109. While the phaC _A-04 gene was insoluble in most expression systems tested, it became soluble when it was expressed as a fusion protein with trigger factor (TF), a ribosome associated bacterial chaperone, under the control of a cold shock promoter. Careful optimization indicates that the cold-shock cspA promoter enhanced phaC_A-04 protein expression and the chaperone function of TF play critical roles in increasing soluble phaC_A-04 protein. Induction strategies and parameters in flask experiments were optimized to obtain high expression of soluble PhaC_A-04 protein with high Y_P/S and PHB productivity. Soluble phaC_A-04 was purified through immobilized metal affinity chromatography (IMAC). The results demonstrated that the soluble phaC_A-04 from pColdTF-phaCAB _A-04 was expressed at a level of as high as 47.4 ± 2.4% of total protein and pColdTF-phaCAB _A-04 enhanced soluble protein formation to approximately 3.09-4.1 times higher than that from pColdI-phaCAB _A-04 by both conventional method and short induction method developed in this study. Cultivation in a 5-L fermenter led to PHB production of 89.8 ± 2.3% PHB content, a Y_P/S value of 0.38 g PHB/g glucose and a productivity of 0.43 g PHB/(L.h) using pColdTF-phaCAB _A-04. The PHB film exhibited high optical transparency and possessed M_w 5.79 × 10⁵ Da, M_n 1.86 × 10⁵ Da, and PDI 3.11 with normal melting temperature and mechanical properties.

SUBMITTER: Boontip T

PROVIDER: S-EPMC8211017 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description: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 phaCA-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.

Dataset Information

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets