Strategies for Poly(3-hydroxybutyrate) Production Using a Cold-Shock Promoter in Escherichia coli
نویسندگان
چکیده
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 promoters. intact C. A-04, an alkaline tolerant isolated in Thailand with a high degree 16S rRNA sequence similarity H16, was subcloned into pGEX-6P-1, pColdI, pColdTF, pBAD/Thio-TOPO, and pUC19 (native promoter) transformed Escherichia coli JM109. While the phaC A–04 gene insoluble most systems tested, it became soluble when expressed as fusion protein trigger factor (TF), ribosome associated bacterial chaperone, under control cold shock promoter. Careful optimization indicates that cold-shock cspA promoter enhanced chaperone function TF play critical roles increasing protein. Induction strategies parameters flask experiments were optimized obtain PhaC Y P/S productivity. Soluble purified through immobilized metal affinity chromatography (IMAC). results demonstrated pColdTF- phaCAB at level 47.4 ± 2.4% total formation approximately 3.09?4.1 times higher than pColdI- both conventional method short induction developed this study. Cultivation 5-L fermenter led 89.8 2.3% content, value 0.38 g PHB/g glucose productivity 0.43 PHB/(L.h) . film exhibited optical transparency possessed M w 5.79 × 10 5 Da, n 1.86 PDI 3.11 normal melting temperature mechanical properties.
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ژورنال
عنوان ژورنال: Frontiers in Bioengineering and Biotechnology
سال: 2021
ISSN: ['2296-4185']
DOI: https://doi.org/10.3389/fbioe.2021.666036