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.

Keywords: Cupriavidus necator; E. coli – Escherichia coli; cold shock; cspA gene; pCold; polyhydroxybutyrate.

FIGURE 1

Schematic of three different induction methods for heterologous expression of the phaCAB_A–04 biosynthesis operon in E. coli JM109 (pColdI-phaCAB_A–04) and E. coli JM109 (pColdTF-phaCAB_A–04). (A) Conventional induction method: the culture was incubated at 37°C and 200 rpm until the OD₆₀₀ reached 0.5, 1.3, 2.1, and 2.4. Then, the cultivation temperature was decreased from 37 to 15°C for 30 min, and the expression of the phaCAB_A–04 operon was induced by the addition of 0.5 mM IPTG. The cultivation temperature was further maintained at 15°C for 24 h. (B) Short-induction method: the culture was incubated at 37°C and 200 rpm until the OD₆₀₀ reached 0.5, 1.3, 2.1, and 2.4. Then, the temperatures were varied at 15, 25, 30, and 37°C for 30 min. Next, the expression of the phaCAB_A–04 operon was induced by adding various concentrations (0.01, 0.05, 0.1, 0.5, and 1.0 mM) of IPTG, and the cultivation temperature was maintained at 37°C for 24 h. (C) Preinduction method: the culture was incubated at 37°C and 200 rpm until the OD₆₀₀ reached 0.5. Next, 0.5 mM IPTG was added into the culture when the temperature was decreased from 37°C to 15°C for 24 h. Then, the induced cells were harvested by centrifugation, the medium was discarded, and the cells were resuspended in an equal volume of fresh LB medium. Finally, the induced cells at 1, 5, or 10% (v/v) were transferred into fresh LB medium supplemented with 100 μg/L ampicillin and 20 g/L glucose and incubated at 37°C and 200 rpm for 24 h.

FIGURE 2

Effect of the growth phase suitable for cold-shock induction on CDM and PHB content (% w/w) under the conventional induction method. The different growth phases were investigated by varying OD₆₀₀ based on cultivation time [0.5 (2 h, early exponential phase), 1.3 (4 h, middle exponential phase), 2.1 (6 h, late exponential phase), and 2.4 (10 h, stationary phase)] for (A) E. coli JM109 (pColdI-phaCAB_A–04) and (B) E. coli JM109 (pColdTF-phaCAB_A–04). A control experiment was performed with 0.0 mM IPTG induction. All the data are representative of the results of three independent experiments and are expressed as the mean values ± standard deviations (SDs). The PhaC_A–04 protein was detected by western blot analysis using anti-His tag antibody as the primary antibody. The band appearing in the western blot at the position corresponding to that of the His-tagged phaC_A–04 protein was 67 kDa in size for pColdI-phaCAB_A–04, and the fusion protein of His-tagged phaC_A–04 and TF was 115 kDa in size. All the data are representative of the results of three independent experiments and are expressed as the mean values ± standard deviations (SDs). Symbols: open squares, CDM (g/L); closed circle, PHB (g/L).

FIGURE 3

Time courses of PHB production (g/L). (A) The insoluble PhaC_A–04 protein was confirmed by SDS-PAGE analysis (20 μg of total protein was loaded in each lane). (B) The soluble PhaC_A–04 protein was confirmed by SDS-PAGE analysis (20 μg of total protein was loaded in each lane). Lane M, Protein molecular weight marker; lane 1, E. coli JM109 (pUC19-nativeP-phaCAB_A–04) under short induction temperature profile, but without addition of IPTG; lane 2, E. coli JM109 pBAD/Thio-TOPO- phaCAB_A–04 under short induction method; lane 3, E. coli JM109 (pGEX-6P-1- phaCAB_A–04) under short induction method; lane 4, E. coli JM109 (pColdI-phaCAB_A–04) under short induction method; lane 5, E. coli JM109 (pColdTF-phaCAB_A–04) under short induction method; lane 6, E. coli JM109 (pColdI-phaCAB_A–04) under conventional induction method; lane 7, E. coli JM109 (pColdTF-phaCAB_A–04) under conventional induction method. The band appearing in the SDS-PAGE at the position corresponding to that of the phaC_A–04 protein was 64 kDa in size for pUC19-nativeP-phaCAB_A–04, His-tagged phaC_A–04 fusion protein was 67 kDa in size for pColdI-phaCAB_A–04, thioredoxin-tagged phaC_A–04 fusion protein was 77 kDa in size for pBAD/Thio-TOPO-phaCAB_A–04, GST-tagged phaC_A–04 fusion protein was 91 kDa in size for pGEX-6P-1-phaCAB_A–04, and the fusion protein of His-tagged phaC_A–04 and TF was 115 kDa in size for pColdTF-phaCAB_A–04.

FIGURE 4

Effect of the growth phase suitable for cold-shock induction on CDM and PHB content (% w/w) under the short-induction method. The different growth phases were investigated by varying OD₆₀₀ based on cultivation time [0.5 (2 h, early exponential phase), 1.3 (4 h, middle exponential phase), 2.1 (6 h, late exponential phase), and 2.4 (10 h, stationary phase)] for (A) E. coli JM109 (pColdI-phaCAB_A–04) and (B) E. coli JM109 (pColdTF-phaCAB_A–04). A control experiment was performed with 0.0 mM IPTG induction. All the data are representative of the results of three independent experiments and are expressed as the mean values ± standard deviations (SDs). Symbols: open squares, CDM (g/L); closed circle, PHB (g/L).

FIGURE 5

Effect of different short-induction temperatures (15, 25, 30, and 37 °C) on the CDM (g/L), RCM (g/L), PHB (g/L), and PHB content (% w/w) of E. coli JM109 (pColdI-phaCAB_A–04). All the data are representative of the results of three independent experiments and are expressed as the mean values ± standard deviations (SDs). Symbols: open circle, CDM (g/L); closed circle, RCM (g/L); open square, PHB (g/L); closed triangle, PHB content (% w/w).

FIGURE 6

Time courses of CDM (g/L), RCM (g/L), PHB (g/L), PHB content (% w/w), and glucose (g/L) and pH during batch cultivation in a 5-L fermenter under the short-induction method in a comparison between (A) E. coli JM109 (pColdI-phaCAB_A–04) and (B) E. coli JM109 (pColdTF-phaCAB_A–04). The band appearing in the western blot at the position corresponding to that of the His-tagged phaC_A–04 protein was 67 kDa in size for pColdI-phaCAB_A–04, and the fusion protein of His-tagged phaC_A–04 and TF was 115 kDa in size. All the data are representative of the results of three independent experiments and are expressed as the mean values ± standard deviations (SDs). Symbols: closed square, CDM (g/L); closed circle, glucose (g/L); asterisks, pH; open square, RCM (g/L); open circle, PHB content (% w/w).

FIGURE 7

Morphology of PHB films produced by (A) C. necator strain A-04, (B) E. coli JM109 (pColdI-phaCAB_A–04), and (C) E. coli JM109 (pColdTF-phaCAB_A–04).