Application of the BPEC pathway for large-scale biotechnological production of poly(3-mercaptopropionate) by recombinant Escherichia coli, including a novel in situ isolation method

Abstract

Metabolically engineered Escherichia coli JM109 harboring plasmid pBPP1 and expressing the nonnatural BPEC pathway for synthesis of thermoplastic polyhydroxyalkanoates (PHA) and novel polythioesters (PTE) to provide suitable substrates of PHA synthase was investigated with respect to biotechnological production of poly(3-mercaptopropionate) [poly(3MP)]. Fed-batch fermentation processes were established at the 30- and 500-liter scales in stirred tank bioreactors to produce kilogram amounts of poly(3MP). Cultivation was done in a modified M9 mineral salts medium containing glucose or glycerol as the carbon and energy source and with 3-mercaptopropionic acid (3MP) as the precursor substrate for poly(3MP) biosynthesis provided from the late exponential growth phase. Approximately 23 g of cell dry matter (CDM) per liter and poly(3MP) cell contents of up to 45% (wt/wt) were the highest cell densities and polymer contents obtained, respectively. At best, 69.1% (wt/wt) of 3MP was converted into poly(3MP), indicating that 3MP was mostly used for poly(3MP) biosynthesis. Furthermore, a novel in situ process for rapid and convenient isolation of poly(3MP) from the cells in the bioreactor was developed. This was achieved by addition of sodium dodecyl sulfate to the cultivation broth immediately after the fermentation, heating to 90 degrees C for 20 min with intensive stirring, and subsequent washing steps. The purity of such in situ isolated poly(3MP) was more than 98%, as revealed by gas chromatographic and elemental sulfur analyses of the material isolated.

FIG. 1.

Time course of fed batch fermentation 5 of E. coli JM109(pBPP1) expressing the BPEC pathway for production of poly(3MP) at the 500-liter scale. Cultivation was done in a 650-liter stirred tank reactor containing 400 liters of modified M9-MSM with 0.6% (wt/vol) glucose and 2 g of NH₄Cl per liter at the beginning of the fermentation. The temperature (37°C) and pH (pH 7.0) were kept constant. The aeration (0.3 to 0.5 vvm) and the stirrer speed (100 to 400 rpm) were adjusted according to the oxygen demand of the culture. During fermentation glucose (solid arrows) and NH₄Cl (dotted arrows) were repeatedly fed before they became limiting at concentrations of 2 to 3 and 0.5 g/liter, respectively. The availability of the carbon source was estimated by monitoring the carbon dioxide concentration in the spent gas and the dissolved oxygen level in the medium. 3MP (514 g) was fed several times in the late exponential phase (horizontal arrow). Table 1 shows the total amounts of medium components used in this fermentation. OD₈₅₀, optical density at 850 nm.

FIG. 2.

Time course of fed batch fermentation 9 of E. coli JM109(pBPP1) expressing the BPEC pathway for production of poly(3MP) at the 30-liter scale. Cultivation was done in a 42-liter stirred-tank reactor containing 18 liters of modified M9-MSM with 3% (wt/vol) glycerol and 2 g of NH₄Cl per liter at the beginning of the fermentation. The temperature (35°C) and pH (pH 7.0) were kept constant. The aeration (0.5 to 1.0 vvm) and agitation (100 to 700 rpm) were adjusted according to the oxygen demand of the culture. Glycerol was used as the sole carbon source. Glycerol (5 to 10 g/liter) and MgSO₄ · 7H₂O (0.9 g/liter) were fed together (solid arrows) and NH₄Cl (0.5 g/liter) (dotted arrows) was fed repeatedly during the fermentation before these compounds became limiting. The availability of the carbon source was estimated by monitoring the carbon dioxide concentration in the spent gas and the dissolved oxygen level in the medium. 3MP (53 g) was fed several times in the late exponential phase (horizontal arrow). Table 1 shows the total amounts of medium components used in this fermentation. OD₈₅₀, optical density at 850 nm.