Effects Of Cascaded VGB Promoters On Poly(hydroxybutyrate) Micro-aerobic Synthesis In Recombinant Escherichia Coli And Halomonas Sp.

Polyhydroxyalkanoates(PHA), as a family of biodegradable and biocompatible thermal polyesters with diverse structures, have the potentials to be developed as enviromentally friendly bioplastics with many applications. However, the high production cost impedes the wide applications of PHA as enviromentally friendly materials. Micro-aeration is a situation that will be encountered in bacterial cell growth especially when the saturated dissolved oxygen level cannot match the demand from cells grown to a high density. Therefore, it is desirable to separate aerobic growth and micro-aerobic product formation into two stages. In order to reduce the cost, we attempted to produce polyhydroxybutyrate(PHB), as a model, in microaerobic cultivation with high effective micro-aerobic promoters to reduce aeration energy cost.To enhance micro-aerobic production of PHB in recombinant Escherichia coli, micro-aerobic promoters were applied to improve the expression level of PHB synthetic genes. Eleven potential low aeration-inducible promoters were cloned and studied for their induction strengths under microaerobic conditions. Of them, Vitreoscilla hemoglobin promoter(Pvgb) was found to be the strongest among all 11 promoters. Recombinant E. coli(pBHR-Pvgb) accumulated 68.48% PHB,13% higher than control.Cascaded Pvgb repeats(Pnvgb) were investigated for enhanced expression level under micro-aerobic growth. The highest PHA production was obtained when a promoter containing eight cascaded Pvgb repeats(P8vgb) was used, and 5.37 g/L CDW containing 90% PHB was obtained from recombinant in E. coli S17-1. The qPCR results of phaC expression level show the positively correlated with PHB content.At the same time, six E. coli hosts harboring poly(R-3-hydroxybutyrate)(PHB) synthesis operon phaCAB were compared for their ability to accumulate poly(hydroxyalkanoates)(PHA). E. coli S17-1 was demonstrated to be the best host achieving a 70% PHB in the cell dry weigh(CDW) after 48 h under microaerobic growth. Cells grew further to 6.30 g/l CDW containing 91% PHB when oxygen-responsive transcription factor Arc A(arcA) was deleted in the same recombinant E. coli S17-1.Finally, 4.5 g/L CDW containing 75% PHB, 35% higher than control, was obtained by co-expressing the gene vgb and PHB synthesis genes(phaCABLS21) with P8 vgb in recombinant Halomonas sp. LS21 grown micro-aerobically. In fermentation, 30 g/L CDW containing 75% PHB was obtained in the same recombinant Halomonas sp. LS21 after 48 h.P8vgb is an effective micro-aerobic promoter, and co-expressing the gene vgb and PHB synthesis genes(phaCABLS21) can improve significantly the CDW and PHB content of Halomonas sp. LS21.