Conversion of glycerol to poly(3-hydroxypropionate) in recombinant Escherichia coli

Abstract

We have developed the conversion of glycerol into thermoplastic poly(3-hydroxypropionate) [poly(3HP)]. For this, the genes for glycerol dehydratase (dhaB1) of Clostridium butyricum, propionaldehyde dehydrogenase (pduP) of Salmonella enterica serovar Typhimurium LT2, and polyhydroxyalkanoate (PHA) synthase (phaC1) of Ralstonia eutropha were expressed in recombinant Escherichia coli. Poly(3HP) was accumulated up to 11.98% (wt/wt [cell dry weight]) in a two-step, fed-batch fermentation. The present study shows an interesting application to engineer a poly(3HP) synthesis pathway in bacteria.

FIG. 1.

Pathway for conversion of glycerol into poly(3HP) in the engineered strain of E. coli.

FIG. 2.

Two-step fermentation of E. coli HMS174(DE3)/pCOLADuet-1::dhaB1B2::pduP::phaC1, with pure (□) or crude (○) glycerol as the sole carbon source. The cultivation of the cells was divided into an aerobic growth phase and an anaerobic production phase and was carried out using Riesenberg medium (19). The content of poly(3HP) derived from pure (▪) or crude (•) glycerol was monitored by GC-MS. CDW, cell dry weight.

FIG. 3.

Proteins in crude extracts of cells of E. coli HMS174(DE3), E. coli HMS174(DE3)/pCOLADuet-1, and E. coli HMS174(DE3)/pCOLADuet-1::dhaB1B2::pduP::phaC1 grown in LB medium. Protein expression was induced with 1 mM IPTG at an optical density at 600 nm of 0.5, and the cells were then cultivated for an additional 8 h. The proteins were separated using 12.5% (wt/vol) SDS-polyacrylamide gels and stained with Coomassie brilliant blue. Lane 1, E. coli HMS174(DE3); lane 2, E. coli HMS174(DE3)/pCOLADuet-1; lane 3, E. coli HMS174(DE3)/pCOLADuet-1::dhaB1B2::pduP::phaC1. The values on the left indicate molecular mass (in kDa).

FIG. 4.

TEM of poly(3HP)-accumulating cells of E. coli HMS174(DE3)/pCOLADuet-1::dhaB1B2::pduP::phaC1. The fermentation was divided into an aerobic growth phase and an anaerobic production phase and was carried out using Riesenberg medium (19). The feeding solution for the anaerobic production phase was supplemented with 0.5 M di-Na⁺-fumarate, 0.5 M K⁺-Na⁺-tartrate, and 1 mM IPTG. Bars, 100 nm.