Engineering of Escherichia coli for direct and modulated biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer using unrelated carbon sources.
While poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] is a biodegradable commodity plastic with broad applications, its microbial synthesis is hindered by high production costs primarily associated with the supplementation of related carbon substrates (e.g. propionate or valerate). Here we report construction of engineered Escherichia coli strains for direct synthesis of P(3HB-co-3HV) from an unrelated carbon source (e.g. glucose or glycerol). First, an E. coli strain with an activated sleeping beauty mutase (Sbm) operon was used to generate propionyl-CoA as a precursor. Next, two acetyl-CoA moieties or acetyl-CoA and propionyl-CoA were condensed to form acetoacetyl-CoA and 3-ketovaleryl-CoA, respectively, by functional expression of β-ketothiolases from Cupriavidus necator (i.e. PhaA and BktB). The resulting thioester intermediates were channeled into the polyhydroxyalkanoate (PHA) biosynthetic pathway through functional expression of acetoacetyl-CoA reductase (PhaB) for thioester reduction and PHA synthase (PhaC) for subsequent polymerization. Metabolic engineering of E. coli host strains was further conducted to enhance total PHA content and the 3-hydroxyvaleryl (3HV) monomer fraction in the copolymer. Using a selection of engineered E. coli strains for batch cultivation with an unrelated carbon source, we achieved high-level P(3HB-co-3HV) production with the 3HV monomer fraction ranging from 3 to 19 mol%, demonstrating the potential industrial applicability of these whole-cell biocatalysts.
Department of Chemical Engineering , University of Waterloo, 200 University Avenue West Waterloo, Ontario N2L 3G1 Canada.
Kajan
K
Srirangan
Department of Chemical Engineering , University of Waterloo, 200 University Avenue West Waterloo, Ontario N2L 3G1 Canada.
Xuejia
X
Liu
Department of Biology, University of Waterloo, Waterloo, Ontario, Canada.
Tam T
TT
Tran
Department of Biology, University of Waterloo, Waterloo, Ontario, Canada.
Trevor C
TC
Charles
Department of Chemical Engineering , University of Waterloo, 200 University Avenue West Waterloo, Ontario N2L 3G1 Canada.
Murray
M
Moo-Young
Department of Chemical Engineering , University of Waterloo, 200 University Avenue West Waterloo, Ontario N2L 3G1 Canada.
C Perry
CP
Chou
D000102:Acetyl-CoA C-Acyltransferase / Q000235:genetics / Q000378:metabolism; D000214:Acyl Coenzyme A / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002244:Carbon / Q000378:metabolism*; D020118:Cupriavidus necator / Q000201:enzymology; D004926:Escherichia coli / Q000378:metabolism*; D005990:Glycerol / Q000378:metabolism; D060847:Metabolic Engineering; D011091:Polyesters / Q000737:chemistry / Q000378:metabolism*; D000094122:Polyhydroxybutyrates
D000102:Acetyl-CoA C-Acyltransferase; D000214:Acyl Coenzyme A; D001426:Bacterial Proteins; D002244:Carbon; D005990:Glycerol; D011091:Polyesters; C436023:3-ketovaleryl-coenzyme A; C052620:poly(3-hydroxybutyrate)-co-(3-hydroxyvalerate); D000094122:Polyhydroxybutyrates; C010667:acetoacetyl CoA; C009061:propionyl-coenzyme A