Engineering of Ralstonia eutropha for production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from fructose and solid-state properties of the copolymer.
Recombinant Ralstonia eutropha capable of producing poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) copolymer [P(3HB-co-3HHx)] from fructose was engineered by introduction of genes for crotonyl-CoA reductase (CCR) from Streptomyces cinnamonensis (ccrSc) and for PHA synthase and (R)-specific enoyl-CoA hydratase from Aeromonas caviae (phaC-JAc). In this recombinant strain, C6-acyl-CoA intermediates were provided via beta-ketothiolase-mediated elongation of butyryl-CoA, which was generated from crotonyl-CoA by the function of CCR. The recombinant strain could accumulate the copolyester up to 48 wt % of dry cell weight with 1.5 mol % of 3HHx fraction from fructose, when the expression of ccrSc under the control of the PBAD promoter was induced with 0.01% L-arabinose. The absence of L-arabinose or the deletion of ccrSc from the plasmid resulted in accumulation of poly(3-hydroxybutyrate) homopolymer, indicating the critical role of CCR in the formation of the 3-hydroxyhexanoate unit. Higher CCR activity obtained by the addition of a larger amount of L-arabinose did not affect the composition but reduced the intracellular content of the copolyester. The P(3HB-co-1.5 mol % 3HHx) copolyester produced from fructose by the recombinant R. eutropha showed relatively lower melting temperatures (150 degrees C and 161 degrees C) and lower crystallinity (48 +/- 5%) compared to those (175 degrees C and 60 +/- 5%) of P(3HB) homopolymer. It has been found that the incorporation of a small amount (1.5 mol %) of 3HHx units into P(3HB) sequences leads to a remarkable change in the solid-state properties of P(3HB) crystals. The present study demonstrates the potential of the engineered pathway for the production of copolyesters having favorable characteristics from inexpensive carbon resources.
Fukui
Toshiaki
T
Polymer Chemistry Laboratory, RIKEN Institute, Hirosawa 2-1, Wako-shi, Saitama 351-0198, Japan.
Abe
Hideki
H
Doi
Yoshiharu
Y
D020155:3-Hydroxybutyric Acid / Q000138:chemical synthesis*; D044944:Acyl-CoA Dehydrogenases; D000217:Acyltransferases / Q000378:metabolism; D001419:Bacteria / Q000201:enzymology; D002208:Caproates / Q000138:chemical synthesis*; D003460:Crystallization; D020118:Cupriavidus necator / Q000235:genetics* / Q000378:metabolism; D004746:Enoyl-CoA Hydratase / Q000378:metabolism; D005632:Fructose / Q000737:chemistry; D005818:Genetic Engineering; D010088:Oxidoreductases / Q000378:metabolism; D011108:Polymers / Q000138:chemical synthesis*; D013696:Temperature; D000094122:Polyhydroxybutyrates
D020155:3-Hydroxybutyric Acid; D044944:Acyl-CoA Dehydrogenases; D000217:Acyltransferases; D002208:Caproates; D004746:Enoyl-CoA Hydratase; D005632:Fructose; D010088:Oxidoreductases; D011108:Polymers; C115940:poly(3-hydroxybutyrate-co-3-hydroxyhexanoate); D000094122:Polyhydroxybutyrates; C100154:acyl-CoA dehydrogenase (NADP+); C077225:poly(3-hydroxyalkanoic acid) synthase; C406162:R-specific trans-2,3-enoylacyl-CoA hydratase