Biosynthesis of poly(4-hydroxybutyric acid) by recombinant strains of Escherichia coli.
The aim of this study was the production of the homopolyester poly(4-hydroxybutyric acid) (poly(4HB)) with recombinant strains of Escherichia coli. Wild-type strains and other widely used non-recombinant strains of E. coli are not able to produce polyhydroxyalkanoic acids (PHA) as storage compounds and cannot utilize 4-hydroxybutyric acid as sole carbon source. Accordingly, hybrid plasmids of pBluescript vectors were constructed which harbored the Alcaligenes eutrophus PHA synthase gene (phaC) and the Clostridium kluyveri orfZ putatively encoding a 4-hydroxybutyric acid-coenzyme A transferase. A 3.5-kb genomic SmaI/ApaI fragment from A. eutrophus, which comprises phaC, and a 1.8-kb genomic ApaI/EcoRI fragment from C kluyveri, which contained orfZ, were inserted into the SmaI and EcoRI sites of the vectors pKS- and pSK-, respectively. The two resulting plasmids pSKSE5.3 and pKSSE5.3 comprising phaC and orfZ colinear or antilinear to lacZ, respectively, were transformed into E. coli XL1-Blue. Recombinant strains synthesized the homopolyester poly(4HB), when the cells were cultivated in Luria-Bertani broth and if glucose and 4-hydroxybutyric acid were provided as carbon sources. If glucose was omitted, a copolyester of 3-hydroxybutyric acid and 4-hydroxybutyric acid was accumulated. The homopolyester poly(4HB) was also accumulated during cultivation of these strains in M9 mineral salts medium containing glucose plus 4-hydroxybutyric acid as carbon sources. Poly(4HB) could amount up to approximately 80% (w/w) of the cell dry matter if E. coli XL1-Blue harboring pKSSE5.3 was cultivated in M9 mineral salts medium and if the cultures were not sufficiently supplied with oxygen. 4HB was also incorporated into PHA if gamma-butyrolactone was used as carbon source. If levulinic acid, 4-hydroxyvaleric acid or gamma-valerolactone were used as carbon sources, only very low amounts of PHA were accumulated which did not contain 4-hydroxyalkanoic acids as constituents.
Hein
S
S
Institut für Mikrobiologie der Westfälischen Wilhelms-Universität Münster, Germany.
Söhling
B
B
Gottschalk
G
G
Steinbüchel
A
A
D015107:4-Butyrolactone / Q000378:metabolism; D000102:Acetyl-CoA C-Acyltransferase / Q000235:genetics; D000217:Acyltransferases / Q000235:genetics; D015256:Coenzyme A-Transferases / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial / Q000235:genetics; D005947:Glucose / Q000378:metabolism; D006885:Hydroxybutyrates / Q000378:metabolism; D007783:Lactones / Q000378:metabolism; D007982:Levulinic Acids / Q000378:metabolism; D011091:Polyesters / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D012978:Sodium Oxybate / Q000378:metabolism*; D013386:Succinates / Q000378:metabolism; D014631:Valerates / Q000378:metabolism; D000094122:Polyhydroxybutyrates
D015107:4-Butyrolactone; D000102:Acetyl-CoA C-Acyltransferase; D000217:Acyltransferases; D015256:Coenzyme A-Transferases; D005947:Glucose; D006885:Hydroxybutyrates; D007783:Lactones; D007982:Levulinic Acids; D011091:Polyesters; D011993:Recombinant Fusion Proteins; D012978:Sodium Oxybate; D013386:Succinates; D014631:Valerates; C107955:poly(4-hydroxybutanoate); C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates; C060970:poly-beta-hydroxybutyrate polymerase; C072231:4-hydroxybutyrate CoA-transferase; C037556:gamma-valerolactone; C032246:levulinic acid