BIOSYNTHESIS OF POLY(4-HYDROXYBUTYRIC ACID) BY RECOMBINANT STRAINS OFESCHERICHIA-COLI

The aim of this study was the production of the homopolyester poly(4-h ydroxybutyric acid) (poly(4HB)) with recombinant strains of Escherichi a coli. Wild-type strains and other widely used non-recombinant strain s of E. coli are not able to produce polyhydroxyalkanoic acids (PHA) a s storage compounds and cannot utilize 4-hydroxybutyric acid as sole c arbon source. Accordingly, hybrid plasmids of pBluescript vectors were constructed which harbored the Alcaligenes eutrophus PHA synthase gen e (phaC) and the Clostridium kluyveri orfZ putatively encoding a 4-hyd roxybutyric acid-coenzyme A transferase. A 3.5-kb genomic SmaI/ApaI fr agment from A. eutrophus, which comprises phaC, and a 1.8-kb genomic A paI/EcoRI fragment from C. kluyveri, which contained orfZ, were insert ed into the SmaI and EcoRI sites of the vectors pKS(-) and pSK(-), res pectively. The two resulting plasmids pSKSE5.3 and pKSSE5.3 comprising phaC and orfZ colinear or antilinear to lacZ, respectively, were tran sformed into E. coli XL1-Blue. Recombinant strains synthesized the hom opolyester 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 ac id and 4-hydroxybutyric acid was accumulated. The homopolyester poly(4 HB) was also accumulated during cultivation of these strains in M9 min eral salts medium containing glucose plus ct-hydroxybutyric acid as ca rbon 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 cultiva ted in M9 mineral salts medium and if the cultures were not sufficient ly supplied with oxygen. 4HB was also incorporated into PHA if gamma-b utyrolactone was used as carbon source. If levulinic acid, 4-hydroxyva leric acid or gamma-valerolactone were used as carbon sources, only ve ry low amounts of PHA were accumulated which did not contain 4-hydroxy alkanoic acids as constituents.