METABOLIC PATHWAY FOR BIOSYNTHESIS OF POLY(3-HYDROXYBUTYRATE-CO-4-HYDROXYBUTYRATE) FROM 4-HYDROXYBUTYRATE BY ALCALIGENES-EUTROPHUS

Various aerobic Gram-negative bacteria have been examined for their ab ility to use 4-hydroxybutyrate and 1,4-butanediol as carbon source for growth. Alcaligenes eutrophus strains H16, HF39, PHB(-)4 and Pseudomo nas denitrificans 'Morris' were not able to grow with 1,4-butanediol o r 4-hydroxybutyrate. From A. eutrophus HF39 spontaneous primary mutant s (e. g. SK4040) were isolated which grew on 4-hydroxybutyrate with do ubling times of approximately 3 h. Tn5::mob mutagenesis of mutant SK40 40 led to the isolation of two phenotypically different classes of sec ondary mutants which were affected in the utilization of 4-hydroxybuty rate. Mutants exhibiting the phenotype 4-hydroxybutyrate-negative did not grow with 4-hydroxybutyrate, and mutants exhibiting the phenotype 4-hydroxybutyrate-leaky grew at a significantly lower rate with 4-hydr oxybutyrate. Hybridization experiments led to the identification of a 10-kbp genomic EcoRI fragment of A. eutrophus SK4040, which was altere d in mutants with the phenotype 4-hydroxybutyrate-negative, and of two 1-kbp and 4.5-kbp genomic EcoRI fragments, which were altered in muta nts with the phenotype 4-hydroxybutyrate-leaky. This 10-kbp EcoRI frag ment was cloned from A. eutrophus SK4040, and conjugative transfer of a pVDZ'2 hybrid plasmid to A. eutrophus H16 conferred the ability to g row with 4-hydroxybutyrate to the wild type. DNA-sequence analysis of this fragment, enzymic analysis of the wild type and of mutants of A. eutrophus as well as of recombinant strains of Escherichia coli led to the identification of a structural gene encoding for a 4-hydroxybutyr ate dehydrogenase which was affected by transposon mutagenesis in five of six available 4-hydroxybutyrate-negative mutants. Enzymic studies also provided evidence for the presence of an active succinate-semiald ehyde dehydrogenase in 4-hydroxybutyrate-grown cells. This indicated t hat degradation of 4-hydroxybutyrate occurs via succinate semialdehyde and succinate and that the latter is degraded by the citric acid cycl e. NMR studies of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) accumul ated from 4-hydroxy [1-C-13]butyrate or 4-hydroxy[2-C-13]butyrate as s ubstrate gave no evidence for a direct conversion of 4-hydroxybutyrate into 3-hydroxybutyrate and therefore supported the results of enzymic analysis.