Azotobacter vinelandii is proposed to contain a single beta -ketothiolase a
ctivity participating in the formation of acetoacetyl-CoA, a precursor for
poly-beta -hydroxybutyrate (PHB) synthesis, and in beta -oxidation (Manchak
, J., Page, W.J., 1994. Control of polyhydroxyalkanoate synthesis in Azotob
acter vinelandii strain UWD. Microbiology 140, 953-963). We designed a dege
nerate oligonucleotide from a highly conserved region among bacterial beta
-ketothiolases and used it to identify bktA, a gene with a deduced protein
product with a high similarity to beta -ketothiolases. Immediately downstre
am of bktA, we identified a gene called hbdH, which encodes a protein exhib
iting similarity to beta -hydroxyacyl-CoA and beta -hydroxybutyryl-CoA dehy
drogenases. Two regions with homology to bktA were also observed. One of th
ese was cloned and allowed the identification of the phbA gene, encoding a
second beta -ketothiolase. Strains EV132, EV133, and GM1 carrying bktA, hbd
H and phbA mutations, respectively, as well as strain EG1 carrying both bkt
A and phbA mutations, were constructed. The hbdH mutation had no effect on
beta -hydroxybutyryl-CoA dehydrogenase activity or on fatty acid assimilati
on. The bktA mutation had no effect on beta -ketothiolase activity, PHB syn
thesis or fatty acid assimilation, whereas the phbA mutation significantly
reduced beta -ketothiolase activity and PHB accumulation, showing that this
is the beta -ketothiolase involved in PHB biosynthesis. Strain EG1 was fou
nd to grow under beta -oxidation conditions and to possess beta -ketothiola
se activity. Taken together, these results demonstrate the presence of thre
e genes coding for beta -ketothiolases in A. vinelandii. (C) 2000 Elsevier
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