SYNTHESIS OF POLY(3-HYDROXYALKANOATES) IN ESCHERICHIA-COLI EXPRESSINGTHE PHA SYNTHASE GENE PHAC2 FROM PSEUDOMONAS-AERUGINOSA - COMPARISON OF PHAC1 AND PHAC2
Qs. Qi et al., SYNTHESIS OF POLY(3-HYDROXYALKANOATES) IN ESCHERICHIA-COLI EXPRESSINGTHE PHA SYNTHASE GENE PHAC2 FROM PSEUDOMONAS-AERUGINOSA - COMPARISON OF PHAC1 AND PHAC2, FEMS microbiology letters, 157(1), 1997, pp. 155-162
In order to obtain functional expression of PHA synthase gene phaC2 fr
om Pseudomonas aeruginosa in Escherichia coli, the coding region of ph
aC2 was subcloned, including the ribosomal binding site, into pBluescr
ipt SK- collinear to the lac promoter. This plasmid pBHR71-C2 enabled
functional expression of phaC2 in E. coli LS1298 (fadB) under Inc prom
oter control, leading to PHA accumulation, when grown in LB medium con
taining 0.5% (w/v) of various fatty acids (C-8-C-14) The strongest acc
umulation of PHA was observed, when dodecanoate was provided as carbon
source, and PHA contributed to 15% of cell dry weight, which was comp
osed of 35 mol% 3-hydroxydodecanoate, 60 mol% 3-hydroxydecanoate and 5
mol% 3-hydroxyoctanoate. Plasmid pBHR78, which contained both genes p
haC1 and phaC2 from P. aeruginosa under Inc promoter control in pBlues
cript SK- led in E. coli LS1298 to PHA accumulation, which contributed
to 13% of cell dry weight, when cells were grown on decanoate. Only s
light differences in PHA composition compared with either PhaC1 or Pha
C2 were obtained. The weight average molecular masses of PHA purified
from decanoate-grown cells of E. coli LS1298 expressing PhaC1 or PhaC2
alone or both PHA synthases, were 106 x 10(3), 70 x 10(3) or 67 x 10(
3), respectively. This study clearly demonstrated that both PHA syntha
ses from P. aeruginosa exhibit very similar properties resulting in si
milar extent of PHA accumulation, similar composition and molecular ma
ss, when expressed in E. coli and that fatty acid beta-oxidation provi
des substrates for both PHA synthases.