Synthesis of poly(3-hydroxyalkanoates) in Escherichia coli expressing the PHA synthase gene phaC2 from Pseudomonas aeruginosa: comparison of PhaC1 and PhaC2.
In order to obtain functional expression of PHA synthase gene phaC2 from Pseudomonas aeruginosa in Escherichia coli, the coding region of phaC2 was subcloned, including the ribosomal binding site, into pBluescript SK- collinear to the lac promoter. This plasmid pBHR71-C2 enabled functional expression of phaC2 in E. coli LS1298 (fadB) under lac promoter control, leading to PHA accumulation, when grown in LB medium containing 0.5% (w/v) of various fatty acids (C8-C14). The strongest accumulation of PHA was observed, when dodecanoate was provided as carbon source, and PHA contributed to 15% of cell dry weight, which was composed of 35 mol% 3-hydroxydodecanoate, 60 mol% 3-hydroxydecanoate and 5 mol% 3-hydroxyoctanoate. Plasmid pBHR78, which contained both genes phaC1 and phaC2 from P. aeruginosa under lac promoter control in pBluescript SK- led in E. coli LS1298 to PHA accumulation, which contributed to 13% of cell dry weight, when cells were grown on decanoate. Only slight differences in PHA composition compared with either PhaC1 or PhaC2 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 synthases from P. aeruginosa exhibit very similar properties resulting in similar extent of PHA accumulation, similar composition and molecular mass, when expressed in E. coli and that fatty acid beta-oxidation provides substrates for both PHA synthases.
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Institute of Microbiology of Shandong University, Jinan, PR China.
Rehm
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Steinbüchel
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D000217:Acyltransferases / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005227:Fatty Acids / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D010957:Plasmids; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics; D013379:Substrate Specificity
D001426:Bacterial Proteins; D005227:Fatty Acids; D011994:Recombinant Proteins; D000217:Acyltransferases; C077225:poly(3-hydroxyalkanoic acid) synthase; C067032:poly(3-hydroxyalkenoate)polymerase