A new metabolic link between fatty acid de novo synthesis and polyhydroxyalkanoic acid synthesis. The PHAG gene from Pseudomonas putida KT2440 encodes a 3-hydroxyacyl-acyl carrier protein-coenzyme a transferase.
To investigate the metabolic link between fatty acid de novo synthesis and polyhydroxyalkanoic acid (PHA) synthesis, we isolated mutants of Pseudomonas putida KT2440 deficient in this metabolic route. The gene phaG was cloned by phenotypic complementation of these mutants; it encoded a protein of 295 amino acids with a molecular mass of 33,876 Da, and the amino acid sequence exhibited 44% amino acid identity to the primary structure of the rhlA gene product, which is involved in the rhamnolipid biosynthesis in Pseudomonas aeruginosa PG201. S1 nuclease protection assay identified the transcriptional start site 239 base pairs upstream of the putative translational start codon. Transcriptional induction of phaG was observed when gluconate was provided, and PHA synthesis occurred from this carbon source. No complementation of the rhlA mutant P. aeruginosa UO299-harboring plasmid pBHR81, expressing phaG gene under lac promoter control, was obtained. Heterologous expression of phaG in Pseudomonas oleovorans, which is not capable of PHA synthesis from gluconate, enabled PHA synthesis on gluconate as the carbon source. Native recombinant PhaG was purified by native polyacrylamide gel electrophoresis from P. oleovorans-harboring plasmid pBHR81. It catalyzes the transfer of the acyl moiety from in vitro synthesized 3-hydroxydecanoyl-CoA to acyl carrier protein, indicating that PhaG exhibits a 3-hydroxyacyl-CoA-acyl carrier protein transferase activity.
Rehm
B H
BH
Institut für Mikrobiologie, Westfälische Wilhelms-Universität Münster, Corrensstrabetae 3, D-48149, Münster, Germany.
Krüger
N
N
Steinbüchel
A
A
D000217:Acyltransferases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon; D017931:DNA Primers; D005230:Fatty Acids, Nonesterified / Q000096:biosynthesis; D005816:Genetic Complementation Test; D005942:Gluconates / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009604:Nitrosoguanidines; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011549:Pseudomonas / Q000235:genetics; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
D003062:Codon; D017931:DNA Primers; D005230:Fatty Acids, Nonesterified; D005942:Gluconates; D009604:Nitrosoguanidines; D011994:Recombinant Proteins; D000217:Acyltransferases; C114682:3-hydroxyacyl-CoA-acyl carrier protein transferase; C030691:gluconic acid