Role of fatty acid de novo biosynthesis in polyhydroxyalkanoic acid (PHA) and rhamnolipid synthesis by pseudomonads: establishment of the transacylase (PhaG)-mediated pathway for PHA biosynthesis in Escherichia coli.
Since Pseudomonas aeruginosa is capable of biosynthesis of polyhydroxyalkanoic acid (PHA) and rhamnolipids, which contain lipid moieties that are derived from fatty acid biosynthesis, we investigated various fab mutants from P. aeruginosa with respect to biosynthesis of PHAs and rhamnolipids. All isogenic fabA, fabB, fabI, rhlG, and phaG mutants from P. aeruginosa showed decreased PHA accumulation and rhamnolipid production. In the phaG (encoding transacylase) mutant rhamnolipid production was only slightly decreased. Expression of phaG from Pseudomonas putida and expression of the beta-ketoacyl reductase gene rhlG from P. aeruginosa in these mutants indicated that PhaG catalyzes diversion of intermediates of fatty acid de novo biosynthesis towards PHA biosynthesis, whereas RhlG catalyzes diversion towards rhamnolipid biosynthesis. These data suggested that both biosynthesis pathways are competitive. In order to investigate whether PhaG is the only linking enzyme between fatty acid de novo biosynthesis and PHA biosynthesis, we generated five Tn5 mutants of P. putida strongly impaired in PHA production from gluconate. All mutants were complemented by the phaG gene from P. putida, indicating that the transacylase-mediated PHA biosynthesis route represents the only metabolic link between fatty acid de novo biosynthesis and PHA biosynthesis in this bacterium. The transacylase-mediated PHA biosynthesis route from gluconate was established in recombinant E. coli, coexpressing the class II PHA synthase gene phaC1 together with the phaG gene from P. putida, only when fatty acid de novo biosynthesis was partially inhibited by triclosan. The accumulated PHA contributed to 2 to 3% of cellular dry weight.
Rehm
B H
BH
Institut für Mikrobiologie, Westfälische Wilhelms-Universität Münster, D-48149 Münster, Germany. rehm@unimuenster.de
Mitsky
T A
TA
Steinbüchel
A
A
D064431:3-Oxoacyl-(Acyl-Carrier-Protein) Reductase; D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism*; D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism; D004251:DNA Transposable Elements; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH); D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D054889:Fatty Acid Synthase, Type II; D005227:Fatty Acids / Q000096:biosynthesis* / Q000235:genetics; D006017:Glycolipids / Q000096:biosynthesis*; D006836:Hydro-Lyases / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D011091:Polyesters / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism*; D016958:Pseudomonas putida / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism
D004251:DNA Transposable Elements; D029968:Escherichia coli Proteins; D005227:Fatty Acids; D006017:Glycolipids; D011091:Polyesters; D011994:Recombinant Proteins; C418382:rhamnolipid; D010088:Oxidoreductases; D000429:Alcohol Oxidoreductases; D064431:3-Oxoacyl-(Acyl-Carrier-Protein) Reductase; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH); C493924:fabI protein, E coli; D000217:Acyltransferases; C114682:3-hydroxyacyl-CoA-acyl carrier protein transferase; D006836:Hydro-Lyases; C047118:3-hydroxyacyl-(acyl-carrier-protein) dehydratase; D054889:Fatty Acid Synthase, Type II