Microbial production of 3-hydroxydodecanoic acid by pha operon and fadBA knockout mutant of Pseudomonas putida KT2442 harboring tesB gene
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To produce extracellular chiral 3-hydroxyacyl acids (3HA) by fermentation, a novel pathway was constructed by expressing tesB gene encoding thioesterase II into Pseudomonas putida KTOY01, which was a polyhydroxyalkanoate (PHA) synthesis operon knockout mutant. 3HA mixtures of 0.35 g/l consisting of 3-hydroxyhexanoate, 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydroxydodecanoate (3HDD) were produced in shake-flask study using dodecanoate as a sole carbon source. Additional knockout of fadB and fadA genes encoding 3-ketoacyl-CoA thiolase and 3-hydroxyacyl-CoA dehydrogenase in P. putida KTOY01 led to the weakening of the β-oxidation pathway. The fadBA and PHA synthesis operon knockout mutant P. putida KTOY07 expressing tesB gene produced 2.44 g/l 3HA, significantly more than that of the β-oxidation intact mutant. The 3HA mixture contained 90 mol% 3HDD as a dominant component. A fed-batch fermentation process carried out in a 6-l automatic fermentor produced 7.27 g/l extracellular 3HA containing 96 mol% fraction of 3HDD after 28 h of growth. For the first time, it became possible to produce 3HDD-dominant 3HA monomers.
KeywordsPolyhydroxyalkanoates PHA 3-hydroxyalkanoic acid 3HA 3-hydroxydodecanoic acid 3HDD Pseudomonas putida 3-Ketoacyl-CoA thiolase 3-Hydroxyacyl-CoA dehydrogenase Thioesterase II TesB fadBA
We are very grateful for the kind donation of plasmid pBBR1MCS-2 from Dr. Philip Green of Procter & Gamble (Cincinnati, OH, USA). P. putida KT2442 was a gift from Professor B. Witholt (ETH Zurich, Switzerland). This study was supported by the National High Tech 863 Grant (project no. 2006AA02Z242 and 2006AA020104), as well as the State Basic Science Foundation 973 (2007CB707804).
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