Microbial production of 3-hydroxydodecanoic acid by pha operon and fadBA knockout mutant of Pseudomonas putida KT2442 harboring tesB gene

Appl Microbiol Biotechnol (2009) 83:513519 DOI 10.1007/s00253-009-1919-6

APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY

Microbial production of 3-hydroxydodecanoic acid by pha operon and fadBA knockout mutantof Pseudomonas putida KT2442 harboring tesB gene

Ahleum Chung & Qian Liu & Shao-Ping Ouyang &

Qiong Wu & Guo-Qiang Chen

Received: 8 January 2009 /Revised: 16 February 2009 /Accepted: 16 February 2009 /Published online: 7 March 2009 # Springer-Verlag 2009

Abstract 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 -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 -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.

Keywords Polyhydroxyalkanoates . PHA . 3-hydroxyalkanoic acid . 3HA . 3-hydroxydodecanoic acid . 3HDD . Pseudomonas putida . 3-Ketoacyl-CoA thiolase . 3-Hydroxyacyl-CoA dehydrogenase . Thioesterase II . TesB . fadBA

Introduction

Polyhydroxyalkanoates (PHA) are biodegradable and bio-compatible polyesters synthesized by many microorganisms; 3-hydroxyalkanoic acids (3HA) are common monomers of PHA (Steinbchel and Fchtenbusch 1998). As chiral molecules, 3HA have many potential applications including starting materials for synthesis of antibiotics, vitamins, aromatics, and pheromones (Chen and Wu 2005). Especially, medium-chain-length (mcl) polyhydroxyalkanoate monomers have been shown to have potential pharmaceutical values (Takahashi et al. 1994). Therefore, the development of a technology for production of mcl-3HA has become increasingly attractive.

Due to the difficulty of synthesizing chiral 3HA, chemical synthesis is economically unfeasible, only a few cases describing chemical synthesis were reported (Jaipuri et al. 2004). In comparison, degradation of biosynthesized polyhydroxyalkanoates is a more efficient method. 3HA could be produced...