Inactivation of Isocitrate Lyase Leads to Increased Production of Medium-Chain-Length Poly(3-Hydroxyalkanoates) in Pseudomonas putida

Abstract

Medium-chain-length (mcl) poly(3-hydroxyalkanoates) (PHAs) are storage polymers that are produced from various substrates and accumulate in Pseudomonas strains belonging to rRNA homology group I. In experiments aimed at increasing PHA production in Pseudomonas strains, we generated an mcl PHA-overproducing mutant of Pseudomonas putida KT2442 by transposon mutagenesis, in which the aceA gene was knocked out. This mutation inactivated the glyoxylate shunt and reduced the in vitro activity of isocitrate dehydrogenase, a rate-limiting enzyme of the citric acid cycle. The genotype of the mutant was confirmed by DNA sequencing, and the phenotype was confirmed by biochemical experiments. The aceA mutant was not able to grow on acetate as a sole carbon source due to disruption of the glyoxylate bypass and exhibited two-to fivefold lower isocitrate dehydrogenase activity than the wild type. During growth on gluconate, the difference between the mean PHA accumulation in the mutant and the mean PHA accumulation in the wild-type strain was 52%, which resulted in a significant increase in the amount of mcl PHA at the end of the exponential phase in the mutant P. putida KT217. On the basis of a stoichiometric flux analysis we predicted that knockout of the glyoxylate pathway in addition to reduced flux through isocitrate dehydrogenase should lead to increased flux into the fatty acid synthesis pathway. Therefore, enhanced carbon flow towards the fatty acid synthesis pathway increased the amount of mcl PHA that could be accumulated by the mutant.Many bacteria are able to accumulate poly(3-hydroxyalkanoates) (PHAs) as carbon and energy reserves. Because of their potential use as biodegradable thermoplastics and as biopolymers that can be produced from renewable resources, PHAs have been extensively studied by academic and industrial groups (2, 6, 30). Pseudomonads synthesize mainly mediumchain-length (mcl) PHAs, which consist of monomers containing 6 to 14 carbon atoms (8,14,31). Although a few PHAs have been developed commercially and marketed (6, 11), widespread use of these polymers has been hindered by high production costs (1,19). Reduction of these costs could be achieved by several means, including increasing the product yield (19) or using transgenic plants for PHA production, provided that PHA levels can be brought to 20 to 40% of the plant dry weight (23,24,32).During the past few years we have constructed several sets of mutants of Pseudomonas putida KT2442 with the goal of altering the carbon flux towards mcl PHAs. While most of these mutants contain clearly reduced mcl PHA levels, a few have exhibited increased PHA production. Preliminary analysis of one of these mutants (P. putida KT217) showed that its glyoxylate pathway was affected.From studies on P. putida KT2442, it is known that PHA precursors can be produced via the following three main pathways: ␤-oxidation, de novo fatty acid biosynthesis, and elongation of 3-hydroxyalkanoates by acetyl coenzyme A (acetylCoA) molecules (12, 32). The ␤-oxidation pathway is...

The polyhydroxyalkanoate metabolism controls carbon and energy spillage in Pseudomonas putida

“…However, we did not observe differences in ICDH between both strains neither at the enzymatic nor the transcriptomic level (qRT‐PCR fold change of 1.3 ± 0.2). These data confirm ICL as key factor connecting central metabolism to PHA accumulation yields (Klinke et al ., 2000). In agreement with a high CoA demand by high acetyl‐CoA synthesis, PP_4700 as part of the panBC putative operon responsible for pantothenate and CoA synthesis was also upregulated 2.7‐fold.…”

Two phenotypically compensating isocitrate dehydrogenases inRalstonia eutropha

“…However, mutations of icd1 or icd2 did not significantly affect poly(3HB) formation from gluconate or acetate. This indicates that: (i) NADPH from dehydrogenation of isocitrate catalyzed by either NADP + ‐dependent IDH isoenzyme is sufficient for poly(3HB) formation in R. eutropha ; or (ii) that the total amount of acetyl‐CoA for poly(3HB) biosynthesis is not increased, although the metabolic flux through TCA cycle might be reduced due to the decrease of IDH activity [25,26]. However, it is unknown to which extent NADPH is formed from NADH by a transhydrogenase in R. eutropha under conditions permitting accumulation of poly(3HB).…”

Proteome reference map of Pseudomonas putida strain KT2440 for genome expression profiling: distinct responses of KT2440 and Pseudomonas aeruginosa strain PAO1 to iron deprivation and a new form of superoxide dismutase

“…Cases et al ., 2001; Kasai et al ., 2001; Llamas et al ., 2003) and for the development of biotechnological applications (e.g. Klinke et al ., 2000; Ronchel and Ramos, 2001; Bühler et al ., 2002); it is also a certified biosafety strain (Bagdasarian et al ., 1981; Federal Register, 1982) for recombinant DNA experiments and applications. The recent determination of its genome sequence (Nelson et al ., 2002) provides a new framework for the elucidation of the genomic and physiological basis of the metabolic versatility and environmental success of this fascinating bacterium.…”