The Improvement of PHA Production in Pseudomonas Putida KTQQ20, the Biosynthesis and Characterization of Poly (3-hydroxy-decanoate-co-3-hydroxy-undecenoate)
|Course||Biochemistry and Molecular Biology|
|Keywords||fatty acid de novo pathway βoxidation Polyhydroxyalkanoate PHDHUe 10-undeconic acid glucose|
Pseudomonas Putida KTQQ20, whose fatty acid beta-oxidation was impeded, is derived from P. Putida KT2442. It is able to synthesize PHA containing high ratio of long-length monomer or even long-chain-length homopolymer. But, the CDW and PHA content were very low. In order to improve the PHA production, on one hand, glucose was added to LB medium. On the other hand, enes coding for 3-ketoacyl-CoA pp3280 and pp4636 alone or together were knocked out from KTQQ20 and two mutant strains were obtained KTQQ21 and KTQQ22, respectively. As a result, the biomass and PHA content of KTQQ20 added with glucose were found to increase significantly while the mole ratio of 3HD to 3HDD was kept constant, which demonstrated that glucose was able to improve biomass and PHA production in KTQQ20. When added with glucose, KTQQ22, in which pp4636 was deleted, could accumulate PHA as much as 86.57wt%, which was significantly higher than that produced by KTQQ20. This suggested that the lack of gene pp4636 further impairedβoxidation and thus more PHA precursors were directed towards the PHA synthesis pathway.We attempted to use 10-undecenoate acid (10-UD) and glucose as the carbon sources to synthesize the homoploymer P(3-hydroxy-undecenoate) (PHUe). But the GC and GC-MS results showed that two monomers were present, including 3-hydroxy-decanoate (3HD) and 3-hydroxy-undecenoate (3HUe). According to our data, it was found that 3HUe was derived from 10-UD while 3HD was from glucose. We presumed that there was another PHA synthesis pathway since the PhaG linking pathway was blocked. The metabolic intermediate of glucose entered the fatty acid de novo biosynthesis pathway and the newly synthesized fatty acid went into theβoxidation pathway. The resultant monomer 3-hydxoyacyl was incorporated into PHA. When 10-UD and glucose were served as the cosubstrates, we found that such presumptive pathway had minor contribution in PHA production.When supplemented with 4.5 g/L 10-UD and 15 g/L glucose, KTQQ22 was able to produce PHDHUe containing 4.45 mol% 3HD and 95.5mol% 3HUe. The glass transition temperature of PHDHUe was approximately -53℃, which was significantly lower than that of mcl PHA. The DSC thermograms also showed a clear endothermic transition at approximately 52℃with a heat of fusion of 19 J/g, which was near to those of other mcl PHA.