Kinetics of medium-chain-length polyhydroxyalkanoate production by a novel isolate of Pseudomonas putida LS46

Abstract

Six bacteria that synthesize medium-chain-length polyhydroxyalkanoates (mcl-PHAs) were isolated from sewage sludge and hog barn wash and identified as strains of Pseudomonas and Comamonas by 16S rDNA gene sequencing. One isolate, Pseudomonas putida LS46, showed good PHA production (22% of cell dry mass) in glucose medium, and it was selected for further studies. While it is closely related to other P. putida strains (F1, KT2440, BIRD-1, GB-1, S16, and W619), P. putida LS46 was genetically distinct from these other strains on the basis of nucleotide sequence analysis of the cpn60 gene hypervariable region. PHA production was detected as early as 12 h in both nitrogen-limited and nitrogen-excess conditions. The increase in PHA production after 48 h was higher in nitrogen-limited cultures than in nitrogen-excess cultures. Pseudomonas putida LS46 produced mcl-PHAs when cultured with glucose, glycerol, or C(6)-C(14) saturated fatty acids as carbon sources, and mcl-PHAs accounted for 56% of the cell dry mass when cells were batch cultured in medium containing 20 mmol/L octanoate. Although 3-hydroxydecanoate was the major mcl-PHA monomer (58.1-68.8 mol%) in P. putida LS46 cultured in glucose medium, 3-hydroxyoctanoate was the major monomer produced in octanoate medium (88 mol%).

Synthesis and Physical Properties of Polyhydroxyalkanoate Polymers with Different Monomer Compositions by Recombinant Pseudomonas putida LS46 Expressing a Novel PHA SYNTHASE (PhaC116) Enzyme

“…Characteristics References P. putida LS46 P. putida isolated from waste water [13] P. putida LS461 phaC1-phaZ-phaC2 deletion mutant of P. putida LS46 This study P. putida LS46123 P. putida LS461 carrying plasmid pJC123; Tc R This study E. coli DH5α ∆lacZ, ∆M15, ∆(lacZYA-argF), U169, recA1, endA1, hsdR17(rK-mK+), supE44, thi-1, gyrA96, relA1 Qiagen, Hilden, Germany pRK7813 IncP oriT cos lacZα, Tc R [30] pJC123 Derivative of pRK7813 carrying phaC1 16 gene from clone 16; Tc R [31] pK18mobsacB Narrow host-range cloning vector; sacB, Km R [32] pRK2013 Helper plasmid pRK290 derivative; Km R [33] pPHAC1C2 pK18mobsacB carrying 840 bp from phaC1 and 857 bp from phaC2 for operon knockout This study The PHA synthesis gene operon of P. putida LS46 was identified from the complete genome sequence (http//www.ncbi.nlm.nih.gov/nucore/ALPV02000008) coordinate 202310-197359; 13) and encoded the following genes: phaC1phaZphaC2phaD. A deletion mutant, designated P. putida LS461, was constructed by deleting the 3 -phaC1, phaZ and 5 -phaC2 genes from the P. putida LS46 pha operon.…”

“…All tests were conducted with three independently replicated cultures (i.e., three biological replicates). Cell biomass, PHA production (% cell dry weight, cdw), and PHA monomer composition were determined as described earlier [13].…”

“…The cell pellet was washed twice in 0.9% NaCl and dried at 60 • C for 48 h to measure cell dry mass. PHA composition was determined by gas chromatography-mass spectrometry (GC-MS) analysis, as described [13].…”

Colonization and degradation of polyhydroxyalkanoates by lipase-producing bacteria

“…P. chlororaphis PA23-63-1 was kindly provided by All bacteria used in this study were grown and purified on LB plates incubated at 30 o C for 24 hours (h). PHA degradation was studied in Ramsay's minimal medium (RMM) and PHAs with different monomer composition were used as a sole carbon source (Sharma et al 2012). For solid RMM, 15 g/L agar was added to the medium.…”

Draft Genome Sequence of Medium-Chain-Length Polyhydroxyalkanoate-Producing Pseudomonas putida Strain LS46

“…P. putida LS46 can utilize glucose, glycerol (including biodiesel-derived glycerol), fatty acids, vegetable oils, and waste fryer oils as carbon sources and synthesizes mcl-PHAs consisting of 3-hydroxy fatty acids with 6 to 14 carbon atoms (6). …”