In vivo and in vitro depolymerizations of intracellular medium-chain-length poly-3-hydroxyalkanoates produced by Pseudomonas putida Bet001

ABSTRACTSaturation point mutagenesis was carried out at position 479 in the polyhydroxyalkanoate (PHA) synthase fromChromobacteriumsp. strain USM2 (PhaCCs) with specificities for short-chain-length (SCL) [(R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyvalerate (3HV)] and medium-chain-length (MCL) [(R)-3-hydroxyhexanoate (3HHx)] monomers in an effort to enhance the specificity of the enzyme for 3HHx. A maximum 4-fold increase in 3HHx incorporation and a 1.6-fold increase in PHA biosynthesis, more than the wild-type synthase, was achieved using selected mutant synthases. These increases were subsequently correlated with improved synthase activity and increased preference of PhaCCsfor 3HHx monomers. We found that substitutions with uncharged residues were beneficial, as they resulted in enhanced PHA production and/or 3HHx incorporation. Further analysis led to postulations that the size and geometry of the substrate-binding pocket are determinants of PHA accumulation, 3HHx fraction, and chain length specificity.In vitroactivities for polymerization of 3HV and 3HHx monomers were consistent within vivosubstrate specificities. Ultimately, the preference shown by wild-type and mutant synthases for either SCL (C4and C5) or MCL (C6) substrates substantiates the fundamental classification of PHA synthases.

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Swapping of Phasin Modules To Optimize the In Vivo Immobilization of Proteins to Medium-Chain-Length Polyhydroxyalkanoate Granules in Pseudomonas putida

Biomacromolecules ◽

10.1021/bm4008937 ◽

2013 ◽

Vol 14 (9) ◽

pp. 3285-3293 ◽

Cited By ~ 22

Author(s):

Nina Dinjaski ◽

M. Auxiliadora Prieto

Keyword(s):

Pseudomonas Putida ◽

Chain Length ◽

Medium Chain ◽

Medium Chain Length

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

Canadian Journal of Microbiology ◽

10.1139/w2012-074 ◽

2012 ◽

Vol 58 (8) ◽

pp. 982-989 ◽

Cited By ~ 24

Author(s):

Parveen K. Sharma ◽

Jilagamazhi Fu ◽

Nazim Cicek ◽

Richard Sparling ◽

David B. Levin

Keyword(s):

Pseudomonas Putida ◽

Chain Length ◽

Saturated Fatty Acids ◽

Dry Mass ◽

Nucleotide Sequence Analysis ◽

Glucose Medium ◽

Medium Chain ◽

Medium Chain Length ◽

Pha Production ◽

Nitrogen Excess

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 C6–C14 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%).

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