Novel polyhydroxyalkanoate copolymers produced in Pseudomonas putida by metagenomic polyhydroxyalkanoate synthases

Abstract

Bacterially produced biodegradable polyhydroxyalkanoates (PHAs) with versatile properties can be achieved using different PHA synthases (PhaCs). This work aims to expand the diversity of known PhaCs via functional metagenomics and demonstrates the use of these novel enzymes in PHA production. Complementation of a PHA synthesis-deficient Pseudomonas putida strain with a soil metagenomic cosmid library retrieved 27 clones expressing either class I, class II, or unclassified PHA synthases, and many did not have close sequence matches to known PhaCs. The composition of PHA produced by these clones was dependent on both the supplied growth substrates and the nature of the PHA synthase, with various combinations of short-chain-length (SCL) and medium-chain-length (MCL) PHA. These data demonstrate the ability to isolate diverse genes for PHA synthesis by functional metagenomics and their use for the production of a variety of PHA polymer and copolymer mixtures.

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

“…The polymers synthesized by P. putida LS46123 contained 40%-50% of 3HB. P. putida LS46123 recombinant accumulated higher 3HB in PHAs than a recombinant of P. putida KT2440, indicating the role of the host and some other accessary genes in the fatty acid metabolism pathways [31]. Generally, polymers with high 3HB content have high melting temperature and toughness.…”

“…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.…”

“…This plasmid was transferred to P. putida LS46 by triparental mating and transconjugants were selected on LB plates containing chloramphenicol (200 µg/mL) and kanamycin (50 µg/mL) [31]. Two kanamycin-resistant clones were selected and grown in 10 mL LB broth at 30 • C for 18 h.…”

“…The phaC1 16 gene was expressed from constitutive lacZ promoter [31]. Pseudomonas putida LS461 with deletion in phaC1phaZphaC2 was used to express a heterologous phaC1 16 gene.…”

Lactic Acid Containing Polymers Produced in EngineeredSinorhizobium melilotiandPseudomonas putida

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14This study demonstrates that novel polymer production can be achieved by introducing pTAM, a 15 broad-host-range plasmid expressing codon-optimized genes encoding Clostridium propionicum 16 propionate CoA transferase (Pct Cp ) and a modified Pseudomonas sp. MBEL 6-19 17 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1 ), into phaC mutant strains of the native 18 polymer producers Sinorhizobium meliloti and Pseudomonas putida.

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