Quantitative ‘Omics Analyses of Medium Chain Length Polyhydroxyalkanaote Metabolism in Pseudomonas putida LS46 Cultured with Waste Glycerol and Waste Fatty Acids

Abstract

Transcriptomes and proteomes of Pseudomonas putida LS46 cultured with biodiesel-derived waste glycerol or waste free fatty acids, as sole carbon sources, were compared under conditions that were either permissive or non-permissive for synthesis of medium chain length polyhydroxyalkanoates (mcl-PHA). The objectives of this study were to elucidate mechanisms that influence activation of biopolymer synthesis, intra-cellular accumulation, and monomer composition, and determine if these were physiologically specific to the carbon sources used for growth of P. putida LS46. Active mcl-PHA synthesis by P. putida LS46 was associated with high expression levels of key mcl-PHA biosynthesis genes and/or gene products including monomer-supplying proteins, PHA synthases, and granule-associated proteins. ‘Omics data suggested that expression of these genes were regulated by different genetic mechanisms in P. putida LS46 cells in different physiological states, when cultured on the two waste carbon sources. Optimal polymer production by P. putida LS46 was primarily limited by less efficient glycerol metabolism during mcl-PHA synthesis on waste glycerol. Mapping the ‘Omics data to the mcl-PHA biosynthetic pathway revealed significant variations in gene expression, primarily involved in: 1) glycerol transportation; 2) enzymatic reactions that recycle reducing equivalents and produce key mcl-PHA biosynthesis pathway intermediates (e.g. NADH/NADPH, acetyl-CoA). Active synthesis of mcl-PHAs was observed during exponential phase in cultures with waste free fatty acids, and was associated with the fatty acid beta-oxidation pathway. A putative Thioesterase in the beta-oxidation pathway that may regulate the level of fatty acid beta-oxidation intermediates, and thus carbon flux to mcl-PHA biosynthesis, was highly up-regulated. Finally, the data suggested that differences in expression of selected fatty acid metabolism and mcl-PHA monomer-supplying enzymes may play a role in determining the monomer composition of mcl-PHA polymers. Understanding the relationships between genome content, gene and gene product expression, and how these factors influence polymer synthesis, will aid in optimization of mcl-PHA production by P. putida LS46 using biodiesel waste streams.

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.…”

“…The phaC1 16 gene of metagenomic clone 16 was amplified by PCR and cloned as a HindII-EcoRI fragment into cosmid pRK7813 to give plasmid pJC123. The phaC1 16 gene was expressed from constitutive lacZ promoter [31].…”

“…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.…”

“…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. Cosmid pJC123 was transferred into P. putida LS461 by triparental mating by using pRK2013 as a helper plasmid [33,34].…”

Biorefinery for Glycerol Rich Biodiesel Industry Waste

“…Subsequently, the enzymes responsible for supplying monomers were also found to express differentially on waste glycerol and waste free fatty acids. PhaJ1 gene seems to be critical in this process especially the supply of C8 monomers [89]. C. necator, a well known PHA producing organism was tested on a range of fatty acid rich wastes.…”