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

Jilagamazhi Fu,Brian Fristensky,Parveen Sharma,David B Levin,Xiangli Zhang,Richard Sparling,Nazim Cicek,Oleg V Krokhin,Vic Spicer

doi:10.1371/journal.pone.0142322

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.

Highlights

Medium chain length polyhydroxyalkanoates are mostly produced by bacteria in the genus Pseudomonas as reserve sources of carbon and energy under conditions of nutritional stress [1]
The mcl-PHA content of the cells started to increase gradually up to 24 h pi, and increased at a much greater rate after nitrogen concentrations in the medium were exhausted (Fig 1B). This suggested that mcl-PHA biosynthesis was not restricted to nitrogen-limitation under waste fatty acids (WFA) culture, the maximum productivity was triggered by nitrogen depletion
To study and predict the potential genetic targets that regulate mcl-PHA biosynthesis profile of P. putida LS46 grown under biodiesel derived waste carbon sources, Multi-level ‘Omics analyses were used to correlate the observed variations in mclPHA synthesis with differences in gene expression profiles with respect to: 1) mcl-PHA biosynthesis activation; 2) intracellular accumulation of mcl-PHA polymers; and 3) differences in monomer composition of the polymers

Summary

Introduction

Medium chain length polyhydroxyalkanoates (mcl-PHAs) are mostly produced by bacteria in the genus Pseudomonas as reserve sources of carbon and energy under conditions of nutritional stress [1]. A number of studies have explored the use of microorganisms to convert agro-industrial waste streams into value-added PHA polymers [3,4]. The by-products from industrial biodiesel production, such as biodiesel-derived glycerol and biodiesel-derived free fatty acids, contain certain amount of impurities making them less useful for other downstream industrial applications. Waste glycerol contains methanol, residual free fatty acids, sodium or potassium soaps derived from the catalysts used to synthesize biodiesel, and numbers of identified heavy metals [5,6], Waste glycerol is normally refined in order to use in food, cosmetics, and pharmaceutical industrial. Biological conversion of biodiesel derived waste carbon sources into highvalue added product, such as synthesis of mcl-PHA by Pseudomonas putida [9,10], is of great interests currently. Understanding the effects of these low cost “waste” carbon sources on the metabolism of P. putida in general, and mcl-PHA synthesis pathways in particular, will provide a rational basis for optimization of fermentation strategies for large-scale mcl-PHA production

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