The metabolic response of P. putida KT2442 producing high levels of polyhydroxyalkanoate under single- and multiple-nutrient-limited growth: Highlights from a multi-level omics approach

Ignacio Poblete-Castro,Vítor Ap Martins Dos Santos,Dietmar Schomburg,Christian Jäger,Jacek Puchalka,Carolyn Chi Lam,Isabel F Escapa,María Prieto

doi:10.1186/1475-2859-11-34

Abstract

BackgroundPseudomonas putida KT2442 is a natural producer of polyhydroxyalkanoates (PHAs), which can substitute petroleum-based non-renewable plastics and form the basis for the production of tailor-made biopolymers. However, despite the substantial body of work on PHA production by P. putida strains, it is not yet clear how the bacterium re-arranges its whole metabolism when it senses the limitation of nitrogen and the excess of fatty acids as carbon source, to result in a large accumulation of PHAs within the cell. In the present study we investigated the metabolic response of KT2442 using a systems biology approach to highlight the differences between single- and multiple-nutrient-limited growth in chemostat cultures.ResultsWe found that 26, 62, and 81% of the cell dry weight consist of PHA under conditions of carbon, dual, and nitrogen limitation, respectively. Under nitrogen limitation a specific PHA production rate of 0.43 (g·(g·h)-1) was obtained. The residual biomass was not constant for dual- and strict nitrogen-limiting growth, showing a different feature in comparison to other P. putida strains. Dual limitation resulted in patterns of gene expression, protein level, and metabolite concentrations that substantially differ from those observed under exclusive carbon or nitrogen limitation. The most pronounced differences were found in the energy metabolism, fatty acid metabolism, as well as stress proteins and enzymes belonging to the transport system.ConclusionThis is the first study where the interrelationship between nutrient limitations and PHA synthesis has been investigated under well-controlled conditions using a system level approach. The knowledge generated will be of great assistance for the development of bioprocesses and further metabolic engineering work in this versatile organism to both enhance and diversify the industrial production of PHAs.

Highlights

Pseudomonas putida KT2442 is a natural producer of polyhydroxyalkanoates (PHAs), which can substitute petroleum-based non-renewable plastics and form the basis for the production of tailor-made biopolymers
Employing 2-D gels, we identified a particular modulation of another important enzyme in the cycle, isocitrate dehydrogenase ICD, which can be repressed by various mechanisms together with the low expression of ICD, two enzymes of the glyoxylate shunt, isocitrate lyase and malate synthase, were significantly downregulated at strict nitrogen limitation
By combining measurements of transcriptomics, proteomics, and metabolomics, under well-controlled nutrient limitations, we have shown that the underlying cellular wiring is remarkably different when two nutrients are limiting at the same time, as compared to those under single limitation

Summary

Introduction

Pseudomonas putida KT2442 is a natural producer of polyhydroxyalkanoates (PHAs), which can substitute petroleum-based non-renewable plastics and form the basis for the production of tailor-made biopolymers. Despite the substantial body of work on PHA production by P. putida strains, it is not yet clear how the bacterium re-arranges its whole metabolism when it senses the limitation of an inorganic (N, S, P, or O) nutrient and the excess of fatty acids as carbon source, resulting in a large accumulation of PHAs within the cell. Three distinct growth regimes were recognized: namely carbon-, carbon-nitrogen-, and strict nitrogen-limiting growth By applying those fermentation strategies, P. putida GPo1 (formerly known as P. oleovorans) was investigated for its capacity to accumulate PHAs from different carbon sources [9,14,15], proving the high metabolic flexibility of GPo1 which was reflected in part by the broad dual-limiting area between the two single-nutrient limitation (for excellent review, see [16]). As this results in less amounts of carbon used for comparable levels of PHA, this can substantially reduce the production costs

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Conclusion