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

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.

Batch Cultivation Model for Biopolymer Production

“…Active biomass dynamics of strains are presented in equation (7). The specific growth rate of biomass with glucose is defined according to the "two substrates" kinetic equation (known as "double Monod" relation).…”

“…The cells rapidly consume the carbon substrate and exhibit exponential growth as long as ammonium is present in the medium 3,6 . The second phase is characterized by the transition from a feast to a famine period, which allows the high synthesis of mcl-PHA in batch reactors 3,6,7 . Nevertheless, the amount of bacterial production of mcl-PHA is insufficient compared to the demand and the reduced quantity of production, influencing the price of bioplastics in the market.…”

Polyhydroxyalkanoates

“…These findings demonstrate that PHA metabolism in Pseudomonads plays a critical role in synchronizing the activity of central metabolic pathways with the availability of nutritional resources. The physiological responses of P. putida KT2442 to PHA accumulation were also systematically investigated by using a multi-omic approach to highlight metabolic differences between single-and multiple-nutrientlimited growth in chemostat cultures (Poblete-Castro et al, 2012). The dilution rate was set at D ¼ 0.1 h À1 to ensure PHA formation, and decanoate and NH 4 Cl were used as the sole carbon and nitrogen sources.…”

Strategies for Automated Control of the Bioproduction of Mcl-PHA Biopolymers

“…overall specific growth rate of the biomass, productivity, conversion yield -see Table 1). More specifically, the concurrent use of NH 4 OH as base for pH control and octanoic acid as carbon source meant that, by changing the carbon source feeding ratio, it was possible to directly influence carbon/nitrogen (C/N) ratio in the feed (positive linear dependency, see Figure 4), which is well known to be an important factor in mcl-PHAs production 12,13,14 . Likewise, a close positive linear correlation was also observed between the carbon source feeding ratios and the overall specific growth rate of the biomass ( Figure 5).…”