Abstract

BackgroundThe substitution of plastics based on fossil raw material by biodegradable plastics produced from renewable resources is of crucial importance in a context of oil scarcity and overflowing plastic landfills. One of the most promising organisms for the manufacturing of medium-chain-length polyhydroxyalkanoates (mcl-PHA) is Pseudomonas putida KT2440 which can accumulate large amounts of polymer from cheap substrates such as glucose. Current research focuses on enhancing the strain production capacity and synthesizing polymers with novel material properties. Many of the corresponding protocols for strain engineering rely on the rifampicin-resistant variant, P. putida KT2442. However, it remains unclear whether these two strains can be treated as equivalent in terms of mcl-PHA production, as the underlying antibiotic resistance mechanism involves a modification in the RNA polymerase and thus has ample potential for interfering with global transcription.ResultsTo assess PHA production in P. putida KT2440 and KT2442, we characterized the growth and PHA accumulation on three categories of substrate: PHA-related (octanoate), PHA-unrelated (gluconate) and poor PHA substrate (citrate). The strains showed clear differences of growth rate on gluconate and citrate (reduction for KT2442 > 3-fold and > 1.5-fold, respectively) but not on octanoate. In addition, P. putida KT2442 PHA-free biomass significantly decreased after nitrogen depletion on gluconate. In an attempt to narrow down the range of possible reasons for this different behavior, the uptake of gluconate and extracellular release of the oxidized product 2-ketogluconate were measured. The results suggested that the reason has to be an inefficient transport or metabolization of 2-ketogluconate while an alteration of gluconate uptake and conversion to 2-ketogluconate could be excluded.ConclusionsThe study illustrates that the recruitment of a pleiotropic mutation, whose effects might reach deep into physiological regulation, effectively makes P. putida KT2440 and KT2442 two different strains in terms of mcl-PHA production. The differences include the onset of mcl-PHA production (nitrogen limitation) and the resulting strain performance (growth rate). It remains difficult to predict a prioriwhere such major changes might occur, as illustrated by the comparable behavior on octanoate. Consequently, experimental data on mcl-PHA production acquired for P. putida KT2442 cannot always be extrapolated to KT2440 and vice versa, which potentially reduces the body of available knowledge for each of these two model strains for mcl-PHA production substantially.

Highlights

  • The substitution of plastics based on fossil raw material by biodegradable plastics produced from renewable resources is of crucial importance in a context of oil scarcity and overflowing plastic landfills

  • We demonstrated that P. putida KT2440 and KT2442 produced medium-chain-length polyhydroxyalkanoates (mcl-PHA) from the fatty acid octanoate with similar efficiency but that P. putida KT2442 had a strongly reduced productivity on gluconate because of a more than 3-fold smaller growth rate

  • P. putida KT2442 exhibits reduced specific growth rate and production of mcl-PHA on gluconate compared to its parent KT2440 P. putida KT2440 and KT2442 were cultivated at 30°C in shake flasks containing mineral medium and either octanoate, gluconate, or citrate as carbon source

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Summary

Introduction

The substitution of plastics based on fossil raw material by biodegradable plastics produced from renewable resources is of crucial importance in a context of oil scarcity and overflowing plastic landfills. Many of the corresponding protocols for strain engineering rely on the rifampicinresistant variant, P. putida KT2442 It remains unclear whether these two strains can be treated as equivalent in terms of mcl-PHA production, as the underlying antibiotic resistance mechanism involves a modification in the RNA polymerase and has ample potential for interfering with global transcription. Pseudomonas putida KT2440 and KT2442 belong to the best-known producers of mcl-PHA They accumulate polymer from both PHA-related carbon sources Much effort is spent on engineering these strains in order to increase their accumulation capacity, for instance by deletion of depolymerases [9], and in modifying the pathways involved in PHA synthesis so as to get polymers with modified compositions and improved material properties [10,11,12,13]. Rifampicin-resistant mutants have an altered b-subunit of RNA polymerase [15,16] and their transcription profiles and physiology can be significantly affected

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