Improved Thermotolerance of Genome-Reduced Pseudomonas putida EM42 Enables Effective Functioning of the PL /cI857 System.

Abstract

The higher intracellular ATP levels of genome-edited strains of P. putida that result from deleting various energy-consuming functions has been exploited for expanding the window of thermal tolerance of this bacterium. Unlike instant growth halt and eventual death of the naturally occurring strain P. putida KT2440 at 42 °C, the EM42 variant maintained growth and viability of most of the population at the higher temperature for at least 6 h. The authors took advantage of this quality for implementing a robust thermo-inducible heterologous expression device in this species. To this end, the cI857/PL pair of the lambda phage of Escherichia coli was reshaped as a functional cargo that followed the SEVA (Standard European Vector Architecture) format. Quantitation of the transcriptional output of the resulting expression device with GFP reporter technology in various gene dosages identified conditions of unprecedented induced/uninduced ratios (>300 folds) and very high total transcriptional capacity in this bacterial host. The broad-host range nature of the cognate replication origins makes expression vectors pSEVA2214 (low plasmid copy number), pSEVA2314 (medium), and pSEVA2514 (high) to cover a wide range of heterologous expression needs in P. putida and possibly other Gram-negative species.