Investigation of monoterpenoid resistance mechanisms in Pseudomonas putida and their consequences for biotransformations

Monoterpenoids are widely used in industrial applications, e.g. as active ingredients in pharmaceuticals, in flavor and fragrance compositions, and in agriculture. Severe toxic effects are known for some monoterpenoids making them challenging compounds for biotechnological production processes. Some strains of the bacterium Pseudomonas putida show an inherent extraordinarily high tolerance towards solvents including monoterpenoids. An understanding of the underlying factors can help to create suitable strains for monoterpenoids de novo production or conversion. In addition, knowledge about tolerance mechanisms could allow a deeper insight into how bacteria can oppose monoterpenoid containing drugs, like tea tree oil. Within this work, the resistance mechanisms of P. putida GS1 were investigated using selected monoterpenoid-hypertolerant mutants. Most of the mutations were found in efflux pump promoter regions or associated transcription factors. Surprisingly, while for the tested monoterpenoid alcohols, ketone, and ether high efflux pump expression increased monoterpenoid tolerance, it reduced the tolerance against geranic acid. However, an increase of geranic acid tolerance could be gained by a mutation in an efflux pump component. It was also found that increased monoterpenoid tolerance can counteract efficient biotransformation ability, indicating the need for a fine-tuned and knowledge-based tolerance improvement for production strain development. Key points • Altered monoterpenoid tolerance mainly related to altered activity of efflux pumps. • Increased tolerance to geranic acid surprisingly caused by decreased export activity. • Reduction of export activity can be beneficial for biotechnological conversions. Electronic supplementary material The online version of this article (10.1007/s00253-020-10566-3) contains supplementary material, which is available to authorized users.


time [h]
WT WT_Cin200 CR_Cin200 TR_Cin200 GR_Cin200 GAR_Cin200 VR1_Cin200 VR2_Cin200 B A Figure S7. Growth of P. putida GS1 WT and mutants + pMiS4-eGFP without and in the presence of 100 mM geraniol. Tolerance assays were conducted in a microbioreactor system over 48 h. Biomass formation was monitored every 10 -15 minutes via (A) scattered light signal intensity (absorbance at 620 nm) and (B) GFP fluorescence signal intensity (excitation filter: 488 nm; emission filter: 520 nm). The data points represent the mean values of three biological replicas. For variations between the replicas of each strain see Figure S23.

time [h]
WT WT_Terp200 CR_Terp200 TR_Terp200 GR_Terp200 GAR_Terp200 VR1_Terp200 VR2_Terp200 B A Figure S10. Growth of P. putida GS1 WT and mutants + pMiS4-eGFP without and in the presence of 60 mM α-terpineol. Tolerance assays were conducted in a microbioreactor system over 48 h. Biomass formation was monitored every 10 -15 minutes via (A) scattered light signal intensity (absorbance at 620 nm) and (B) GFP fluorescence signal intensity (excitation filter: 488 nm; emission filter: 520 nm). The data points represent the mean values of two (VR1) or three biological replicas. For variations between the replicas of each strain see Figure S26.
Growth curves of cultures with 60 mM α-terpineol are shown. Tolerance assays were conducted in a microbioreactor system over 48 h. Biomass formation was monitored every 10 -15 minutes via (A) scattered light signal intensity (absorbance at 620 nm) and (B) GFP fluorescence signal intensity (excitation filter: 488 nm; emission filter: 520 nm). The data points represent the mean values of three biological replicas. For variations between the replicas of each strain see Figure S32. Supplementary note 1 The growth curve figures Fig. 2, Fig. 3 and Fig. 5 as well as Figure S1 - Figure S15 show mean values from cultivations of three biological replicates. To show the variations within the experiments, Figure S17 - Figure S32 present the corresponding individual growth curves.

time [h]
WT CR TR GR GAR Figure S30. Verification of ttgR involvement in α-terpineol-hypertolerance phenotype by deletion and complementation. Growth curves of three individual cultures with 60 mM α-terpineol are shown. _pMiS4_ = strains contain the pMiS4 empty plasmid, _ttgR_ = strains contain the pMiS4-ttgR vector. Tolerance assays were conducted in a microbioreactor system over 48 h. Biomass formation was monitored every 10 -15 minutes via (A) scattered light signal intensity (absorbance at 620 nm) and (B) GFP fluorescence signal intensity (excitation filter: 488 nm; emission filter: 520 nm).
Growth curves of three individual cultures with 60 mM α-terpineol are shown. Tolerance assays were conducted in a microbioreactor system over 48 h. Biomass formation was monitored every 10 -15 minutes via (A) scattered light signal intensity (absorbance at 620 nm) and (B) GFP fluorescence signal intensity (excitation filter: 488 nm; emission filter: 520 nm).