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

Abstract

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.

Keywords: Monoterpenes; Monoterpenoids; Pseudomonas putida GS1; Resistance; Tolerance; Ttg efflux pumps.

Fig. 1

Chemical structures of monoterpenes and monoterpenoids used in this study. Red: linear, green: monocyclic, blue: bicyclic structures

Fig. 2

Growth of P. putida GS1 (WT) + pMiS4-eGFP without and in the presence of different monoterpenes and monoterpenoids. Terp200: γ-terpinene (200 mM), Cin200: 1,8-cineole (200 mM), aTer60: α-terpineol (60 mM), Ger100: geraniol (100 mM), GA200: geranic acid (200 mM), Ver35: verbenone (35 mM), Ter4ol80: terpinen-4-ol (80 mM). Tolerance assays were conducted in a microbioreactor system over 48 h. Biomass formation was monitored every 10–15 min via GFP fluorescence signal intensity (excitation filter 488 nm; emission filter 520 nm). The data points represent the mean values of three biological replicates. For variations between the replicates of each strain, see Online Resource Fig. S18

Fig. 3

Growth of P. putida GS1 WT and mutants + pMiS4-eGFP in the presence of a 35 mM verbenone (Ver35) or b 200 mM geranic acid (GA200). Tolerance assays were conducted in a microbioreactor system over 48 h. Biomass formation was monitored every 10–15 min via 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 Online Resource Fig. S24 and Fig. S28

Fig. 4

Further characterization of mutant strains (CR, TR, GR, GAR, VR1, and VR2). a Localization of identified mutations in different mutant strains. b Nucleotide sequence of the ttgABC-ttgR intergenic region. Deleted region in TR mutant is highlighted in green. The putative TtgR palindromic recognition site is indicated by the arrows; ttgABC and ttgR + 1 (arrowheads), − 10, and − 35 points are marked according to Terán et al. (2003). c Relative expression levels of ttg genes in mutants compared with P. putida GS1 wild type (2^−ΔΔCt). d Resazurin efflux pump activity of P. putida GS1 mutant strains relative to wild type. Slope mean values of fluorescence increase over time of mutant strains normalized to wild type are given. Cells were grown in LB medium until mid-exponential phase and resuspended in PBS buffer with resazurin. Efflux activity was monitored by measuring the fluorescence intensity of resazurin reduction product resorufin (excitation filter 530 nm; emission filter 590 nm). While a slow increase in fluorescence indicates high efflux activity, a high slope shows low efflux activity. In c und d, results represent the mean values and standard deviations of three biological replicates. The asterisks indicate a significant difference for the mutant strains compared with wild-type GS1 according to van der Waerden normal score test with Benjamini-Yakutiel p value adjustment (p < 0.05)

Fig. 5

Geranic acid production (a) and growth (b) of P. putida GS1 WT and mutants (CR, TR, GR, GAR) in a geraniol to geranic acid biotransformation. The experiment was conducted in a microbioreactor system over 38 h. To determine geranic acid concentration, samples were taken at time points t = 0, 12, 21, and 38 h and analyzed via HPLC. The data points represent the mean values and standard deviations of three biological replicas. The asterisks indicate a significant difference for the mutant strains as compared with wild-type GS1 according to van der Waerden normal score test with Benjamini-Yakutiel p value adjustment (p < 0.05). Biomass formation was monitored every 10–15 min via scattered light signal intensity (absorbance at 620 nm). The data points represent the mean values of three biological replicas. For variations in growth between the replicas of each strain, see Online Resource Fig. S29