Chemicals and media
All chemicals were purchased from Sigma-Aldrich (Taufkirchen, Germany), Carl-Roth (Karlsruhe, Germany), or Merck Millipore (Darmstadt, Germany).
Monoterpenoids were acquired with different purities: 1,8-cineole (≥ 99%), citral (≥ 98%), geranic acid (≥ 85%), geraniol (≥ 99%), geranyl acetate (≥ 99%), geranyl formate (≥ 95%), linalool (≥ 97%), myrcene (≥ 95%), α-terpinene (≥ 95%), γ-terpinene (≥ 98.5%), (+)-terpinen-4-ol (≥ 98.5%), (+)-α-terpineol (≥ 97%), α-terpinyl acetate (≥ 97%), and (1S)-(−)-verbenone (≥ 99%).
For cultivation of E. coli and P. putida, lysogeny broth (LB) or terrific broth (TB) medium was used. For solid media, 17 g l−1 agar-agar was added. Antibiotics and other supplements were used at the following concentrations, if required: kanamycin (Km) 50 μg ml−1, gentamicin (Gm) 25 μg ml−1, tetracycline (Tet) 50–150 μg ml−1, and L-rhamnose 2 mg ml−1.
Strains, plasmids, and oligonucleotides
All strains, plasmids, and oligonucleotides used in this study are listed in Online Resource Table S1.
P. putida cells were routinely grown at 30 °C and E. coli cells at 37 °C. All oligonucleotides were ordered from Sigma-Aldrich (Taufkirchen, Germany).
Strain and plasmid construction
Deletion mutants of ttgR, ttgT, and 10 nucleotides of ttgABC-5′-UTR were obtained following the method published by Martínez-García and de Lorenzo (2011). For this purpose, plasmids pEMG-ΔttgR, pEMG-ΔttgT, and pEMG-Δ10 nt-ttgABC were constructed using primer P1–P4, P5–P8, or P9–P12, respectively. The correct deletion was checked by colony PCR and sequencing of the obtained PCR fragments.
Plasmids pMiS4-ttgR and pMiS4-ttgT were constructed by amplifying the ttgR and the ttgT genes together with their native promoter regions via PCR using P. putida GS1 gDNA as template and primer P13 and P14 or P15 and P16, respectively.
All plasmids used in this study were constructed using Gibson isothermal assembly (Gibson et al. 2009) and chemical competent E. coli cells (Inoue et al. 1990). The correct and sequenced plasmids were electroporated into P. putida GS1 strains following a protocol by Choi et al. (2006).
Generation of mutant library
Transposon mutagenesis of P. putida GS1 was performed following methods published by Klebensberger et al. (2007) and Li et al. (2010). Introduction of plasmid pALMAR-3, harboring a Himar1 mariner transposon (Lampe et al. 1999), into P. putida GS1 was performed by bi-parental mating with E. coli S17-1 λpir as donor. For E. coli S17-1 λpir harboring pALMAR-3, 20 ml LB medium containing 30 μg ml−1 kanamycin was inoculated with an OD600 of 0.1 from an overnight pre-culture. For P. putida GS1, 100 ml LB medium was inoculated with an OD600 of 0.05. After reaching OD600 of 2 or 0.3, respectively, 8 ml of LB culture of the E. coli donor strain and 100 ml of the recipient strain P. putida GS1 were harvested, washed twice with 8 ml pre-warmed LB medium, and resuspended in 400 μl pre-warmed LB medium each. Subsequently, the cell suspensions were mixed in a 1:1 volume ratio (cell ratio E. coli to P. putida around 1:2) and placed on LB agar. After incubation for 24 h at 30 °C, the cells were resuspended from the plate with 2 ml 0.9% (w/v) NaCl solution. One hundred microliter aliquots of the cell suspension were spread on Pseudomonas isolation agar plates (King et al. 1954) containing 150 μg ml−1 tetracycline to select for P. putida transposon mutants. After incubation for 24 h at 30 °C, colonies were washed from the plates with 2 ml LB medium containing 20% glycerol and stored at − 80 °C.
Growth selection of monoterpenoid-hypertolerant mutants
For growth selection under different monoterpenoid stress conditions, both P. putida GS1 wildtype (WT) cells and the transposon mutant library were used as a mixture to ensure large cell variety at the starting point. The monoterpenoids were added directly to the bacteria cultures in different, partly increasing concentrations: 1,8-cineole 20, 40, and 60 mM; geranic acid 35 and 90 mM; geraniol 32 and 65 mM; α-terpineol 17.5 mM; and verbenone 35 mM. The cryo stocks were thawed on ice and grown overnight at 30 °C in shake flasks with 20 ml TB medium containing 50 μg ml−1 tetracycline.
For the selection of 1,8-cineole-hypertolerant mutants, the preculture was diluted to an OD600 of 0.1 with TB medium. The cells were cultivated with the stressor for 24 h at 30 °C in a microbioreactor system (BioLector®) at 1000 rpm using 1.5 ml culture volume in 48-well Flowerplates® (m2p-labs GmbH, Baesweiler, Germany) covered by gas-permeable sealing foil. From the stationary phase culture, new TB cultures were inoculated to an OD600 of 0.1 and again cultivated in the microbioreactor system with 1,8-cineole. This procedure was followed for five cultivation steps. After each round, cultures with improved tolerance properties were selected for the next selection round. Each culture was cultivated in triplicates. Only for the first cultivation 50 μg ml−1 tetracycline was added to the medium. After cultivation 2, 3, and 4, the cells were spread on LB-agar plates and incubated over night at 30 °C or for 3 days at room temperature. Subsequently, the cells were washed from the agar plates with 3 ml NaCl (0.9%), and 1 ml of the cell suspension was used to inoculate the new culture.
For the selection of geranic acid, geraniol, α-terpineol, and verbenone-hypertolerant mutants 200 μl preculture was transferred into 20 ml TB medium each and the respective stressor was added. The cultures were incubated in shake flasks for around 24 h at 30 °C. Between five and seven sequential cultivation rounds were conducted for each monoterpenoid. For each further round, the main culture was inoculated to a start OD600 of 0.1. Before the last cultivation round for all monoterpenoids, the cells were spread out on LB agar to obtain single colonies for inoculation.
Based on the growth performance of the different mutants in the last cultivation round, for each selection substance one (or in the case of verbenone two) candidate strains were selected for further characterization.
Genetic characterization of monoterpenoid-hypertolerant mutants
In order to determine the genotype of the selected mutant strains, the mutation sites were mapped via splinkerette PCR (Devon et al. 1995; Mikkers et al. 2002) (CR mutant) or genome sequencing (TR, GR, GAR, VR1, VR2 mutant).
For the 1,8-cineole selected mutant (CR), gDNA was extracted using the GenElute Bacterial Genomic DNA kit (Sigma-Aldrich, Taufkirchen, Germany). Of total gDNA, 1.5 μg was digested with BamHI. Subsequently, hybridized splinkerette adapter (P17 and P18, 1.2 pmol) was ligated to 300 ng of the DNA fragments using T4-Ligase (NEB, Frankfurt am Main, Germany). Ligation products were isolated using the DNA Clean & Concentrator™-5 kit (Zymo Research, Freiburg, Germany). Transposon-chromosome junctions were amplified by PCR with a primer specific for the adapter (P19) and a primer specific for the mariner transposon (P20). After purification of the PCR product with the DNA Clean & Concentrator™-5 kit (Zymo Research, Freiburg, Germany), a second PCR with the first PCR product as template using primer P21 and P22 was conducted. Finally, the obtained PCR product was purified as described before and sequenced.
To localize the transposon integration site, the sequences adjacent to the transposon were mapped to the genome sequence of P. putida GS1. The latter was provided by the company GenXPro (Frankfurt am Main, Germany) using the SMRT method and with the annotation software Prokka (v1.11) (Torsten Seemann 2014).
For the genotypic characterization of the mutants GAR, GR, TR, VR1, and VR2, for each strain, 200 ml TB medium was inoculated to an OD600 of 0.2 from an overnight culture and incubated at 30 °C and 180 rpm. When cultures reached an OD600 between 2.5 and 5, cells were harvested, washed with 20 ml H2O, and frozen with liquid nitrogen to store at − 80 °C. Genomic DNA preparation and genome sequencing via SBS (sequencing by synthesis)-Illumina approach (Illumina, San Diego, USA) was conducted by the company GenXPro (Frankfurt am Main, Germany). Reads obtained from sequencing were mapped against the P. putida GS1 genome using the software Geneious (Biomatters Ltd., Auckland, New Zealand). In order to reduce errors occurring from the sequencing method, the P. putida GS1 WT genome was resequenced in parallel with the Illumina approach.
Monoterpenoid tolerance assays
Pre-cultures of P. putida GS1 WT and mutant strains grown in TB medium were used to inoculate TB cultures to an OD600 of 0.1, supplemented with appropriate antibiotics if required. The respective monoterpenoid was added directly after inoculation. The concentrations tested for each compound, without consideration of purity, were 1,8-cineole 40, 60, 100, and 200 mM; citral 25 and 100 mM; geranic acid 100 and 200 mM of substance with 85% geranic acid; geraniol 40, 65, and 100 mM; geranyl acetate 25 and 100 mM; geranyl formate 25 and 100 mM; linalool 25 and 100 mM; myrcene 17.5, 35, 60, 100, and 200 mM; α-terpinene 25 and 200 mM; γ-terpinene 17.5, 35, 60, 100, and 200 mM; (+)-terpinen-4-ol 80 mM; (+)-α-terpineol 17.5, 35, and 60 mM; α-terpinyl acetate 25 and 100 mM; (1S)-(−)-verbenone 15, 25, and 35 mM.
Cells exposed to the different chemicals were incubated for 48 h at 30 °C in a microbioreactor system (BioLector®) at 1000 rpm and 85% humidity using 1 ml culture volume in 48-well Flowerplates® (m2p-labs GmbH) covered with gas-permeable sealing foil. Biomass formation was monitored via scattered light signal intensity at 620 nm. If the strains contained the pMiS4-eGFP plasmid, growth was monitored additionally via GFP fluorescence signal intensity, using an excitation filter of 488 nm and an emission filter of 520 nm. For induction of GFP expression, L-rhamnose was added to a final concentration of 0.2% (w/v) directly after inoculation. All growth comparison experiments in Flowerplates® were performed at least in triplicates. Culture samples were distributed randomly on the plate.
For quantification of verbenone concentration in the cell suspensions and in cell-free medium over time, cells were cultivated as described above but with 1.5 ml culture volume. Verbenone (35 mM) was added at inoculation. Samples of 180 μl were taken at time points t = 0, 15, 22, 40, and 45 h. To the samples, 20 μl 1 M H2SO4 was added and analytes were extracted using 200 μl ethyl acetate. Samples were centrifuged (16,000×g, 2 min), and the organic phase was analyzed by GC-MS (GC-17A with QP5050A detector, Shimadzu) with a VB-5 column (30 m × 0.25 mm × 0.25 μm, ValcoBond® (Valco Instruments Co. Inc. and VICI AG)). Measurements were conducted as follows: helium as carrier gas, split ratio 35, injections at 250 °C, and a column flow of 2.6 ml min−1. The column temperature was programmed as follows: 80 °C for 3 min, 7 °C min−1 up to 150 °C followed by 150 °C for 2 min. Absolute concentration of verbenone was calculated from chromatogram peak areas by comparison to a calibration curve prepared by measuring a dilution series of verbenone standard with known concentrations.
RNA extraction, cDNA synthesis, and quantitative polymerase chain reaction
In order to quantify differences in expression of the efflux pump genes between P. putida GS1 WT and mutants, total RNA was harvested from growing cultures following a protocol modified from Otto et al. (2019). Therefore, pre-cultures grown in TB medium were used to inoculate TB medium to an OD600 of 0.1. Cultures were cultivated at 30 °C and 200 rpm until mid-exponential growth phase (OD600 0.9–1.1). Samples of 1 ml cell suspension were harvested (13,000×g, 2 min), resuspended in 800 μl RNA/DNAShield solution (Zymo Research Europe GmbH), and stored at − 80 °C until further analysis. RNA was extracted from cells using Quick RNA™ Miniprep Plus Kit (Zymo Research Europe GmbH) following manufacturer’s instructions including in-column DNase treatment. cDNA was prepared from purified RNA using the IScript™ gDNA Clear cDNA Synthesis Kit (Bio-Rad Laboratories, Inc.). The expression levels of different efflux pump genes were analyzed using primers designed by an online Realtime PCR tool (http://www.idtdna.com/scitools/Applications/RealTimePCR). Primers are listed in Table S1 (P23–34). As reference gene, rpoD was used and amplified with primers described previously (Franden et al. 2018). Quantitative PCR was performed using QuantiTec SYBR Green PCR Kit (Qiagen) on a PikoReal™ Real-Time PCR System (Thermo Scientific). The reaction conditions were 15 min at 95 °C, 45× (15 s at 94 °C, 30 s at 60 °C, and 30 s at 72 °C), followed by melting curve analysis (30 s starting at 50 °C, increasing 0.2 °C per cycle, ending at 95 °C). Experiments were performed with biological triplicates. No-template controls were run for every primer pair, and no-RT (reverse transcriptase) controls were run for every RNA sample. Transcript levels of ttg genes were estimated by comparing their Ct (cycle threshold) values to the Ct value of the housekeeping gene rpoD (Wang and Nomura 2010). Final expression levels were averaged for each mutant strain and normalized to the expression level of the P. putida GS1 wild-type strain using the following formula:
ΔΔCt = ΔCtE − ΔCtC
with ΔCtE = Ct (ttg gene mutant) − Ct (rpoD gene mutant).
and ΔCtC = Ct (ttg gene wild type) − Ct (rpoD gene wild type).
Gene expression fold change was calculated as follows: Fold change = 2−ΔΔCt.
For statistical analysis, Shapiro-Wilk normality test, followed by pairwise comparison using van der Waerden normal score test with Benjamini-Yakutiel p value adjustment, was applied on ΔCt values.
Efflux activity assay
Efflux pump activity of P. putida wild type and mutants was determined via resazurin accumulation assay described by Vidal-Aroca et al. (2009). Ten milliliter LB medium in 100-ml Erlenmeyer flasks was inoculated from overnight cultures to an OD600 of 0.1 and grown at 30 °C and 180 rpm to mid-exponential phase (OD600 around 1). Cells were harvested by centrifugation (12,000×g, 3 min). The cell pellets were washed two times with 1× PBS buffer and resuspended in PBS + 0.4% glucose. Fluorescence signal was measured with an Infinite® 200 PRO microtiter plate reader (Tecan). Experiments were performed with biological triplicates. The slope of the fluorescence increase was averaged for each strain over 50 min of the experiment and compared to the wild-type value. For statistical analysis, Shapiro-Wilk normality test, followed by pairwise comparison using van der Waerden normal score test with Benjamini-Yakutiel p value adjustment, was applied on slope mean values of fluorescence measurements.
Biotransformation experiments
An approach modified from Mi et al. (2014) was used. Pre-cultures of P. putida GS1 WT and mutant strains grown in TB medium were diluted with TB medium to an OD600 of 0.1. Cultures were divided into 1.5 ml aliquots in 48-well Flowerplates® (m2p-labs GmbH, Baesweiler, Germany), and 35 mM geraniol as biotransformation substrate was added directly after inoculation. Cells were incubated for 38 h at 30 °C in a microbioreactor system (BioLector®) at 1000 rpm covered with gas-permeable sealing foil. Biomass formation was monitored via scattered light signal intensity at 620 nm. Geranic acid concentrations at different time points (0, 12, 21, and 38 h) were determined by HPLC analysis. All strains were tested in triplicates, distributed randomly on the plate.
To P. putida culture samples of 150 μl, 15 μl 1 M HCl was added and analytes were extracted using 165 μl hexane containing 1 mM thymol as an internal standard. Samples were centrifuged (16,000×g, 5 min), and the organic phase was analyzed by HPLC, consisting of a diode array detector and a C18 column (Alltech Alltima, C18, 5 μm, 250 × 4.6 mm; C18 Precolumn, Grace GmbH and Co. KG, Worms, Germany). Substances were separated isocratically using acetonitrile/acidified water (containing 0.05% (v/v) 3 M phosphoric acid) in a ratio of 45:55 (v/v) as mobile phase. After each run, the column was washed with 90:10 mixture of acetonitrile/acidified water.
For statistical analysis, Shapiro-Wilk normality test, followed by pairwise comparison using van der Waerden normal score test with Benjamini-Yakutiel p value adjustment, was applied.