Fluorescence lifetime imaging of red yeast Cystofilobasidium capitatum during growth

Carotenogenic yeast strain Cysofilobasidium capitatum CCY 10-1-2 was obtained from Culture Collection of Yeasts (CCY; Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovak Republic) and preserved on malt agar, stored at 4 °C in darkness.

Cystofilobasidium capitatum (CCY-10-1-2) was cultivated in 2l bioreactor Biostat® B plus (Sartorius) on glucose medium (60 g/l). Two-step inoculation was performed in Erlenmeyer flasks in the optimal inoculation medium (in g/l: glucose 40.0, (NH₄)-₂SO₄) 5.0, KH₂PO₄ 5, MgSO₄·7H₂O 0.696, yeast extract 7.0). The first inoculum (50 ml) was cultivated for 24 h at 28 °C under continuous lighting and shaking. Inoculum I was then transferred into 0.25 l of fresh inoculum II, which was grown under the same conditions as inoculum I. After 24 h, inoculum II was transferred into a fermentor containing 1.5 l of sterile production medium (in g/l: glucose 60.0, (NH₄)₂SO₄) 5.0, KH₂PO₄ 5.0, MgSO₄·7H₂O 0.696, yeast extract 7.0). Batch cultivation in a fermentor was carried out at 28 °C, pH 5.5, constant aeration (2 l/min) and under permanent light exposure (11 W fluorescent bulb on the outer side of the fermentor) and stirring (DOT set to 30%). As an initial screening, samples were collected after 6, 22, 30, 46 and 52 hours of cultivation for FLIM, gravimetric determination of biomass and RP-HPLC/PDA determination of pigments.

For comparison, cultivation on agar plate was performed on very microscope stage, thus providing the possibility to measure cells with higher frequency. Flow cell chamber FCS2 (Bioptechs) was used for cultivation on agar. Constitution of agar medium was the same as inoculation medium (see above), but with addition of 20 g agar l^-1.

To compare FLIM images and coupled data with model systems, FLIM analysis was performed on micelles of SDS:ergosterol and SDS:coenzyme Q with different content of ergosterol and coenzyme Q, respectively, and with constant addition of β-carotene. Liposomes lecithin:ergosterol:β-carotene were investigated too. Liposomes were prepared by thin layer evaporation (TLE). Ergosterol, coenzyme Q, lecithin and β-carotene were purchased from Sigma-Aldrich.

Micelles were prepared by dissolution of components in chloroform, mixing them up in required ratio and then chloroform was evaporated. Thereafter, dry thin layer was hydrated with distilled water on laboratory ultra-sonicator (Powersonic PS 02000) for 15 min at power of 40 W. Concentration of β-carotene was held constant at 0.1 mM and concentration of SDS was set to 12 mM for all solutions. Modification of these micelles was done by addition of coenzyme Q with concentration ranging from 0 to 1.25 mM, or by addition of ergosterol with concentration ranging from 0 to 1.25 mM.

Liposomes were prepared by TLE of primary mixture of above mentioned components dissolved in chloroform. Then the hydration was done the same manner like mentioned above.

Concentration of β-carotene was held constant at 0.6 μM and concentration of lecithin was set to 1.2 mM for all solutions. Modification of liposomes was done by addition of coenzyme Q with concentration ranging from 0.012 to 0.5 mM.

FLIM was performed on MicroTime 200 machine (PicoQuant GmbH, Germany), with 467 nm laser diode head for excitation (80 MHz pulse rate), 520/35 nm emission filter (Semrock, USA), using inverted water immersion objective Olympus UPLSAPO 60XW (60x mag, NA 1.2).

For data processing we used SymPhoTime software supplied by manufacturer of the machine. Data were analyzed pixel by pixel to obtain each fluorescent species lifetime, amplitude and intensity.

The model systems samples and samples from fermentor were measured directly on the cover glass placed on the objective. Semi-continuous monitoring of culture development was performed on agar in flow cell chamber FCS2 (Bioptechs, USA). This equipment is appropriate for long course measurement right on the microscope stage while protecting culture from negative effects of surrounding environment.

During culture growth in fermentor 50 ml samples were collected for carotenoid extraction and analysis. Samples were then centrifuged at 4500 RCF for 10 min (Sigma 3-15, Sigma Laborzentrifugen GmbH, Germany), washed in distilled water and centrifuged again. The pellet was then re-suspended in acetone and mechanically disrupted by pestle and mortar. After saponificantion by 20% ethanolic solution of KOH at 90°C extraction by diethylether was performed and the extract was dried on rotary evaporator. Dry extract was dissolved in 1-2 ml of UV-VIS grade ethanol.

Samples were then filtered using 0.45 μm PTFE filters and 10 μl of each sample was injected onto column Kinetex C18 5 μm, 150 × 4.6 mm (Phenomenex, USA) with guard column 5 μm, 30 × 4.6 mm, both equilibrated to 45°C with methanol as elution solvent (flowrate 1 ml/min), on Thermo Finnigan Surveyor HPLC system. Xcalibur software was used for chromatography data analysis. Carotenoids content was evaluated according to previous study (15), i.e. individual carotenoids were identified using RP-HPLC/PDA and their content was evaluated using calibration curve constructed with β-carotene standard solutions with concentration ranging 10-100 μg/mL. Β-carotene standard was purchased from Sigma-Aldrich.

Investigation of model systems showed the presence of four different lifetimes (see Table 1), one of them (≈40 ps) with negative value of amplitude due to excited state kinetics of slowly rotating molecules in environment with high viscosity. Considering the nature of model systems – liposomes and micelles – it should be stated that rotation and diffusion is also constrained in such systems. No significant variance of lifetimes values with concentration of ergosterol or coenzyme Q (CoQ) was observed (data not shown), with exception of the longest lifetime (see Fig. 1). It should be mentioned that this lifetime was significantly longer in liposomes than in micelles and occurs more frequently in liposomes (9% vs 0.03% - see Table 1). This indicates substantially higher level of excited state stabilization in liposomes.

Figure 1

Although lifetimes generally do not change with varying concentration of trigs (ergosterol or CoQ) in model lipidic structures, relative abundances (derived as relative amplitudes) changed moderately (see Fig. 2 and Fig. 3 for comparison of ergosterol influence in micelles and liposomes, respectively). Both in micelles and liposomes the shortest lifetime was overwhelmingly abundant (around 80%). In micelles, significantly abundant was also negative-amplitude lifetime while the other lifetimes were rare. In liposomes there was found more equality in contribution to overall fluorescence between all other lifetimes than the shortest one.

Figure 2

Figure 3

Based on data evaluation, in red yeast cells only two carotenoid lifetimes were significant – the longest one, assigned as Carotenoids I, and the shortest one, assigned as Carotenoids II. The third lifetime with negative values of amplitude does occur only in specific phases of culture development. Simultaneously with carotenoid fluorescence, the NADP(H) autofluorescence (16) was observed when measured in green region of spectra. Variable relative intensity of this NADP(H) fluorescence ranging up to 25% (see Fig. 4) was observed.

Figure 4

Carotenoids I lifetimes show very distinct time pattern. When focused on the start of cultivation, a sudden drop was observed when cells move from lag to exponential phase. This drop was observed both in lifetimes and relative abundances (Fig. 4 and Fig. 5). This drop can be seen as disappearing of Carotenoids I, the membrane form (Fig. 6 and Fig. 7). A dropped value of Carotenoids I remain even if the culture move from exponential to stationary phase, up to 56^th hour, when the observation was stopped.

Figure 5

Figure 6

Figure 7

When considering relative abundance (Fig. 4), Carotenoids II, which occurs in lipid bodies inside cells, increased mildly with entering to exponential phase (from 40 % to 45%), where remain constant. This is a bit contradictory to our belief that storage lipids should be consumed during exponential phase as a source of energy and carbon for biosynthesis. When culture moves to stationary phase, this form typical for storage lipid bodies rapidly drops in cell content but another form of carotenoids, assigned as Carotenoids III (lifetime ≈0.3 ns, in range 0.2 - 0.4 ns respectively), rapidly grows. When comparing with model systems, we hypothesized that this form exists in cells as a micelles or transport vesicles of lipids containing bunch of carotenoids, and these micelles/vesicles are metabolically active forms of cell lipids. Furthermore, NADP(H) fluorescence and Carotenoids I fluorescence also increased, supporting the idea of synthesizing and transporting carotenoids into cytoplasmic membrane or other organelle membranes, where it serves as a protective system.

Simple overview of culture development monitored by FLIM is in Fig. 8 as a screening for method application. Lifetimes of Carotenoids I during cultivation were roughly comparable with that of agar cultivation. Only Carotenoids I, Carotenoids II and NADP(H) fluorescence was observed, not the micellar form – Carotenoids III. Relative abundances (see Fig. 9) shows massive change of Carotenoids I: Carotenoids II ratio during start of stationary phase, right the other way as it was when cultivating on solid medium. It should be hypothesized that during deep stationary phase the ratio will be shifted in the other way to protect cells from harm. Longer cultivation and sampling for examination this hypothesis should be an object of further research.

Figure 8

Figure 9

Summed fluorescence intensities of both observed forms of carotenoids (data not shown) show similarity with total carotenoids content determined by HPLC and suggests kind of quadratic dependence on cultivation time, while beta-carotene content enlarge in a linear way.