Real-time monitoring of recombinant GFP expression in single-cell Komagataella phaffii through filamentous pelletization technology

Xiao Zheng; Wenjie Cong; Hualan Zhou; Jianguo Zhang

doi:10.1515/revac-2023-0059

Article Open Access

Real-time monitoring of recombinant GFP expression in single-cell Komagataella phaffii through filamentous pelletization technology

Xiao Zheng , Wenjie Cong , Hualan Zhou and Jianguo Zhang

Published/Copyright: October 11, 2023

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From the journal Reviews in Analytical Chemistry Volume 42 Issue 1

Abstract

Komagataella phaffii is an important cell factory for recombinant protein production under methanol induction. Tracking of recombinant protein expression by single K. phaffii cell is a hot topic to identify the variation of expression level in submerge cultivation system. In this study, a platform system harnessing Aspergillus niger pellets for K. phaffii cell immobilization in fabricated glass plate was developed to measure the green fluorescent protein (GFP) expression of single cell using fluorescence microscope. This system was optimized through pellets preparation, K. phaffii cell absorption, inducer comparison, methanol concentration, flow velocity of medium, and obtaining a successful platform to track GFP expression of single K. phaffii cell. This system provided an on-line analytical technology to discover the heterogeneous capability of various K. phaffii cells.

Keywords: on-line single cell analysis; cell immobilization; fungal pellets; Komagataella phaffii

1 Introduction

Filamentous fungal pellet is a fascinating morphology at certain condition of submerged liquid cultivation [1]. Inspired by the traditional application of Aspergillus niger pellets for citric acid production, filamentous fungal pellets were applied into many fields, such as microalgae harvest [2], metal absorption [3], waste water treatment and biofuel production [4], immobilization of microbial cells [5], and sustainable food production [6]. This morphology was formed through twist of spores and hyphae which were driven by estimated forces of electrostatic interactions, hydrophobicity, and specific interactions of polysaccharides [7]. During submerge cultivation, these forces were influenced by cultivation conditions, such as pH, ion concentration, rotate speed, etc.; therefore, cultivation optimization attracted many researchers to demonstrate pellet formation using various fungal species [8]. Among filamentous fungal species, A. niger was a generally regarded as safe, and a typical filamentous species of pellet formation, which was also used for mechanism of pellet formation investigation [9]. The formation forces of electrostatic interactions and specific interactions of polysaccharides were confirmed by Wargenau et al. [10] and Miyazawa et al. [11], respectively. The surface hydrophobicity of A. nidulans was also reported by Dynesen and Nielsen [12]. The absorption of microbial cell on A. niger hyphae led to A. niger hypha as a natural carrier without any chemicals, such as glutaraldehyde which might interfere microbial metabolism [13]. Therefore, A. niger pellet was considered as a potential carrier to investigate the physiological status of microbial cells via its immobilization [14].

Komagataella phaffii is a famous methylotrophic yeast for its capability of high-level recombinant production expression under alcohol oxidase 1 promoter at the condition of methanol induction [15], which could express as high as 22 g·L⁻¹ recombinant collagen intracellularly [16]. The physiological response of K. phaffii at the condition of methanol induction attracted attention of researchers to reveal fluctuation of recombinant protein expression [17]. Currently, yield of recombinant protein by recombinant K. phaffii was measured using fermentation broth which harbored K. phaffii population. Therefore, the heterogeneous capability of various K. phaffii cells was covered, which provided a clue of recombinant K. phaffii diversity during fermentation phase [18]. Single-cell technology for recombinant protein monitoring showed the heterogeneous capability of various K. phaffii cells, which benefited cell analysis in detail. This study developed a platform of single cell analysis for K. phaffii expressing heterologous protein using green fluorescent protein as a model of heterologous protein because of its easy detection in real-time by fluorescence microscope. This platform showed the heterogeneous K. phaffii cells and provided a base for physiological investigation of K. phaffii.

2 Materials and methods

2.1 Strains and chemicals

A. niger ATCC 1015 was purchased from American Type Culture Collection (Manassas, VA, USA). K. phaffii-gfp was constructed in our previous work [15].

Potato dextrose agar (PDA) medium contained 37 g·L⁻¹ PDA.

Medium A contained 10 g·L⁻¹ glucose, 0.04 g·L⁻¹ K₂HPO₄·3H₂O, 0.075 g·L⁻¹ MgSO₄·7H₂O, 0.036 g·L⁻¹ CaCl₂·2H₂O, 0.006 g·L⁻¹ citric acid, 0.006 g·L⁻¹ ammonium ferric citrate, 0.001 g·L⁻¹ EDTA, 1.5 g·L⁻¹ NaNO₃, 0.02 g·L⁻¹ Na₂CO₃, 1 mL trace metal solution, and was prepared by sterilization at 115℃ for 20 min for use.

Medium B contained 1.0 g·L⁻¹ KNO₃, 0.075 g·L⁻¹ KH₂PO₄, 0.1 g·L⁻¹ K₂HPO₄, 0.5 g·L⁻¹ MgSO₄·7H₂O, 0.0625 g·L⁻¹ CaCl₂·4H₂O, 0.01 g·L⁻¹ FeSO₄·7H₂O, 0.5 g·L⁻¹ yeast extract, 1 mL trace metal solution, and was prepared by at 115°C for 20 min for use.

Medium C contained 10.0 g·L⁻¹ glucose, 2.0 g·L⁻¹ NH₄Cl, 2.0 g·L⁻¹ KH₂PO₄, 0.5 g·L⁻¹ MgSO₄, 0.01 g·L⁻¹ FeSO₄·7H₂O, 1 mL trace metal solution, and was prepared by sterilization at 115°C for 20 min for use.

Yeast extract peptone dextrose (YPD) medium contained 10 g·L⁻¹ yeast extract, 20 g·L⁻¹ peptone, and 20 g·L⁻¹ glucose (20 g·L⁻¹ agar for plate medium).

Buffered glycerol-complex (BMGY) medium consisted of 1% yeast extract, 2% tryptone, 1% glycerol, 1.34% YNB, 4 × 10⁻⁵% biotin, 0.8 g NaCl, 0.02 g KCl, and 0.363 g Na₂HPO₄‧12H₂O, pH 6.0.

Trace metal solution contained 2.86 g·L⁻¹ H₃BO₃, 1.81 g·L⁻¹ MnCl₂·4H₂O, 0.222 g·L⁻¹ ZnSO₄·7H₂O, 0.39 g·L⁻¹ Na₂MoO₄·2H₂O, 0.079 g·L⁻¹ CuSO₄·5H₂O, and 0.05 g·L⁻¹ CoCl₂·6H₂O, was adjusted to pH 7.1 using 1 mol·L⁻¹ HCl solution.

2.2 A. niger spores and pellets preparation

A. niger spores were stored in 25% glycerol at −80°C, and were activated by cultivation on PDA at 28°C for 7 days. Then, 10 min sterilized deionized water was applied to wash the spores on PDA plate three times. Finally, 30 mL spore solution was obtained for counting through hemocytometer method under microscope (CX40, Olympus, Tokyo, Japan).

A. niger pellets were prepared by following procedure. One microliter (1 × 10⁸ spore‧mL⁻¹, OD 1.26) A. niger spores were transferred into 100 mL medium A, medium B, and medium C for 48 h cultivation at 28°C, 150 rpm shaking in 250 mL flask. Pellets were harvested through filtration and washed twice finally.

2.3 K. phaffii cultivation

K. phaffii was transferred from 25% glycerol stock tube to YPD medium, and cultivated at 30°C, 200 rpm shaking for 72 h. Then, K. phaffii in YPD medium was transferred into BMGY medium with 1% inoculation ratio for another 72 h cultivation at 30°C, 200 rpm shaking, followed by centrifugation at 1,700g for 10 min repeated twice (5804R Thermo Fisher Scientific, Waltham, MA, USA), and deionized water washing.

2.4 K. phaffii absorption by A. niger pellets

A. niger pellets in 100 mL medium were harvested through filtration, and mixed with recombinant K. phaffii to obtain a pH 4.0, 100 mL 10 mmol‧L⁻¹ PBS solution containing 6.48 × 10¹⁰ L⁻¹ K. phaffii cells. After 120 min shaking at 30°C, 200 rpm, samples were taken to calculate the K. phaffii cell number in the solution.

2.5 Experimental equipment set up for single-cell green fluorescent protein (GFP) monitoring

A glass plate was fabricated to obtain a Ф12 mm sinking-mode of circle as shown in Figure 1a. First A. niger pellets with K. phaffii cells were transferred to this sinking circle. The sinking circle was then filled with BMMY medium containing 1% methanol and covered by cover glass. The side view of the glass plate is shown in Figure 1b with a 0.53 mm depth sinking-mode of circle in the 1 mm thickness glass. This system was monitored by fluorescence microscope (CX40, Olympus, Tokyo, Japan) for cell picture capture and analysis.

Figure 1

Schematic diagram of equipment for single-cell GFP monitoring: (a) planform view and (b) side view.

2.6 Optimization of GFP expression by K. phaffii in monitoring system through A. niger pellet sizes, carbon sources, methanol concentration, and flow velocity of medium in sequence

After setting up this system with A. niger pellets and K. phaffii cells, the system was optimized through A. niger pellet sizes, carbon sources, methanol concentration, and flow velocity of medium in sequence. Pellets of different sizes with K. phaffii cells were transferred into the monitoring system. The initial condition was that BMMY containing 1% methanol was fed with 0.9 mL·min⁻¹ for GFP expression. This system was monitored by fluorescence microscope for cell picture capture and analysis. For carbon source optimization, 1% methanol, 1% formate sodium, and 1% sorbitol was used. For methanol concentration optimization, BMMY containing 0.2%, 0.4%, 0.6%, 0.8%, and 1% methanol was fed. BMMY containing 1% methanol, was fed with 0.09, 0.18, 0.3, 0.9, and 1.8 mL·min⁻¹ to optimize the flow velocity of the medium.

2.7 Cell density measurement

Cell density of K. phaffii was determined using spectrophotometer (721, Shanghai INESA Scientific Instruments Co., Ltd, Shanghai, China) at 600 nm after appropriate dilution.

2.8 Harvest ratio of K. phaffii calculation

The number of absorbed K. phaffii cells on A. niger hypha was obtained as the total K. phaffii cells monitoring the K. phaffii cells in the solution. The absorption ratio of K. phaffii was calculated as percentage of absorbed K. phaffii cells in the total K. phaffii cells.

2.9 Fluorescence intensity of K. phaffii cells determination

Fluorescence intensities of ten K. phaffii cells on hyphae in photograph were quantified using software associated with the fluorescence microscope.

3 Results

3.1 Effects of A. niger pellets of different sizes on its absorption of K. phaffii in monitoring system

Figure 2 shows the morphologies of A. niger pellets and its absorption with K. phaffii cells. The diameters of A. niger pellets in medium A, medium B, and medium C were calculated as 5–8, 3–5, and 1–3 mm according to the ruler comparison. After mixing for 120 min with K. phaffii cells, the absorption ratios of A. niger pellets which were formed in medium A, medium B, and medium C were 47.32%, 61.46%, and 82.93%, respectively. The highest absorption ratio of K. phaffii cells was obtained at the condition of A. niger pellets in medium C. It was concluded roughly that absorption ratio was inversely proportional to the diameter of A. niger pellets, at least in this study.

Figure 2

Absorption ratios of K. phffii on A. niger pellets of different sizes: (a) morphology of A. niger pellets and (b) absorption ratios of K. phffii.

3.2 Effects of A. niger pellet sizes on GFP expression of K. phaffii in monitoring system

The fluorescence photos of K. phaffii cells and A. niger pellets hypha of different sizes are shown in Figure 3a. GFP of recombinant K. phaffii was expressed successfully because the fluorescence of K. phaffii cells became observable significantly with the increasing of induction time. The intensities of GFP were quantified with the results as shown in Figure 3b. The intensity of K. phaffii cell increased and reached its highest value of 171 a.u. after 24 h induction at the condition of A. niger pellets of medium B. K. phaffii GFP intensities of A. niger from two other media also increased with the results of 154 and 113 a.u.

Figure 3

Effects of A. niger pellets of different sizes on GFP expression of K. phaffii in monitoring system: (a) fluorescence photos and (b) GFP intensity.

3.3 Effects of carbon sources on GFP expression by K. phaffii in monitoring system

Pellets formatted in medium B and mixed with K. phaffii in PBS solution for methanol and formate sodium induction led to results as shown in Figure 4. The fluorescence of K. phaffii immobilized on A. niger pellets hyphae became remarkable as time went on. The fluorescence of K. phaffii at the condition of 1% methanol induction was brighter than that of formate sodium. These results were confirmed by the fluorescence intensities as shown in Figure 4b although both the fluorescence intensities of K. phaffii cells at two inducers increased significantly. The highest fluorescence intensities of K. phaffii cells at 1% methanol and 1% formate were obtained as 239 and 161 a.u. after 24 h induction.

Figure 4

Effects of carbon sources on GFP expression by K. phaffii in monitoring system: (a) fluorescence photos and (b) GFP intensity.

3.4 Effects of methanol concentration on GFP expression by K. phaffii in monitoring system

Methanol concentration was an important factor for recombinant protein expression by K. phaffii. The effects of methanol concentration on GFP expression by immobilized K. phaffii are shown in Figure 5. Immobilized K. phaffii cells expressed GFP successfully when 0.2–1.0% methanol was fed into this system. The fluorescence intensities of all K. phaffii cells at different methanol concentrations increased, and reached their maximum after 24 h, which were 235, 184, 184, 248, and 172 a.u. at 0.2–1.0% methanol, respectively. Therefore, 0.8% methanol was suitable for GFP expression by K. phaffii in this system.

Figure 5

Effects of methanol concentration on GFP expression by K. phaffii in monitoring system: (a) fluorescence photos and (b) GFP intensity.

3.5 Effects of flow velocity of medium on GFP expression by K. phaffii in monitoring system

The flow velocity of medium influenced the GFP expression by immobilized K. phaffii cells with results as shown in Figure 6. GFP was expressed successfully at all the grades of flow velocity (Figure 6a). The fluorescence intensities of K. phaffii increased as the induction time went on and reached the highest values at 24 h induction. Fluorescence intensities were obtained as 162, 196, 163, 229, and 191 a.u. at 0.09, 0.18, 0.3, 0.9, and 1.8 mL·min⁻¹. The low GFP intensities at low and high concentration of methanol were considered as carbon sources shortage and toxicity of methanol. Additionally, the GFP intensities of ten K. phaffii cells are shown in Figure 6c, which indicated that GFP intensity of single K. phaffii cell could be tracked successfully.

Figure 6

Effects of flow velocity of medium on GFP expression by K. phaffii in monitoring system: (a) fluorescence photos, (b) GFP intensity, and (c) GFP intensities of ten K. phaffii cells.

4 Discussion

To monitor the GFP expression by single cell of K. phaffii in real-time, we fabricated a sinked-circle glass for K. phaffii cells immobilization on the hypha of filamentous fungal pellets sucessfully. Furthermore, this system was optimized through A. niger pellets cultivation medium, methanol concentration, and flow velocity to demostrate the application states of this system. The optimized conditions of this system were Ф3–5 mm A. niger pellets, 0.8% methanol medium with a flow rate of medium of 0.9 mL·min⁻¹. Therefore, a simple platform for single-cell expressing heterologous protein was developed sucessfully avoiding the disadvantages of agar medium plating, mirofluidics, and droplet flow cytometry Raman-activated cell sorting as in Table 1. Agar medium plating was labrious because many agar plates needed to be prepared, and microbes broth after serial dilution also needed to be spreaded continually. Microfluidics, droplet flow cytometry, and Raman-activated cell sorting needed expensive equipments. Therefore, fungal pellets hypha immobilization does not require expensive equipment, and could work in a on-line model, although low-throughout was considered as the only shortage.

Table 1

Summary of single-cell analysis technology

No.	Systems	Advantages	Disadvantages	Reference
1	Agar medium plating	No expensive equipment needed	Laborious, off-line measurement	[20]
2	Mirofluidics	On-line measurement	Expensive equipment needed	[21]
3	Droplet flow cytometry	High-throughout	Expensive equipment needed, off-line measurement	[22]
4	Raman-activated cell sorting	Specific target	Expensive equipment needed, off-line measurement	[23]
5	Fungal pellets hypha immobilization	No expensive equipment needed, on-line measurement	Low-throughout	This study

For monitoring of GFP as a model of heterologous, the on-line model of monitoring overcame the fluoresence quenching of off-line model [19]. The time-course of GFP expression by the single-cell was captured, which benefited to track GFP expression profile by K. phaffii in real time. During methanol induction phase, K. phaffii cells immobilization on A. niger pellets hypha did not release from the hypha, which identified the stability of this system.

This K. phaffii immobilization on A. niger pellets hypha also explored the analytical technology which was a new field for fungal pellets application. In recent years, filamentous fungal pellets were applied into microalgae harvest, and waste water treatment by numerous research articles. Many researchers focused on condition optimization of various microalgae harvest using different filamentous fungal pellets [24], although A. niger pellets were the major species [25]. The interaction between K. phaffii cell and A. niger hypha was attributed to electrostatic neutralization, surface protein interaction, and exopolysaccharide adhesion [26]. Therefore, this cell immobilization via fungal pellet hypha for analytical technology could be applied to other single-cell species using diverse fungal pellets. It is promising to develop more cell immobilization using this technology.

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Funding information: This work was supported by the International Science and Technology Cooperation Foundation of Shanghai (Grant no. 19230742900).
Author contributions: Xiao Zheng: conducted the experiments; Wenjie Cong, Hualan Zhou: analyzed the data and wrote the manuscript; Jianguo Zhang: conceived this research and revised the manuscript.
Conflict of interest: Authors state no conflict of interest.
Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Received: 2023-06-19

Revised: 2023-07-30

Accepted: 2023-08-19

Published Online: 2023-10-11

This work is licensed under the Creative Commons Attribution 4.0 International License.

Articles in the same Issue

https://doi.org/10.1515/revac-2023-0059

Keywords for this article

on-line single cell analysis; cell immobilization; fungal pellets; Komagataella phaffii

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