Materials

Yeast extract and soybean peptone were purchased from Oxoid (Thermo Fisher Scientific, the United States of America). Glucose and corn steep liquor were industrial grade reagents stored at our laboratory. All other chemicals were purchased from Titan Scientific Co., Ltd. (Shanghai, China). 48-deep-well plates were purchased from Labgic Technology Co., Ltd. (Beijing, China).

Strain, medium and culture conditions

The parent strain C. jadinii CCTCC AY 92020 used in this study, which was purchased from China Center for Type Culture Collection (CCTCC).

Solid medium (g/L): glucose 20, yeast extract 10, tryptone 20, agar 15.

Seed medium (g/L): glucose 40, corn steep liquor 15, KH2PO4 2.34, MgSO4 1.2, pH 6.5.

Fermentation medium (g/L): sucrose 50, yeast extract 10, soybean peptone 10, KH2PO4 2.34, MgSO4 1.2, FeSO4 0.01, ZnSO4 0.01, pH 5.5.

Cultivation conditions: a single colony was inoculated into either 48-deep-well plates or a 250 mL flask with 800 μL or 25 mL seed medium. Cultures were conducted at 30 ℃ for 18 h with a rotation speed of 220 rpm. The 48-deep-well plates culture were directly used for RNA analysis, while the flask fermentation was conducted with a 4% (v/v) inoculum in a 250 mL baffle flask containing 50 mL of fermentation medium. The culture was incubated at 30 ℃ for 8 h with a rotation speed of 220 rpm.

ARTP mutation

Cyberlindnera jadinii AY 92020 was treated with ARTP mutagenesis breeding machine (ARTP-IIS, Si Qing Yuan Biotechnology Co., Ltd., Wuxi, China) equipped with a plasma generator, helium gas source, and regulator system. Yeast cells were cultured overnight, adjusted to the concentration of 107 CFU/mL, then exposed to ARTP mutagenesis as follows: the radio frequency powered at 120 W, helium flow rate was maintained at 10 SLM, and the distance between the sample and nozzle was set to 2 mm (Li et al. 2008; Ottenheim et al. 2018). The mutagenized suspension was serially diluted, cultured at 30 ℃ for 2 days. The lethality rates were calculated as follows:

$$}(\% ) = (}_0} - }_1})/}_0} \times 100$$

In which, T0 is the cell number without mutation, while T1 is the cell number after mutation treatment of different time.

Dry cell weigh (DCW) measurement

10 mL of fermentation broth was centrifuged, washed twice, and dried at 80 ℃ until a constant weight. Additionally, a regression equation was established correlating the absorbance at 600 nm (OD600) with DCW as 0.4194 * OD600 + 1.0518.

Extraction and measurement of RNA content

The RNA content was measured via perchloric acid extraction method with some modifications (Chuwattanakul et al. 2011). Following cultivation, the fermentation broth was centrifuged at 4000 rpm for 10 min to collect the cell pellets. After washing with 0.9% NaCl twice, the pellets were resuspended in 0.25 mol/L perchloric acid at 4 ℃ for 15 min, then centrifuged at 4000 rpm for 10 min to collect the pellets, resuspended in 0.5 mol/L perchloric acid at 75 ℃ for 15 min with gentle agitation. After a final centrifugation step, the supernatant was quantified at 260 nm using a microplate reader or ultraviolet–visible spectrophotometer after proper dilution.

The RNA content was calculated using the following equation:

$$}\,(}/}) = (}}_} \times } \times 0.03365 \times }_1})/(} \times }_2})$$

In which, OD260 is the absorbance of extracted supernatant at 260 nm, D is the dilution ratio; V1 is the volume of 0.5 mol/L perchloric acid solution, mL; V2 is the volume of fermentation broth, mL; 0.03365 corresponds to the RNA content in the solution to be tested when the absorbance is 1.0.

Genetic stability of the mutant strain

The mutant strain exhibiting high RNA content was sequentially subcultured up to the 10th generation on agar slant cultures. Subsequently, the mutant strains were cultivated in shaking flasks to measure the RNA content and assess their genetic stability.

Plackett–Burman and central composite designs

Plackett–Burman design is a valuable tool to identify significant factors using less experiments to screen multiple factors simultaneously. In the preliminary experiments, eight factors, including sucrose, yeast extract, soybean peptone, (NH4)2SO4, KH2PO4, MgSO4, FeSO4, and ZnSO4, had impact on the RNA content of C. jadinii. Using Design-Expert 8.0.6 software, a total of 12 experiments were designed as Table 1.

Table 1 Plackett–Burman design and experimental results

Based on the results of Plackett–Burman design, the optimal concentration of significant factors was further examined using the path of steepest ascent. A central composite design with three significant factors was designed to optimize the concentrations of culture medium components.

Scanning electron microscopy (SEM) analysis

The yeast cells collected at the 8-h of the fermentation process were separated by centrifugation and then washed twice with 0.1 mol/L phosphate buffer (pH 7.0). The cells were fixed with 2.5% glutaraldehyde overnight at 4 ℃. Subsequently, the cells were dehydrated using a series of ethanol solutions with increasing concentrations (50%, 70%, 80%, 90%, 95%, and 100%, v/v). Finally, the samples were dried by freeze dryer (FD5-3, GOLD SIM International Co., Ltd., Beijing, China), coated with a layer of gold spray, and observed by scanning electron microscope (Hitachi S3400-N, Hitachi, Tokyo, Japan), which was maintained at approximately 15 kV. The width and length of yeast cells were measured as follows: selecting cells with clear boundaries in the electron microscope images, utilizing a ruler or scale bar, clicking, and dragging the line tool on the image to align it precisely with the cell's boundary, and documenting the measured values.

Quantitative real‑time PCR

The yeast cells were cultivated in fermentation medium and collected after centrifugation for RNA extraction. RNA extraction was performed using UNlQ-10 Column Trizol total RNA isolation kit (Sangon Biotech, Shanghai, China). Reverse transcription was carried out using TransScript® II first-strand cDNA synthesis SuperMix (TransGen Biotech, Beijing, China). Quantitative real-time polymerase chain reaction (qPCR) was conducted using SuperReal PreMix Plus (SYBR Green) (Tiangen Biotech, Beijing, China) and the CFX96 touch real-time PCR detection system (Bio-Rad, Shanghai, China). The primers utilized in this experiment were shown in the Additional file 1: Table S1. The primer pairs UBC6-F and UBC6-R, 18S-F and 18S-R, 25S-F and 25S-R, RPL13-F and RPL13-R, RPS6-F and RPS6-R were employed to amplify genes of UBC6, 18S rRNA, 25S rRNA, RPL13 and RPS6. The qPCR conditions were as follows: 95 ℃ for 15 min, followed by 40 cycles at 95 ℃ for 10 s and 60 ℃ for 32 s. The transcriptional level of gene UBC6 which encodes ubiquitin-conjugating enzyme was used as an internal control (Guo et al. 2020).

Statistical analysis

Three parallel samples were set in each group during the experiment. The data was presented as averages standard deviation. Analysis of variance (ANOVA) was conducted using IBM SPSS Statistics 25 to determine significant differences between the samples (P < 0.05).