Microorganism and culture medium
H. denitrificans FJNU-6, which was used as the initial strain for PQQ production, was isolated from chemical sewage and preserved by the China General Microbiological Culture Collection Center (Accession No. CGMCC 1.12893). For the screening and evaluation of the high-yield PQQ strains, the screening medium contained 4 g/L (NH4)2SO4, 3 g/L KH2PO4, 2 g/L Na2HPO4, 2 g/L MgSO4·7H2O, and 20–80 g/L CH3OH; the initial fermentation medium contained 20 g/L CH3OH, 2 g/L (NH4)2SO4, 1.5 g/L KH2PO4, 3 g/L Na2HPO4, 1.6 g/L MgSO4, 30 mg/L CaCl2·2H2O, 5 mg/L ZnSO4·7H2O, 30 mg/L FeSO4·7H2O, 5 mg/L MnCl2·4H2O, 3.2 mg/L CoCl2·6H2O, and 0.5 mg/L CuSO4·5H2O. The CH3OH and (NH4)2SO4 solutions were filtered to remove bacteria, and the other medium components were autoclaved at 121 °C for 20 min.
ARTP mutagenesis and adaptive directed domestication
The ARTP workflow started with the preparation of 200 μL of the FJNU-6 strain in exponential phase (OD600 = 1.0–1.2) followed by dropping the culture onto a sterilized stainless steel plate from an ARTP biological mutagenesis system (ARTP-IIS, Wuxi Yuanqing Tianmu Biological Technology Co., Ltd., Wuxi, China). The operating parameters were as follows: the distance between the plate and the torch nozzle exit was 2 mm, the radio frequency power input was 120 W, the helium gas flow rate was 10 standard liters per minute and the plasma treatment times ranged from 15 to 120 s (15, 30, 45, 60, 75, 90, 105, and 120 s). Subsequently, the treated cells were resuspended in screening medium (2% methanol) and cultivated with the methanol concentration increasing from 2 to 8% at 30 °C for ALE. The colonies of mutants after each ARTP-ALE selection step were collected for rapid screening in fermentation medium of 96-well deep well plates using a SpectraMax microplate reader (i3x; Molecular Devices, LLC, San Jose, CA, USA) by measuring the absorbance at 330 nm (PQQ concentration) and 650 nm (biomass, OD). The strain with the highest PQQ concentration/OD in the former ARTP-ALE round was used for the next ARTP-ALE cycle, and three ARTP-ALE cycles were conducted. Finally, 30 mutant strains with the highest PQQ concentration/OD were isolated from 864 single colonies and then cultured in fermentation medium to detect PQQ production. Among them, ten strains with the highest PQQ production were detected for their stability after 9 consecutive passages.
Optimization of fermentation medium using one-factor-at-a-time and response surface methodology
Five components of the fermentation medium, including the concentrations of methanol, (NH4)2SO4, KH2PO4, Na2HPO4 and MgSO4, were optimized for PQQ biosynthesis with one-factor-at-a-time approach using the FJNU-A26 strain. Fermentation was conducted in a 250-mL shaker flask, and the five selected medium components were set as follows: methanol concentration (10, 20, 30, 40, and 50 g/L), (NH4)2SO4 (1, 2, 3, 4, and 5 g/L), KH2PO4 (1, 2, 3, 4, and 5 g/L), Na2HPO4 (2, 4, 6, 8, and 10 g/L) and MgSO4 (0.25, 0.5, 1, 1.5, and 2 g/L). All experiments were performed in triplicate, and the statistical analysis was performed using SPSS software (version 22.0, SPSS Inc., Chicago, IL, USA).
Optimization using response surface methodology (RSM) was also performed in a 250-mL shaker flask, and each of the four factors described above, except for MgSO4, was used in three levels according to the results from the single-factor experiments. The respective levels were 10, 20, and 30 g/L for methanol; 1, 2, and 3 g/L for (NH4)2SO4; 1, 2, and 3 g/L for KH2PO4; and 4, 6, and 8 g/L for Na2HPO4. The Box–Behnken design (BBD) of RSM designed using Design-Expert software (version 12.0.3.0; Stat-Ease Inc., Minneapolis, MN, USA) was applied to develop a statistical model for establishing the individual and interactive effects of these factors on the fermentation medium components.
Production of PQQ by fed-batch fermentation in 5-L fermenters
The fermentation of wild and selected mutant strains was performed in 5-L fermentation systems (Shanghai National Center of Bioengineering &Technology, China) containing 2.5 L of fermentation medium optimized based on RSM studies. The optimum fermentation conditions were established as described previously [29] as follows: the temperature was maintained at 30 °C, and a two-stage oxygen supply strategy (60% oxygen supply before 55 h, 40% oxygen supply after 55 h) was used by adjusting the agitator speed, air pressure, and ventilation rate; the pH was maintained at a constant value of 7.0 by streaming NH4OH, and methanol was constantly supplied to ensure that its concentration remained at 1–2 g/L in the medium.
The pH value throughout the whole fermentation process was controlled at 6.0, 6.5, 7.0 and 7.5 with the addition of NH4OH to establish the two-stage pH control strategy. Other conditions were similar to those from the experiment with the constant pH control described above. For the two-stage pH control strategy experiments, the pH was maintained at 6.5 for the first 40 h and then changed to 7.0 for the rest of fermentation. Samples were collected regularly to analyze biomass, methanol consumption, PQQ concentration and other parameters.
Assay of gene transcription
Cells from the ARTP-ALE derived strain FJNU-A26 and the initial strain FJNU-6 were collected at 30 h, 42 h, 76 h, 105 h, and 140 h from 5-L fermenters under constant pH 7.0 conditions to extract their total RNA using an RNA plus Kit (Takara Biotechnology, Dalian, China) and then reverse transcribed into cDNAs using a ProtoScript II First-Strand cDNA Synthesis Kit (Invitrogen, Massachusetts, USA). The qRT-PCR assay was conducted using a three-step protocol with the LightCycler® 96 system (Roche, Maryland, USA) and FastStart Essential DNA Green Master reagent (Roche, Maryland, USA), and the primers used in this assay are listed in Additional file 1: Table S1. Two genes, gapdH and recA, were used as reference genes, and the target genes transcribed from parallel cDNA samples were assayed 3 times. Finally, gene expression was calculated using LightCycler® Software (version 1.1.0.1320).
Specific cell growth rate and specific PQQ production rate
The specific cell growth rate and specific PQQ production rate were estimated from experimental or fitted data of cell growth and PQQ production. The values of the specific cell growth rate (μx) and specific PQQ production rate (μp) were obtained from the following equations:
$${\mu }_{x}\text{=}\frac{1}{X}*\frac{{\text{d}}X}{{\text{d}}t}{ \mu }_{p} = \frac{1}{X}*\frac{{\text{d}}P}{{\text{d}}t}.$$
The plots of cell growth and PQQ production were fitted to a logistic regression equation (R2 > 0.99) using Origin Pro 2021 (version 9.8.0.200; Origin Lab Corporation, Northampton, MA, USA), and the parameters μx and μp were computed from the slope drawn on the plots of semilogarithmic DCW and PQQ concentration versus fermentation time, respectively.
Analytical methods
The biomass was determined with a UV spectrophotometer (UV1800, Shimadzu, Japan) by measuring the absorbance at 650 nm. The biomass was collected by centrifugation at 8000×g and then dried at 105 °C until a constant weight was achieved for the dry cell weight (DCW) determination. High-performance liquid chromatography (Waters e2695) was performed to determine the peak areas of PQQ both in PQQ standard solution and culture media using an XBridge BEH C18 Column (4.6 × 150 mm, 5 μm, Waters) at detection wavelengths of 254 and 330 nm (Additional file 1: Fig. S1). The column temperature was set to 35 °C, the mobile phase was 95:5 water with 1‰ trifluoroacetic acid (TFA):acetonitrile with 1‰ TFA, the flow rate was 1 mL/min, and the injection volume was 10 μL. The PQQ product purchased from Sigma (Sigma-Aldrich-80198) was used as the reference material, and a calibration curve was constructed based on the observed peak areas of PQQ measured by HPLC through a linear regression fitting over the range from 43.9 to 877 mg/L PQQ standard solution with a correlation coefficient of 0.999 (Additional file 1: Fig. S2). Therefore, the PQQ concentration was calculated corresponding to the peak area according to a calibration curve [29, 34]. The concentration of methanol in the medium was analyzed using gas chromatography (Shimadzu, Japan) with a SHIMADZU-Rt-Q-BOND column (30 m × 0.53 mm, DF = 20 μm). Nitrogen was used as the carrier gas, and the initial temperature of the column was set to 110 °C. After holding for 1 min, the gas was heated to 190 °C at a rate of 15 °C/min. Then, the heating rate was changed to 3 °C/min up to 250 °C. The holding time was 4 min, and the injection volume was 1 μL.