Enhancing (2S)-naringenin production in Saccharomyces cerevisiae by high-throughput screening method based on ARTP mutagenesis

Abstract

(2S)-Naringenin, a dihydro-flavonoid, serves as a crucial precursor for flavonoid synthesis due to its extensive medicinal values and physiological functions. A pathway for the synthesis of (2S)-naringenin from glucose has previously been constructed in Saccharomyces cerevisiae through metabolic engineering. However, this synthetic pathway of (2S)-naringenin is lengthy, and the genes involved in the competitive pathway remain unknown, posing challenges in significantly enhancing (2S)-naringenin production through metabolic modification. To address this issue, a novel high-throughput screening (HTS) method based on color reaction combined with a random mutagenesis method called atmospheric room temperature plasma (ARTP), was established in this study. Through this approach, a mutant (B7-D9) with a higher titer of (2S)-naringenin was obtained from 9600 mutants. Notably, the titer was enhanced by 52.3% and 19.8% in shake flask and 5 L bioreactor respectively. This study demonstrates the successful establishment of an efficient HTS method that can be applied to screen for high-titer producers of (2S)-naringenin, thereby greatly improving screening efficiency and providing new insights and solutions for similar product screenings.

Keywords: (2S)-Naringenin; Atmospheric room temperature plasma; Color reaction; High-throughput screening; Saccharomyces cerevisiae.

Fig. 1

Effect of irradiation time on strain HB52 lethality. The lethality rate of S. cerevisiae under different treatment times was compared with untreated strains. Each experiment was repeated three times. After treatment for 90 s and 95 s, the lethality rate reached 96.57% and 96.97%, respectively. Few single colonies survived after treatment for 100 s

Fig. 2

Establishment of high-throughput screening procedure. a (2S)-Naringenin chalcone is generated by the reaction of (2S)-naringenin with strong alkali (KOH). b Correlation curve between (2S)-naringenin concentration and OD₄₁₀ (Different concentrations of (2S)-naringenin were diluted using YPD liquid medium). c Correlation between OD₄₁₀ and (2S)-naringenin titer (Detected by HPLC) in the same strain. S. cerevisiae strains were randomly selected to detect OD₄₁₀ using a microplate reader and (2S)-naringenin titer using HPLC; the distribution range of OD₄₁₀ was 0.3–0.7, and the distribution range of (2S)-naringenin titer was 30–50 mg/L

Fig. 3

Selection of well plate and schematic diagram of high-throughput primary screening. a Titer of (2S)-naringenin in 96-well-plates of the starting strain HB52. 21 strains of S. cerevisiae were randomly selected from 96-well plates to prepare samples, and the titer of (2S)-naringenin was detected by HPLC. b Titer of (2S)-naringenin in 48-well -plates of the starting strain HB52. 16 strains of S. cerevisiae were randomly selected from 48-well plates to prepare samples, and the titer of (2S)-naringenin was detected by HPLC. c Titer of (2S)-naringenin in 24-well plates of the starting strain HB52. 16 strains of S. cerevisiae were randomly selected from 24-well plates to prepare samples, and the titer of (2S)-naringenin was detected by HPLC. d (2S) -Naringenin titer of the starting strain HB52 in a 250 mL shake flask. The (2S)-naringenin titer of the starting strain HB52 increased with time, reaching 430.26 mg/L (extracellular concentration) at 144 h. The black squares represent OD₆₀₀. e Schematic diagram of the high-throughput screening process. f Results of the first round of ARTP mutagenesis screening (OD₄₁₀ value). Different colors represent different OD₄₁₀, gradually increasing from pink to green, with purple representing the control OD₄₁₀ (0.75)

Fig. 4

96-well plate high-throughput primary screening. a–e Results of the first (a), second (b), third (c), fourth (d), and fifth (e) rounds of secondary screening. The strains obtained from the preliminary screening were subjected to 250 mL shake flask secondary screening and the (2S)-naringenin titer (extracellular) obtained were detected using HPLC. f The high-yield strains obtained from the secondary screening were verified by shaking flask fermentation. The optimal strains obtained by secondary screening were cultured in a shake flask, and the intracellular and extracellular (2S)-naringenin concentration was detected; from left to right, they were 565.06 mg/L, 669.52 mg/L, 841.5 mg/L, 628.2 mg/L, 619.53 mg/L, and 617.77 mg/L. The black squares represent OD₆₀₀

Fig. 5

Comparison of wild-type and B7-D9 mutant strains in a 5 L bioreactor. a Culturing of the starting strain HB52 in a 5 L bioreactor. b Changes of mutant strain B7-D9 in a 5 L bioreactor. c The (2S)-naringenin titer and OD₆₀₀ of the strains with integrated uracil synthesis genes were measured in 250 mL shake flasks. Fifteen strains were randomly integrated with uracil synthesis genes and were incubated at 30 °C and 220 rpm for 5 days, and the (2S)-naringenin titer was detected by HPLC. d Changes of strain 5 with integrated uracil synthesis genes in a 5 L bioreactor. The starting strain, mutagenic high-yielding strain and the strain with integrated uracil synthesis genes were cultured in a 5 L bioreactor at 30 °C for 120 h, and the changes of glucose, ethanol, OD₆₀₀ and the titer of (2S)-naringenin were determined with time