2,2-Diphenyl-1-picrylhydrazyl as a screening tool for recombinant monoterpene biosynthesis

Abstract

Background: Monoterpenes are a class of natural C10 compounds with a range of potential applications including use as fuel additives, fragrances, and chemical feedstocks. Biosynthesis of monoterpenes in heterologous systems is yet to reach commercially-viable levels, and therefore is the subject of strain engineering and fermentation optimization studies. Detection of monoterpenes typically relies on gas chromatography/mass spectrometry; this represents a significant analytical bottleneck which limits the potential to analyse combinatorial sets of conditions. To address this, we developed a high-throughput method for pre-screening monoterpene biosynthesis.

Results: An optimised DPPH assay was developed for detecting monoterpenes from two-phase microbial cultures using dodecane as the extraction solvent. The assay was useful for reproducible qualitative ranking of monoterpene concentrations, and detected standard preparations of myrcene and γ-terpinene dissolved in dodecane at concentrations as low as 10 and 15 μM, respectively, and limonene as low as 200 μM. The assay could not be used quantitatively due to technical difficulties in capturing the initial reaction rate in a multi-well plate and the presence of minor DPPH-reactive contaminants. Initially, limonene biosynthesis in Saccharomyces cerevisiae was tested using two different limonene synthase enzymes and three medium compositions. The assay indicated that limonene biosynthesis was enhanced in a supplemented YP medium and that the Citrus limon limonene synthase (CLLS) was more effective than the Mentha spicata limonene synthase (MSLS). GC-MS analysis revealed that the DPPH assay had correctly identified the best limonene synthase (CLLS) and culture medium (supplemented YP medium). Because only traces of limonene were detected in SD medium, we subsequently identified medium components that improved limonene production and developed a defined medium based on these findings. The best limonene titres obtained were 1.48 ± 0.22 mg limonene per L in supplemented YP medium and 0.9 ± 0.15 mg limonene per L in a pH-adjusted supplemented SD medium.

Conclusions: The DPPH assay is useful for detecting biosynthesis of limonene. Although the assay cannot be used quantitatively, it proved successful in ranking limonene production conditions qualitatively and thus is suitable as a first-tier screen. The DPPH assay will likely be applicable in detecting biosynthesis of several other monoterpenes and for screening libraries of monoterpene-producing strains.

Figure 1

The DPPH reaction and monoterpene compounds examined in this study. DPPH exhibits strong absorbance at 517 nm (purple) which decreases proportionately with the loss of its radical (A). The chemical structures of monoterpene compounds (+)-limonene (B), γ-terpinene (C), myrcene (D), and β-pinene (E) are shown.

Figure 2

Detection of monoterpenes by incubation with DPPH. (A) Sample output are shown for 100 μM DPPH incubated with 0 μM (+), 100 μM (♦), 200 μM (Δ), 800 μM (□), and 1600 μM limonene (▼), with an expanded section of this data to 12.5 min shown in (B). The rates of reaction between 100 μM DPPH and varying concentrations of limonene (C), myrcene (D), γ-terpinene (E), and β-pinene (F) were calculated with a linear regression of the data collected in period of the reaction between 7–12 min (n = 3 for each concentration, mean ± 1 SD). Reaction rates for DPPH incubated with monoterpene standards were compared to the relevant negative controls using an unpaired Student’s t-test (* = p <0.05, ** = P <0.01, *** = p < 0.001).

Figure 3

Detection of limonene biosynthesis in a small-scale screen.S. cerevisiae EPY210C expressing the C. limon (CLLS) or M. spicata (MSLS) limonene synthase, or carrying an empty expression vector (Negative) were cultured for 120 h in 5 mL of either SD, YP, or YP + medium (n = 3 biological replicates in each condition). Reaction rates between dodecane extracts and DPPH (100 μM) were calculated and compared to the negative control for each medium composition (mean ± 1 SD, unpaired Student’s t-test, ** = p < 0.01).

Figure 4

Limonene biosynthesis in 50 mL cultures.S. cerevisiae EPY210C expressing the C. limon (CLLS) limonene synthase, M. spicata limonene synthase (MSLS), or carrying an empty expression vector (−ve) were cultured for 120 h in 50 mL of culture medium. Initially strains were cultured in supplemented YP medium (YP+) and the optical density (A) and reaction rate of the dodecane phase with DPPH (B) were recorded. This experiment was repeated with –ve and CLLS cultures in several defined medium compositions (panels C and D): SD medium containing extra metals (SD + M), pH adjusted SD medium (SD(pH)), pH adjusted SD medium with extra metals (SD(pH) + M), extra nitrogen (SD(pH) + N), or extra metals and nitrogen (SD(pH) + MN), or supplemented YP medium (YP+). Limonene titres (E) were calculated following GC-MS analysis of dodecane extracts. All data shown are calculated from n = 3 biological replicates, showing mean ± 1 SD. ND = not detected, NQ = not quantifiable. Results were compared to negative controls using an unpaired Student’s t-test (** = p < 0.01, *** = p < 0.001).