Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia QLD 4072, Australia.
Microb Cell Fact. 2013 Aug 23;12:76. doi: 10.1186/1475-2859-12-76.
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.
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.
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.
单萜是一类具有多种潜在应用的天然 C10 化合物，包括用作燃料添加剂、香料和化学原料。在异源系统中单萜的生物合成尚未达到商业可行的水平，因此是菌株工程和发酵优化研究的主题。单萜的检测通常依赖于气相色谱/质谱法；这是一个显著的分析瓶颈，限制了分析组合条件的潜力。为了解决这个问题，我们开发了一种高通量的方法来预筛选单萜生物合成。
使用正十二烷作为提取溶剂，我们开发了一种优化的 DPPH 测定法，用于检测两相微生物培养物中的单萜。该测定法可用于重现性地定性排列单萜浓度，并且可以检测到溶解在正十二烷中的标准制备的月桂烯和γ-萜品烯，浓度低至 10 和 15 μM，分别和浓度低至 200 μM 的柠檬烯。由于在多孔板中捕获初始反应速率的技术困难和存在少量 DPPH 反应性杂质，因此无法进行定量测定。最初，使用两种不同的柠檬烯合酶和三种培养基成分测试了酿酒酵母中的柠檬烯生物合成。该测定法表明，在补充 YP 培养基中柠檬烯生物合成得到增强，并且柑橘柠檬柠檬烯合酶（CLLS）比薄荷柠檬烯合酶（MSLS）更有效。GC-MS 分析表明，DPPH 测定法正确识别了最佳的柠檬烯合酶（CLLS）和培养基（补充 YP 培养基）。由于在 SD 培养基中仅检测到微量的柠檬烯，因此我们随后确定了可提高柠檬烯产量的培养基成分，并根据这些发现开发了一种确定的培养基。在补充 YP 培养基中获得的最佳柠檬烯浓度为 1.48±0.22mg/L，在 pH 调节的补充 SD 培养基中获得的最佳柠檬烯浓度为 0.9±0.15mg/L。
DPPH 测定法可用于检测柠檬烯的生物合成。尽管该测定法不能进行定量测定，但它成功地对柠檬烯生产条件进行了定性排序，因此适合作为第一级筛选。DPPH 测定法可能适用于检测其他几种单萜的生物合成，并用于筛选单萜产生菌株文库。
