DECHEMA-Forschungsinstitut, Industrial Biotechnology, Frankfurt am Main, Germany.
Faculty Biological Sciences, Goethe University Frankfurt, Frankfurt am Main, Germany.
Appl Environ Microbiol. 2021 Apr 27;87(10). doi: 10.1128/AEM.02873-20.
More than 30,000 tons of menthol are produced every year as a flavor and fragrance compound or as a medical component. So far, only extraction from plant material and chemical synthesis are possible. An alternative approach for menthol production could be a biotechnological-chemical process with ideally only two conversion steps, starting from (+)-limonene, which is a side product of the citrus processing industry. The first step requires a limonene-3-hydroxylase (L3H) activity that specifically catalyzes hydroxylation of limonene at carbon atom 3. Several protein engineering strategies have already attempted to create limonene-3-hydroxylases from bacterial cytochrome P450 monooxygenases (CYPs, or P450s), which can be efficiently expressed in bacterial hosts. However, their regiospecificity is rather low compared to that of the highly selective L3H enzymes from the biosynthetic pathway for menthol in species. The only naturally occurring limonene-3-hydroxylase activity identified in microorganisms so far was reported for a strain of the black yeast-like fungus sp. in South Africa. We have discovered additional fungi that can catalyze the intended reaction and identified potential CYP-encoding genes within the genome sequence of one of the strains. Using heterologous gene expression and biotransformation experiments in yeasts, we were able to identify limonene-3-hydroxylases from and Further characterization of the enzyme demonstrated its high stereospecificity and regioselectivity, its potential for limonene-based menthol production, and its additional ability to convert α- and β-pinene to verbenol and pinocarveol, respectively. (-)-Menthol is an important flavor and fragrance compound and furthermore has medicinal uses. To realize a two-step synthesis starting from renewable (+)-limonene, a regioselective limonene-3-hydroxylase enzyme is necessary. We identified enzymes from two different fungi which catalyze this hydroxylation reaction and represent an important module for the development of a biotechnological process for (-)-menthol production from renewable (+)-limonene.
每年有超过 30000 吨薄荷醇作为香料和香精化合物或作为医药成分被生产。到目前为止，只能通过从植物材料中提取和化学合成来获得。薄荷醇生产的另一种方法可能是一种生物技术-化学过程，理想情况下只需要两个转化步骤，从（+）-柠檬烯开始，（+）-柠檬烯是柑橘加工业的副产物。第一步需要一种柠檬烯-3-羟化酶（L3H）活性，该活性专门催化柠檬烯在碳原子 3 上的羟化。几种蛋白质工程策略已经试图从细菌细胞色素 P450 单加氧酶（CYPs 或 P450s）中创建柠檬烯-3-羟化酶，这些酶可以在细菌宿主中有效地表达。然而，与薄荷醇生物合成途径中高度选择性的 L3H 酶相比，它们的区域特异性较低。迄今为止，在微生物中鉴定出的唯一天然存在的柠檬烯-3-羟化酶活性是报道南非黑酵母样真菌 sp. 的一个菌株。我们已经发现了其他可以催化预期反应的真菌，并在其中一个菌株的基因组序列中鉴定出潜在的 CYP 编码基因。通过在酵母中进行异源基因表达和生物转化实验，我们能够从 和 中鉴定出柠檬烯-3-羟化酶。对 酶的进一步表征表明其具有高立体特异性和区域选择性、用于基于柠檬烯的薄荷醇生产的潜力，以及将α-和β-蒎烯分别转化为马鞭烯醇和 pinocarveol 的额外能力。（-）-薄荷醇是一种重要的香料和香精化合物，此外还具有药用用途。为了从可再生的（+）-柠檬烯开始实现两步合成，需要一种区域选择性的柠檬烯-3-羟化酶酶。我们从两种不同的真菌中鉴定出了催化这种羟化反应的酶，它们代表了从可再生的（+）-柠檬烯生产（-）-薄荷醇的生物技术过程开发的重要模块。
