Biotranformations of natural and inexpensive platform feedstocks to high added value flavour compounds

该项目侧重于利用先前衍生的p450和kred酶板进行相对廉价和丰富的天然单萜烯的生物催化。其中，α蒎烯（占松节油的75-90%）可转化为马鞭草醇、马鞭草酮、桃金娘烯醇和桃金娘烯醛，β蒎烯可转化为松卡维罗醇和松卡维罗酮。马鞭草酚和马鞭草酮已经在微生物系统与黑酵母（激素sp）或黑曲霉生产。然而，由于前者干扰了微生物的代谢，后者的产量在商业上不可行，这些方法尚未商业化。(R)-柠檬烯是另一种廉价的天然单萜，已被用作化学合成香芹酮的起始原料，香芹酮是香芹籽、莳萝籽和绿薄荷的主要挥发性化合物。这种化学合成可以被使用p450和KRED面板的生物催化所取代。其他廉价的天然原料包括松油烯、-3-蒈烯、石竹烯和-松油醇。特别令人感兴趣的是生产所有香料的主要成分-石竹烯醇，从石竹烯中生产石竹烯氧化物，以及从-松油醇中生产卡维罗。其他方法将侧重于从相应的萜烯中生产线性萜烯，例如从法尼烯中生产α -香薰。后者是一种相对昂贵的起始材料，然而，目前除了从含有少量化合物的精油中蒸馏之外，没有其他方法用于商业化生产香薰草。类胡萝卜素与植物中许多挥发性化合物的形成有关（即- ionone， （α, β） damascone， damascenone等），然而，很少有研究利用这些原料生产风味分子。众所周知，类胡萝卜素可以被某些真菌和酵母的外酶降解，从而形成风味化合物。花斑曲霉、scorodonius和Ischnoderma benzoinum已被证明导致胡萝卜素降解93- 98%，而鉴定的最丰富的风味挥发性物质是β -ionone（高达10%的转化率）。最近，一系列类胡萝卜素裂解双加氧酶（CCDs）已经从包括藻类在内的许多来源中分离出来。β -胡萝卜素的中心裂解由β -胡萝卜素加氧酶1催化，产生两分子视黄醛，而β -胡萝卜素非中心双键的偏心裂解由其他酶催化，也可以发生非酶性裂解（通过高锰酸钾氧化裂解）。来自玉米的VP14在11,12双键上特异地切割9-(Z)环氧类胡萝卜素，而其他区域特异性的类胡萝卜素降解酶最近在番茄和矮牵牛花中被表征。结果表明，刺毛霉、解淀粉芽孢杆菌、红绒小芽孢杆菌和土曲霉也能降解叶黄素，生成以7,8 -二氢- β -离子醇和β -离子酮为主要产物的类去甲异戊二烯风味化合物。多功能过氧化物酶是一类新型的过氧化物酶，它结合了木质素过氧化物酶和锰过氧化物酶的催化特性，通常存在于担子菌中。这些反应的非挥发性产物，apocarotenones和apocarotenones，也可以形成平台分子，用于生产一些倍半萜，如α和β sinensal。上述大多数化合物以及最终产物都是疏水分子，相对容易氧化，然而，通过之前参与的一个旨在从亚麻酸中生产醛和醇的可行性项目，已经开发出了与该项目相关的工作流程，同时也常规采用了96孔板快速筛选方法。

The project focuses on the biocatalysis of relatively inexpensive and abundant natural monoterpenes using previously derived enzyme panels of P450s and KREDS. In particular, alpha pinene (75-90% of turpentine oil) could be transformed to verbenol, verbenone, myrtenol and myrtenal while beta-pinene could be transformed to pinocarveol and pinocarvone. Verbenol and verbenone have been previously produced in microbial systems with black yeast (Hormonema sp) or Aspergilus niger. However, those processes have not been commercialized due to, in the first case interferences with the metabolism of the microorganism and in the latter the commercially non-viable yields. (R)-Limonene is another inexpensive natural monoterpene that has been used as the starting material in the chemical synthesis of carvone, a key volatile compound of caraway seeds, dill seeds and spearmint. Such chemical synthesis could be replaced by biocatalysis using P450s and KRED panels. Other inexpensive naturally derived feedstocks include terpinolene, delta-3-carene, caryophyllene and a-terpineol. Of particular interest is the production of beta-caryophyllene alcohol, a major constituent of all spice, and caryophyllene oxide from beta-caryophyllene as well as carveol from alpha-terpineol. Other approaches will focus on the production of linear terpenals from the corresponding terpenes for example alpha-sinensal from farnesene. The latter is a relatively expensive starting material, however currently there is no other process for the commercial production of sinensal other than distillation from essential oils that contain low amounts of the compound. Carotenoids have been linked with the formation of a number of volatile compounds in plants (i.e beta ionone, (alpha, beta) damascone, damascenone etc), however, very few studies exist that make use of such feedstocks for the production of flavour molecules. It is known that carotenoids could be degraded by exoenzymes of certain fungi and yeasts leading to the formation of flavour compounds. Trametes versicolor, Marasmius scorodonius and Ischnoderma benzoinum have been shown to result in carotene degradation of 93-98 % while the most abundant flavour volatile identified was beta-ionone (up to 10% conversion). More recently a series of carotenoid cleavage dioxygenases (CCDs) have been isolated from a number of sources, including algae. Central cleavage of beta-carotene catalyzed by beta carotene oxygenase 1 yields two molecules of retinaldehyde, while eccentric cleavage of beta carotene at non-central double bonds is catalyzed by other enzymes and can also occur non-enzymatically (through oxidative cleavage with potassium permanganate). VP14 from maize cleaves 9-(Z) epoxy carotenoids specifically at the 11,12 double bond while other regiospecific carotenoid degrading enzymes have recently been characterised in tomato and petunia flowers. Trichosporon asahii, Paenibacillus amylolyticus, Peurotus eryngii as well as Geotrichum sp were also shown to degrade lutein leading to norisoprenoid flavour compounds with 7, 8-dihydro-beta-ionol and beta ionone as the main products. Versatile peroxidases are a novel class of peroxidases which combine the catalytic properties of lignin peroxidase and manganese peroxidase, typically found in Basidiomycetes. Non volatile products of these reactions, apocarotenals and apocarotenones, could also form platform molecules for the production of a number of sesquiterpenes such as alpha and beta sinensal. Most of the above compounds as well as the resulting end products are hydrophobic molecules and relatively prone to oxidation, however, through previous participation in a feasibility project that aimed to produce aldehydes and alcohols from linolenic acid have developed workflows that are also pertinent to this project while 96 well plate fast screening approaches are also routinely employed.