Investigation and utilisation of the promiscuity of the sesquiterpene synthase amorpha-4,11-diene

尽管存在几种青蒿素的全合成方法，包括使用工程酵母生产青蒿素，但迄今已证明合成或半合成青蒿素生产在工业规模上提供靶药物分子是成本效益低的。通过合成生物学和流动化学的结合，我们将为青蒿素的生产提供一种具有成本效益的半合成方法，并具有附加值，因为我们的方法也适用于生产传统合成化学不容易获得的新型青蒿素衍生物。青蒿素生产的核心是紫穗槐二烯合酶（ADS），萜烯环化酶，其将水溶性底物法呢基二磷酸（1）转化为水不溶性的烃产物。因此，产物抑制是以时间和成本有效的方式利用此类酶的主要问题，因为产物从酶活性位点扩散缓慢。我们将采用领先的高价值倍半萜烯生产商（Isobionics BV，荷兰）提供的技术，该生产商已开发出使用专有红细菌属的发酵程序。该发酵程序使用已知的环化酶产生大量任何所需的倍半萜烯。我们将ADS基因和编码已知从FDP 1（CYP 124 A1）产生12-羟基法呢基二磷酸的P450细胞色素的基因引入红杆菌属的生产菌株中，并使用其直接从发酵产生双氢青蒿醛。为了降低风险，我们还将使用我们内部开发的酵母菌株或Huvepharma进行相同的合成生物学。Huverpharma是一家成立于2015年的新公司，旨在收购位于意大利加雷西奥的赛诺菲工厂。后一种酵母菌株已被证明能产生大量的青蒿酸。我们将用CYP 124 A1基因转化该菌株以如上所述制备二氢青蒿醛，当然可以直接利用它来产生青蒿酸，并使用我们的流动氧化方法（见下文）来直接产生青蒿素。通过发酵生产二氢青蒿醛减少了青蒿素的必要化学步骤，从而降低了其生产成本。在附加值方面，作为一个长期目标，该系统将是灵活的，因为可以将其他P450细胞色素和/或甲基转移酶添加到该系统中，以产生通过常规合成化学不易获得的全新青蒿素类似物。这些将在流入氧化为青蒿素骨架之前修饰紫穗槐-4，11-二烯的骨架，因此不需要破坏抗疟疾活性所必需的脆弱的氧致密核心的化学反应。这将提供一个可行的方法来打击日益增长的ACT耐药性。

In spite of the existence of several total syntheses of artemisinin including the use of engineered yeast for its production, synthetic or semi-synthetic artemisinin production has so far proven to be cost-ineffective to provide the target drug molecule on an industrial scale. Through a combination of synthetic biology and flow chemistry we will deliver a cost-effective semi-synthetic approach to the manufacturing of artemisinin with added value since our approach will also be adaptable to the generation of novel artemisinin derivatives that are not easily accessible by conventional synthetic chemistry.At the heart of artemisinin production lies the enzyme amorphadiene synthase (ADS), a terpene cyclase that converts the water-soluble substrate farnesyl diphosphate (1) into a hydrocarbon product that is water-insoluble. Hence product inhibition is a major issue for utilizing such enzymes in a time and cost-effective manner as the product is slow to diffuse from the enzyme active site. We will employ technology provided by a leading producer of high-value sesquiterpenes (Isobionics BV, Netherlands) who have developed a fermentation procedure using a proprietary Rhodobacter sp. This generates large quantities of any desired sesquiterpene with a known cyclase. We will introduce both the ADS gene and an gene coding for a P450 cytochrome that is known to generate 12-hydroxyfarnesyl diphosphate from FDP 1 (CYP124A1) into the producing strain of Rhodobacter and use this to generate dihydroartemisinic aldehyde directly from fermentation. To mitigate risk we will also use yeast strains developed in-house by us or from Huvepharma to perform the same synthetic biology. Huverpharma are a new company formed in 2015 created to purchase the Sanofi factory in Garessio, Italy. The latter yeast strain has been demonstrated to generate large quantities of artemisinic acid. We will convert this strain with the CYP124A1 gene to make dihydroartemisinic aldehyde as above and can of course utilize it directly to generate artemisinic acid and use our flow-oxidation methodology (vide infra) to generate artemsinin directly. Production of dihydroartemsinic aldehyde by fermentation reduces the necessary chemical steps to artemisinin and so cuts the cost of its production. In added value, as a longer-term goal, this system will be flexible as other P450 cyctochromes and/or methyltranferases can be added to the system to generate completely new analogues of artemisinin that would not be readily accessible through conventional synthetic chemistry. These will modify the skeleton of amorpha-4,11-diene prior to the in-flow oxidations to the artemisinin skeleton and hence not require chemistry that will disrupt the delicate oxygen-dense core essential to the antimalarial activity. This will provide a viable approach to combat the growing ACT resistance.