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，但到目前为止，合成或半合成青蒿素的生产被证明是成本低的，无法在工业规模上提供靶向药物分子。通过合成生物学和流动化学的结合，我们将提供一种具有成本效益的半合成方法来制造具有附加值的青蒿素，因为我们的方法也将适用于传统合成化学不容易获得的新型青蒿素衍生物的生成。青蒿素生产的核心是氨甲二烯合成酶(ADS)，这是一种萜类环化酶，将水溶性底物法尼基二磷酸(1)转化为不溶于水的碳氢化合物产品。因此，产物抑制是以时间和成本有效的方式利用这些酶的主要问题，因为产物从酶活性部位扩散很慢。我们将采用一家领先的高价值倍半萜生产商(Isobionics BV，荷兰)提供的技术，该生产商已经开发出使用专有红杆菌sp.的发酵程序。这会产生大量具有已知环化酶的倍半萜。我们将把ADS基因和一个编码从FDP 1产生12-羟法尼基二磷酸的P450细胞色素基因(CYP124A1)导入到红细菌产生菌中，并利用它直接从发酵中产生二氢青蒿醛。为了降低风险，我们还将使用我们内部开发的或来自HuvePharma的酵母菌株来执行相同的合成生物学。HuverPharma是一家成立于2015年的新公司，成立目的是收购意大利加雷西奥的赛诺菲工厂。后者的酵母菌株已被证明能产生大量的青蒿酸。我们将把这株带有CYP124A1基因的菌株转化成如上所述的双氢青蒿醛，当然，我们可以直接利用它来生成青蒿酸，并使用我们的流动氧化法(见下文)直接生成青蒿素。发酵法生产二氢青蒿醛减少了青蒿素的必要化学步骤，从而降低了生产成本。在附加值方面，作为一个较长期的目标，这个系统将是灵活的，因为可以在系统中添加其他P450细胞色素和/或甲基转移酶，以产生通过传统合成化学不容易获得的全新的青蒿素类似物。它们将在青蒿素骨架的流动氧化之前修饰紫穗菊-4，11-二烯的骨架，因此不需要破坏抗疟疾活动所必需的微妙的高氧核心的化学作用。这将提供一种可行的方法来对抗日益增长的ACT抵抗。

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.