Methods for bioengineering NRPS/PKS assembly lines delivering peptide natural products with electrophilic warheads.

天然产物（NP）是从微生物和植物中分离出来的分子，这些分子激发了许多领先的抗生素，抗癌药，免疫抑制剂和其他广泛用于临床的基本药物的开发。通常，从天然生物体中分离出来的NP在一开始就不具备先决条件。通常需要进一步的合成修饰以提供最终的药物化合物。然而，NP通常是高度复杂的分子，需要费力的多步化学合成，这是非常昂贵的，污染和越来越不可持续的。与优化的NP变体的合成相关的困难对进行药物开发的制药公司提出了主要障碍。这在制造治疗发展中国家疾病如疟疾、利什曼原虫和恰加斯病所需的药物方面尤其成问题。这些由昆虫传播的单细胞（原生动物）寄生虫引起的高度传染性疾病影响了世界上数十亿最贫穷的人，每年导致50多万人死亡。目前，对这些疾病的有效治疗方法很少。NP青蒿素用于治疗疟疾，但出现了对青蒿素有抗药性的疟疾寄生虫（恶性疟原虫）新菌株。已经确定了用于疟疾和其他相关疾病的有希望的新NP先导物，但是合成衍生物的成本阻碍了新的治疗方法的可用。生产优化的NP衍生物的替代方案是操纵构建亲本NP的微生物中的生物合成装配线（酶）。通过重新编程（工程化）装配线以接受不同的前体，具有改进性质的NP变体可以在更高效、更具成本效益的单步发酵过程中交付。在这个项目中，我们的目标是工程化生物合成途径以生产具有抗原生动物活性的NP衍生物，这些衍生物可以用于对抗疟疾或相关疾病。靶NP是由链霉菌和其他细菌产生的肽，由具有反应性末端官能团（弹头）的氨基酸组成。这些NP将被设计为结合到原生动物如恶性疟原虫的蛋白酶体。蛋白酶体是负责降解细胞中受损或不再需要的其他蛋白质的大型多蛋白质复合物。肽NP的弹头可以与蛋白酶体交联，抑制其功能，导致细胞死亡。我们将使用新的基因编辑和其他方法来设计编码组装含有肽NP的弹头的酶的基因。这将使我们能够改变肽的序列，并包括不同的弹头，以提高它们的活性，选择性和药物开发的其他特性。生物合成装配线包括缩合氨基酸前体的非核糖体肽合成酶（NRPS）。通过替换NRPS内的结构域或亚结构域，可以改变肽产物中的氨基酸序列。在早期合成研究的指导下，我们将创建含有对恶性疟原虫蛋白酶体具有高度选择性的肽的弹头。抑制人类以及寄生虫细胞中蛋白酶体的化合物将是有毒的并且不合适。我们还将设计装配线，以提供含有肽的弹头，这些肽旨在抑制人类癌细胞中的蛋白酶体。这包括合成类似物奥普扎林（oprozaline），目前正在进行治疗多发性骨髓瘤（骨髓癌）的临床试验。通过开发这种类型化合物的工程途径，有可能在一步发酵中生产抗癌药物，如奥普佐米，使其以更低的成本更广泛地获得。我们开发的方法是通用的，可用于生产一系列含有弹头的肽，用于许多其他治疗应用。

Natural products (NP) are molecules isolated from microorganisms and plants that inspired the development of many leading antibiotics, anticancer, immunosuppressive agents and other essential medicines that are widely used in the clinic today. Often the NP that are isolated from the native organism do not possess the prerequisite properties at the outset. Further synthetic modification is typically required to provide the final drug compound. However, NP are typically highly complex molecules requiring laborious multistep chemical synthesis, which is very expensive, polluting and increasingly unsustainable. The difficulty associated with the synthesis of optimised NP variants presents a major barrier to pharma companies undertaking drug development. This is particularly problematic in the manufacture of drugs required to treat diseases of developing world such as malaria, Leishmania and Chagas disease. These highly infectious diseases, caused by single celled (protozoan) parasites transmitted by insects, effect billions of the poorest people in the world and lead to over 500,000 deaths pa. Currently there are very few effective treatments available for these diseases. A NP artemisinin is used to treat malaria, but new strains of the malaria parasite (P. falciparum) have emerged which are resistant to artemisinin. Promising new NP leads have been identified for malaria and other related diseases, but the costs of synthesising derivatives have prevented new treatments being made available. An alternative for producing optimised NP derivatives, is to manipulate the biosynthetic assembly lines (enzymes) in the microorganisms that construct the parent NP. By reprogramming (engineering) the assembly line to accept different precursors, NP variants with improved properties can be delivered in a more efficient, cost-effective single-step fermentation process.In this project we aim to engineer biosynthetic pathways to produce NP derivatives with antiprotozoal activity that could be used to combat malaria or related diseases. The target NP are peptides, composed of amino acids with a reactive terminal functional group (warhead), produced by Streptomyces and other bacteria. These NP will be designed to bind to the proteasome of protozoa such as P. falciparum. Proteasomes are large multi-protein complexes responsible for degrading other proteins in the cell that are either damaged or no longer needed. The warhead of the peptide NP can cross-link with the proteasome inhibiting its function leading to cell death. We will use novel gene editing and other approaches to engineer the genes encoding the enzymes that assemble the warhead containing peptide NP. This will allow us to change the sequence of the peptides and also include different warheads, to improve their activity, selectivity and other properties for drug development. The biosynthetic assembly line includes nonribosomal peptide synthetase (NRPS) enzymes that condense amino acid precursors. By replacing domains, or subdomains, within the NRPS it is possible to change the sequence of the amino acids in the peptide products. Guided by earlier synthetic studies, we will create warhead containing peptides that are highly selective for the P. falciparum proteasome. Compounds that inhibit proteasomes in human as well as the parasite cell, would be toxic and unsuitable. We will also engineer assembly lines that deliver warhead containing peptides designed to inhibit the proteasomes in human cancer cells. This includes oprozomib a synthetic analogue, which is in clinical trials for treatment of multiple myeloma (bone marrow cancer). By developing an engineered pathway to this type compound, it may be possible to produce anticancer drugs, like oprozomib, in a single-step fermentation making them more widely available at lower costs. The methods we develop are generic and can be used to produce a range of warhead containing peptides for a number of other therapeutic applications.