GEN2NCE - a synthetic biology platform for natural product discovery

微生物天然产物及其合成衍生物广泛应用于医药、农化等行业。在1981-2006年进入市场的所有小分子新化学实体(NCEs)中，34%是天然产品或半合成衍生物。特别是，天然产品构成了大量新的抗菌剂(占NCE的68%)、抗癌(占NCE的54%)和农用化学品(占NCE的40%)的基础。然而，在过去的20年里，由于使用传统的基于生物活性筛选的方法重新发现已知化合物的比率很高，天然产物已经不再是发现新的药物和农用化学品先导的资源。在过去的五年里，基因组测序技术有了很大的进步，现在有可能以低于GB 500的成本获得完整的细菌基因组序列。对这类基因组序列的生物信息学分析表明，许多细菌具有组装比实验室培养中通常观察到的数量多得多的潜在有用自然产品的能力。放线菌尤其如此，它通常含有20-50条生物合成途径，但只产生少数相应的代谢产物。其根本原因似乎是编码这些途径的基因在实验室生长条件下表达不佳。在文献中已经报道了几种诱导这些基因簇表达的方法，但似乎很少是通用的。沉默生物合成基因簇激活的一般方法的发展有望产生丰富的新的生物活性天然产物，在医学和农业中具有潜在的应用。2011年，我们报道了一种激活放线杆菌沉默生物合成基因簇的方法，该方法涉及特定类型转录激活基因的结构性表达。在BBSRC资助的后续工作中，我们试图确定它是否具有广泛的适用性。通过一个战略性的更长或更大的奖项(SLoLa)，我们已经证明这种类型的激活基因通常与放线杆菌中的天然产物生物合成基因簇相关。此外，我们已经证明，这些激活基因的结构性表达能够启动各种生物合成的基因簇，这些基因簇在实验室培养中表达很差，导致了几种新的天然产物的发现。在作为华威综合合成生物学(WISB)中心“工程生物合成途径”主题的其他工作中，我们开发了快速克隆整个生物合成基因簇的有效方法。这为我们的基因簇激活技术在异种宿主中的应用开辟了道路。我们最近完成了创新英国大学研究和商业化项目以及BBSRC探路者项目，这些项目展示了对新颖的结构多样化的天然产品的需求，以满足农化和制药发现管道的需求。现在已经为我们的科学发展成为生产新的天然产品库的商业技术奠定了基础。然而，要做到这一点，我们需要增加我们方法的吞吐量。这将有助于生产适合与高通量屏幕一起使用的更大的库。我们的目标是开发异源宿主，在可诱导的或生长阶段依赖的启动子的控制下，允许生物合成基因簇和激活基因的共同表达。我们还打算通过使用WISB提供的机器人来自动化我们的基因簇激活技术的吞吐量。到项目结束时，我们的目标是建立一个足够大的化合物文库，作为筛选资源提供给早期采用者。

Microbial natural products and their synthetic derivatives are widely used in the pharmaceutical and agrochemical industries. Of all small-molecule new chemical entities (NCEs) that entered the market from 1981-2006, 34% were natural products or semisynthetic derivatives. In particular, natural products formed the basis for a large number of new antimicrobial (68 % of NCE), anticancer (54 % of NCE) and agrochemical (40% of NCE) agents. However, natural products have become de-prioritised as resource for the discovery of new pharmaceutical and agrochemical leads over the last two decades due to the high rate of rediscovery of known compounds using traditional bioactivity screening-based approaches.Over the past five years, genome-sequencing technologies have improved greatly and it is now possible to obtain a complete bacterial genome sequence for less than £500. Bioinformatics analyses of such genome sequences have revealed that many bacteria harbour the capability to assemble a far a greater number of potentially useful natural products than are typically observed in laboratory cultures. This is particularly true of Actinobacteria, which typically contain 20-50 biosynthetic pathways, but only produce a handful of the corresponding metabolic products. The underlying reason for this appears to be that the genes encoding such pathways are poorly expressed under laboratory growth conditions. Several methods for inducing the expression of these gene clusters have been reported in the literature, but few appear to be general. The development of general methods for the activation of silent biosynthetic gene clusters promises to yield a wealth of novel bioactive natural products with potential applications in medicine and agriculture.In 2011, we reported a method for activating silent biosynthetic gene clusters in Actinobacteria involving the constitutive expression of particular type of transcriptional activator gene. In follow up work funded by the BBSRC, we have sought to establish whether it has broad applicability. Through a strategic longer or larger award (sLoLa) we have shown that this type of activator gene is commonly associated with natural product biosynthetic gene clusters in Actinobacteria. Moreover, we have shown that constitutive expression of such activator genes is able to turn on various biosynthetic gene clusters that express poorly in laboratory cultures, leading to the discovery of several novel natural products. In other work carried out as part of the "engineering biosynthetic pathways" theme of the Warwick Integrative Synthetic Biology (WISB) Centre, we have developed efficient methods for rapid cloning of entire biosynthetic gene clusters. This opens the path for using our gene cluster activation technology in heterologous hosts.We have recently completed Innovate UK Innovation and Commercialisation of University Research and BBSRC Pathfinder projects, which have demonstrated a demand for novel structurally diverse natural products to feed agrochemical and pharmaceutical discovery pipelines. The stage is now set for development of our science into a commercial technology for the production of novel natural product libraries. To do this, however, we need to increase the throughput of our approach. This will facilitate the production of larger libraries that are suitable for use with high-throughput screens. We aim to develop heterologous hosts that allow co-expression of a biosynthetic gene cluster and an activator gene under the control of an inducible or growth-phase dependent promoter. We also aim increase the throughput of our gene cluster activation technology by employing robotics available through WISB to automate it. By the end of the project, we aim to have produced a compound library of sufficient size to supply to early adopters as a screening resource.