Sandpit: Synthetic integrons for continuous directed evolution of complex genetic ensembles

合成生物学的一个重大挑战是需要能够在生物系统中构建新的和复杂的功能的技术。当这些功能涉及多个基因的表达和协调时，例如通过工程代谢或组装元基因组文库中的cDNA来合成新的小分子，通过迭代的方法构建它们是费力和困难的。然而，大自然已经进化出了处理这种复杂性的机制。在这里，我们建议开发一个合成系统，它利用多种自然机制的力量，使合成生物学家能够产生、多样化和提炼复杂的多基因功能。我们技术的核心将基于细菌整合子，这是一种自然的克隆和表达系统，它组装了多个开放阅读框架(基因盒)，使用定点重组，并通过内部启动子的表达转化为功能基因。捕获不同的单个基因并将它们物理连接到适合共同表达的阵列中的能力是这些遗传元素所独有的特征。结果是一组功能协调的基因有助于新表型的快速进化。我们建议开发一种新的技术平台，通过利用整合子的力量进行持续定向进化来革命性地设计复杂的多基因功能。具体地说，我们将1)构建和表征用于持续定向进化的基于合成整合子的系统(SYINGRON)，2)通过原理验证实验和计算优化开发有效使用SYINGRON技术的原理，以及3)使用SYNERGRON技术来组装和优化天然产物(例如紫杉醇)的复杂的、多基因的生物合成途径。创建整合子的关键步骤将是产生一个具有良好特性的整合子整合酶工具箱，该工具箱具有高效、可控的重组频率和一系列不同但特定的插入位点。为了进一步提高融合基因系统的多样性，我们将开发一种可调节、可诱导的横向基因转移技术，该技术基于质粒的接合交换和转导介导的噬菌体转移。我们将使用两种互补的方法来开发植物中整合子的部署策略1)鉴定和开发植物中微生物整合子平台的功能等价物2)利用基于叶绿体的技术测试和利用植物中的微生物整合子元件。生物信息学方法将用于表征我们的定向进化整合子(DES)系统的组件和该系统的实验产品。系统工程的一个基本原则是，随着系统变得更加复杂，设计者选择的参数数量增加，直觉崩溃和计算机辅助设计(CAD)变得必不可少。这一原理在合成生物学领域才刚刚开始受到重视。在这个项目中，我们的目标是在拟议的实验框架的核心嵌入计算建模和优化工具，从而在这个方向上做出一步的改变。SynIntegron技术的主要用途之一将是构建代谢途径，以改善已知途径的代谢流量和未知的代谢途径。为了证明SynIntegrons的有效性，我们将从一个已知的具有挑战性的代谢途径开始--基于甲氧戊酸的类异戊二烯生物合成途径。在确定整合子系统在模型应用中的功能后，我们将尝试进化一种新的多基因功能-在细菌中生物合成具有经济和医学价值的分子紫杉醇，展示同步整合子平台在进化各种复杂的多基因功能方面的效用，这些功能是其他方法无法构建或发现的。

A grand challenge in synthetic biology is the need for technologies that enable the construction of novel and complex functions in biological systems. When these functions involve the expression and coordination of multiple genes e.g engineering metabolism or assembly of cDNAs from a metagenomic library to synthesize novel small molecules, building them by an iterative approach is laborious and difficult. Nature has however evolved mechanisms to deal with such complexity. Here we propose to develop a synthetic system that harnesses the power of multiple natural mechanisms to enable synthetic biologists to generate, diversify, and refine complex multigenic functions. The core of our technology will be based on a bacterial integrons, which are natural cloning and expression systems that assemble multiple open reading frames (gene cassettes), using site-specific recombination and conversion to functional genes by expression from an internal promoter. The ability to capture disparate individual genes and physically link them in arrays suitable for co-expression is a trait unique to these genetic elements. The result is an assembly of functionally coordinated genes facilitating the rapid evolution of new phenotypes.We propose to develop a novel technology platform to revolutionise the process of engineering complex multigenic functions by harnessing the power of integrons for continuous directed evolution. Specifically, we will 1) construct and characterise a synthetic integron-based system (syntegron) for continuous directed evolution, 2) develop principles for effectively using syntegron technology via proof-of-principle experiments and computational optimisation, and 3) use synegron technology to assemble and optimise complicated, multi-gene, biosynthetic pathways for natural products (e.g., Taxol). A key step in the creation of syntegrons will be the generation of a toolbox of well-characterized integron integrases with efficient, controllable recombination frequencies and a range of diverse but specific insertion sites. In order to further enhance the potential diversification of the syntegron system we will develop a tunable, inducible lateral gene transfer technology based around conjugative exchange of plasmids and transduction-mediated transfer of phagemid. We will use two complementary approaches to develop strategies for deployment of syntegrons in plants 1) Iidentification and exploitation of functional equivalents of microbial integron platforms in plants 2) test and utilize microbial syntegron elements in plants using plastid-based technology.Bioinformatic approaches will be used to characterize components of our directed evolution in syntegrons (DES) system and the experimental products of this system. A fundamental principle of Systems Engineering is that, as a system become more complex and the number of parameters chosen by the designer increases, intuition breaks-down and computer-aided design (CAD) becomes essential. This principle is just beginning to be appreciated in the field of Synthetic Biology. In this project, we aim to make a step change in this direction, by embedding computational modeling and optimization tools in the heart of the proposed experimental framework.One of the primary uses of the Syntegron technology will be the construction of metabolic pathways to improve known pathway metabolic flux and unknown metabolic pathways. To demonstrate the usefulness of Syntegrons, we will begin with a known challenging metabolic pathway - the mevalonate-based isoprenoid biosynthetic pathway. After establishing that the syntegron system functions in model applications, we will attempt to evolve a novel multigenic function - the biosynthesis of the economically and medicinally valuable molecule Taxol in bacteria demonstrating the utility of the syntegron platform for evolving a wide variety of complex multigenic functions that could not feasibly be constructed or discovered by other means.