Designing a Mutagenesis Circuit for the Directed Evolution of a Target Gene In Vivo

定向进化已经成为生物技术行业的一个重要工具，因为它倾向于开发生物催化剂和利用生物体生产消费品。生物催化剂和细胞基础系统提供了某些优于化学生产方法的优势1。它们提供高效率和底物特异性，同时在生理pH和温度下操作，而不是可能需要极端反应条件的化学方法。目前生物催化剂的使用是有限的，因为一种酶等于一种功能，并且生产某些产品所需的许多酶和代谢途径在自然界中没有发现或不存在。定向进化使我们能够修改现有酶的特性，并产生具有自然界中未发现的功能的新酶。这是通过加速达尔文进化论的自然过程来实现的2，3。到目前为止，像易错PCR这样的体外方法一直是进行定向进化最流行的方法。易错PCR具有极高的通量，可以产生1010个靶基因突变体的文库4。然而，将所有这些突变体转化到用于筛选和表征的所需底盘中可能是麻烦的，并且需要大量的时间。这些突变体中的许多可以是非功能性的并且对底盘有害。已经开发了体内定向进化方法以克服筛选如此大的突变体文库的转化瓶颈5。目前最流行的单基因定向进化方法是噬菌体辅助连续进化（PACE），它将两个宿主之间的突变和选择分离开来;大肠杆菌和噬菌体6（图1）。急诊大肠杆菌宿主含有一个突变质粒，导致全局突变，最终影响其适应性。通过噬菌体的成功再感染进行选择，使E。克服了连续突变造成的适应度损失。这是通过将T7 RNA聚合酶从产生选择信号的T3启动子序列突变为有活性来进行的。PACE的使用受限于将选择与转录激活联系起来的能力，除非这是可能的，否则噬菌体将无法感染宿主细胞，从而使筛选成为不可能。此外，优化选择的条件可能是棘手的。如果泻湖的流速太高，噬菌体会在流出物中被冲走。在某些情况下，可能需要遗传漂变来产生一个多样化的基因库，以进行持续的进化和选择7。我们希望设计一个体内连续进化系统，以靶向单个基因，如PACE，但具有更简单的选择策略，即突变和选择都可以在单个宿主中发生。该系统将应用在淋巴细胞中发现的体细胞超突变的概念来产生变体抗原结合区域8。在这个过程中，特定的基因被靶向突变，同时保持整个基因组的保真度。我们希望应用这种严格调控的突变来进化大肠杆菌中的lasR基因。coli底盘，并通过选择标记和荧光输出选择功能突变体。该系统将被设计为进行连续进化，并在最少人为干预的情况下在复制细胞池中选择所需的突变体。

Directed evolution has emerged as an important tool in the Biotechnology industry as it gravitates towards development of biocatalysts and utilising living organisms to produce consumer products. Biocatalysts and cell bases systems offer certain advantages over chemical methods for production1. They provide high efficiencies and substrate specificities, while operating at physiological pH and temperature as opposed to chemical methods that may require extreme reaction conditions. The use of biocatalysts at the moment is limited as one enzyme equals one function and many of the enzymes and metabolic pathways required to produce certain products are not found in nature or do not exist. Directed evolution allows us to modify the characteristics of existing enzymes and generate novel enzymes with function not found in nature. This is done by accelerating the natural process of Darwinian Evolution2,3. So far, in vitro methods like error-prone PCR have been the most popular for conducting Directed evolution. Error-prone PCR is extremely high-throughput and can generate a library of 1010 mutants of a target gene4. However, transforming all these mutants into the desired chassis for screening and characterisation can be cumbersome and requires a significant amount of time. Many of these mutants can be non-functional and deleterious to the chassis. In vivo directed evolution methods have been developed to overcome the transformation bottle-neck of screening such a large library of mutants5. The most popular method for single gene directed evolution is Phage-assisted Continuous Evolution (PACE), which separates the mutation and selection between two hosts; E. coli and a bacteriophage6 (Fig. 1). The E. coli host contains a mutator plasmid that leads to global mutation that ultimately affects its fitness. Selection via successful re-infection by the bacteriophage enables the E. coli host to be recycled and overcomes the loss of fitness due to continuous mutation. This was performed by mutating T7 RNA polymerase to be active from a T3 promoter sequence that produced the selection signal. The use of PACE is limited by the ability to link selection to transcriptional activation.Unless this is possible, the phage will be unable to infect the host cells, making screening impossible. Also, optimising the conditions for selection can be tricky. If the flow rate of the lagoon is too high, the phage get washed out in the outflow. In some cases, genetic drift might be required to generate a diverse gene pool for continuous evolution and selection7. We hope to design an in vivo continuous evolution system to target a single gene like PACE, but with a simpler selection strategy, whereby both mutation and selection can take place in a single host. The system would apply the concept of somatic hypermutation found in lymphocytes to generate variant antigen-binding regions8. During this process, specific genes are targeted for mutations, while maintaining the fidelity of the overall genome. We hope to apply this tightly regulated mutation to evolve the lasR gene in the E. coli chassis and select for functional mutants via a selectable marker and fluorescent output. The system will be designed to perform continuous evolution and select for desired mutants in a pool of replicating cells with minimal human intervention.