A High-Throughput Continuous Evolution System for in vivo Biosensor Engineering

 描述（由申请人提供）：进化是生物学背后的最终设计算法，如果加速，可能在生物工程中具有巨大的实用性。我提出了一种独特的策略，称为正交复制（OrthoRep），以实现快速和可扩展的靶向基因在体内的进化，使进化过程可以常规应用于生物分子工程。这个想法是创造一个细胞，从酵母开始，它有第二个复制系统，由一个特殊的DNA质粒由一个专用的DNA聚合酶（DNAP）复制。第二个系统将与基因组复制正交，使得专用DNAP（ortho-DNAP）仅复制特殊质粒（ortho-plasmid）而不复制宿主基因组。然后，可以对ortho-DNAP进行工程改造，使ortho-plasmid以远远超过基因组所能容忍的速率发生突变。我们的分析表明，OrthoRep可以极大地加速进化，因为在正向质粒上编码的基因原则上可以比在酵母基因组上编码的基因更快地多样化~106倍。我们已经成功地在酿酒酵母中建立了OrthoRep，并且我们将通过工程化高度易错的ortho-DNAP来继续其发展，以达到无性基因进化的最大速率。我们还将把有性进化加入到正向质粒中，这是由于我们偶然观察到了正向质粒的自然重组倾向。最后，我们将工程控制正向质粒的拷贝数，使更广泛的选择功能，特别是负选择。一旦OrthoRep被开发出来，我建议将其应用于体内生物传感器的快速发展。代谢工程在生物医学方面具有巨大的潜力，因为它有望将多基因生物合成途径从复杂的天然来源合成复杂的药物，并将其转化为廉价的微生物生产宿主。然而，当多基因生物合成途径被转移到微生物中时，不可避免地需要一系列合理的组合优化步骤。优化需要能够检测产品生产，但没有高通量测定能够做到这一点。我们的OrthoRep系统将消除代谢工程中的这一关键障碍，通过发展体内生物传感器，用于几乎按需的感兴趣的小分子。我们将发展四种分子的体内生物传感器，紫杉二烯，casbene，amorphadiene和parthenoprotein，它们在酵母中的有效生产为代谢工程带来了不同的挑战。这些生物传感器不仅将直接用于这些药物和药物前体的微生物生产，所吸取的经验教训将帮助我们巩固OrthoRep作为一种潜在的变革性进化工程技术，可以广泛应用。

 DESCRIPTION (provided by applicant): Evolution is the ultimate design algorithm behind biology and if sped up, could have enormous utility in bioengineering. I propose a unique strategy, called orthogonal replication (OrthoRep), to achieve fast and scalable targeted gene evolution in vivo so that the evolutionary process can be routinely applied for biomolecular engineering. The idea is to create a cell, starting with yeast, that has a second replication system consisting of a special DNA plasmid replicated by a dedicated DNA polymerase (DNAP). The second system would be orthogonal to genomic replication such that the dedicated DNAP (ortho-DNAP) only replicates the special plasmid (ortho-plasmid) and not the host genome. The ortho-DNAP could then be engineered to mutate the ortho-plasmid at rates far exceeding what the genome could tolerate. Our analysis suggests that OrthoRep could accelerate evolution by enormous amounts, as a gene encoded on the ortho-plasmid could in principle be forced to diversify ~106-fold faster than if it were encoded on the genome of yeast. We have successfully established OrthoRep in Saccharomyces cerevisiae, and we will continue its development by engineering highly error- prone ortho-DNAPs to reach maximum rates of asexual gene evolution. We will also add sexual evolution to ortho-plasmid, spurred by fortuitous observations of our ortho-plasmids' natural tendency to recombine. Finally, we will engineer copy number control for the ortho-plasmid to enable a broader array of selectable functions, especially negative selections. Once OrthoRep is developed, I propose to apply it to the rapid evolution of in vivo biosensors. Metabolic engineering has great potential for biomedicine, as it promises to move multi-gene biosynthetic pathways that synthesize complex drugs from difficult natural sources into cheap microbial production hosts. However, when a multi-gene biosynthesis pathway is transferred into a microbe, an array of rational and combinatorial optimization steps are inevitably needed. Optimization requires the ability to detect product production, but there are no high-throughput assays capable of this. Our OrthoRep system will remove this key roadblock in metabolic engineering by evolving in vivo biosensors for small molecules of interest nearly on-demand. We will evolve in vivo biosensors for four molecules, taxadiene, casbene, amorphadiene, and parthenolide, whose efficient production in yeast offer different challenges to metabolic engineering. Not only will these biosensors be directly useful for microbial production of these drug and drug precursors, lessons learned will help us solidify OrthoRep as a potentially transformative evolutionary engineering technology that can be broadly applied.