Synthetic Biology is a rapidly growing interdisciplinary field that is primarily built upon foundational advances in molecular biology combined with engineering design principles such as modularity and interoperability. The field considers living systems as programmable at the genetic level and has been defined by the development of new platform technologies and methodological advances. A key concept driving the field is the Design-Build-Test-Learn cycle which provides a systematic framework for building new biological systems. One major application area for synthetic biology is biosynthetic pathway engineering that requires the modular assembly of different genetic regulatory elements and biosynthetic enzymes. In this review we provide an overview of modular DNA assembly and describe and compare the plethora of in vitro and in vivo assembly methods for combinatorial pathway engineering. Considerations for part design and methods for enzyme balancing are also presented, and we briefly discuss alternatives to intracellular pathway assembly including microbial consortia and cell-free systems for biosynthesis. Finally, we describe computational tools and automation for pathway design and assembly and argue that a deeper understanding of the many different variables of genetic design, pathway regulation and cellular metabolism will allow more predictive pathway design and engineering.

合成生物学是一个快速发展的跨学科领域，主要建立在分子生物学的基础进步与工程设计原则（如模块化和互操作性）相结合的基础上。该领域认为生命系统在基因水平上是可编程的，并且已经通过新平台技术的发展和方法论的进步来定义。驱动该领域的一个关键概念是设计-构建-测试-学习循环，它为构建新的生物系统提供了系统框架。合成生物学的一个主要应用领域是生物合成途径工程，它需要不同基因调控元件和生物合成酶的模块化组装。在这篇综述中，我们概述了模块化 DNA 组装，并描述和比较了多种用于组合通路工程的体外和体内组装方法。还介绍了零件设计和酶平衡方法的注意事项，我们简要讨论了细胞内途径组装的替代方案，包括用于生物合成的微生物聚生体和无细胞系统。最后，我们描述了用于通路设计和组装的计算工具和自动化，并认为对遗传设计、通路调节和细胞代谢的许多不同变量的更深入理解将允许更具预测性的通路设计和工程。我们简要讨论了细胞内途径组装的替代方案，包括用于生物合成的微生物聚生体和无细胞系统。最后，我们描述了用于通路设计和组装的计算工具和自动化，并认为对遗传设计、通路调节和细胞代谢的许多不同变量的更深入理解将允许更具预测性的通路设计和工程。我们简要讨论了细胞内途径组装的替代方案，包括用于生物合成的微生物聚生体和无细胞系统。最后，我们描述了用于通路设计和组装的计算工具和自动化，并认为对遗传设计、通路调节和细胞代谢的许多不同变量的更深入理解将允许更具预测性的通路设计和工程。