Modular Cloning by Golden Gate Assembly and Possible Application in Pathway Design

Preparation of expression vectors using conventional cloning strategies is laborious and not suitable for the design of metabolic pathways or enzyme cascades, which usually requires the preparation of a vector library to identify productive clones. Recently, Modular Cloning as a novel cloning technique in synthetic biology has been developed. Modular Cloning relies on Golden Gate assembly and supports preparation of individual expression vectors in one-step and one-pot reactions, thus allowing rapid generation of vector libraries. A number of Modular Cloning toolkits for specific applications has been established, providing a collection of distinct genetic elements such as promoters, ribosome binding sites and tags, that can be combined individually in one-step using defined fusion sites. Modular Cloning has been successfully applied to generate various strains for producing value-added compounds. This was achieved by orchestrating complex pathways involving up to 20 enzymes. Due to the novelty of the genetic approach, industrial applications are still rare. In addition, some applications are limited due to the lack of high-throughput screening methods. This shifts the bottleneck from library preparation to screening capacity and needs to be addressed by future developments to pave the path for the establishment of Modular Cloning in industrial applications.

Automated summary

The conventional cloning strategies for preparing expression vectors are time-consuming and laborious, which are not suitable for designing metabolic pathways or enzyme cascades. However, Modular Cloning, a novel cloning technique in synthetic biology, provides a rapid and efficient method for preparing individual expression vectors using Golden Gate assembly. This technique has successfully been used to generate strains capable of producing complex compounds involving up to 20 enzymes. Although industrial applications of Modular Cloning are still rare, the lack of high-throughput screening methods limits its potential. Future developments should address the bottleneck in screening capacity to promote the establishment of Modular Cloning in industrial applications.