(142f) Enabling Microbial Cell Factories: Synthetic Biology Tools for Efficient Pathway Assembly and Integration

Advances in DNA sequencing has unveiled an immense treasure trove of biological parts and biosynthetic pathways encoded in genomic information, and the emergence of synthetic biology over the past decade has accelerated our ability use these components to systematically design microbial cell factories for the production of a wide variety of useful compounds. As our understanding of biological parts and systems improve, genetic circuit and pathway designs invariably become more complex. As a result, the sizes of DNA fragments encoding these designs are ever-increasing. However, the speed of uncovering genetic components and designing synthetic biological systems has far outstripped our ability to build and test these systems. Despite recent development in DNA synthesis methods, the quick and reliable assembly of individual DNA components into complete pathways remains one of the limiting factors in the implementation of the design-build-test cycle of synthetic biology. Moreover, large DNA fragments encoding the constructed pathways often suffer from genetic instability in production hosts, necessitating the need to integrate them into the host genome for genetic stability, which is a challenge as the integration efficiency of DNA fragment decreases precipitously with increasing DNA size.

Here, we present our recent progress in developing synthetic biology tools to address these challenges. We developed a scarless, sequence-independent, and enzyme free method of assembling PCR amplified DNA fragments, named Twin-Primer Assembly (TPA). TPA is capable of the assembly of up to a 31 kb plasmid with high fidelity. Even without the use of enzymes, the performance of TPA is on par or better with some of the best in vitro assembly methods currently available. The absence of an enzymatic assembly step in can simplify workflow, reduce human intervention and make TPA amendable to the automation process. To stably propagate the genetic pathways assembled and maximize their gene expression, we also a developed simple platform for high-efficiency, single-step, markerless, and multi-copy chromosomal integration of full biochemical pathways in Saccharomyces cerevisiae. This Di-CRISPR (delta integration CRISPR-Cas) platform is mediated by the use of CRISPR-Cas to generate double strand breaks at multiple delta sites in the S. cerevisiae genome, allowing simultaneous integration of multiple copies of linearized donor DNA containing large biochemical pathways. With the Di-CRISPR platform, we were able to attain highly efficient and markerless integration of large biochemical pathways and achieve an unprecedented 18-copy genomic integration of a 24 kb combined xylose utilization and (R,R)-2,3-butanediol (BDO) production pathway in a single step, thus generating a strain that was able to produce BDO directly from xylose. These tools developed provide simple and highly efficient methods that have the potential to streamline the central design-test-build tenet of synthetic biology and translate the product of this endeavour into industrially applicable microbial cell factories.