Directed evolution combined with synthetic biology strategies expedite semi-rational engineering of genes and genomes

Abstract

Owing to our limited understanding of the relationship between sequence and function and the interaction between intracellular pathways and regulatory systems, the rational design of enzyme-coding genes and de novo assembly of a brand-new artificial genome for a desired functionality or phenotype are difficult to achieve. As an alternative approach, directed evolution has been widely used to engineer genomes and enzyme-coding genes. In particular, significant developments toward DNA synthesis, DNA assembly (in vitro or in vivo), recombination-mediated genetic engineering, and high-throughput screening techniques in the field of synthetic biology have been matured and widely adopted, enabling rapid semi-rational genome engineering to generate variants with desired properties. In this commentary, these novel tools and their corresponding applications in the directed evolution of genomes and enzymes are discussed. Moreover, the strategies for genome engineering and rapid in vitro enzyme evolution are also proposed.

Keywords: DNA assembly; HTS, high-throughput screening; LCR, Ligase Cycling Reaction; MAGE, multiplex automated genome engineering; directed evolution; dsDNA, double-stranded DNA; enzyme; genome engineering; metabolic engineering; recombineering; ssDNA, single-stranded DNA; synthetic biology.

Figure 1.

Schematic overview of multiplex genome engineering and DNA-assembly methods. (A) Recombineering with synthetic double-stranded or single-stranded DNA fragments. Short segments containing different mutation sites were designed and synthesized. After multiple rounds of transformation and recombination, the variants with desired phenotypes were isolated by high-throughput screening methods. (B) Illustration of the Gibson, ligase cycling reaction, and yeast-dependent DNA-assembly methods.

Figure 2.

Combinatorial recombineering to optimize the biosynthesis pathway of interest. To construct a balanced synthetic pathway to the end product, different libraries of pathway functional genes and regulatory elements—including promoters, intergenic spacers, and ribosome-binding sites were designed and synthesized. Applying these recombineering tools, all of the above segments can be combinatorially optimized.

Figure 3.

Illustration of a rapid in vitro directed evolution technique for enzyme engineering. The native gene of interest is divided into several segments. The designed potential mutation sites are introduced during amplification. Subsequently, the mutant fragments are assembled and overexpressed in the host expression strains. With high-throughput screening approaches, the variants with desirable phenotypes are quickly isolated.