Programming cells by multiplex genome engineering and accelerated evolution

Abstract

The breadth of genomic diversity found among organisms in nature allows populations to adapt to diverse environments1,2. However, genomic diversity is difficult to generate in the laboratory and new phenotypes do not easily arise on practical timescales3. Although in vitro and directed evolution methods4–9 have created genetic variants with usefully altered phenotypes, these methods are limited to laborious and serial manipulation of single genes and are not used for parallel and continuous directed evolution of gene networks or genomes. Here, we describe multiplex automated genome engineering (MAGE) for large-scale programming and evolution of cells. MAGE simultaneously targets many locations on the chromosome for modification in a single cell or across a population of cells, thus producing combinatorial genomic diversity. Because the process is cyclical and scalable, we constructed prototype devices that automate the MAGE technology to facilitate rapid and continuous generation of a diverse set of genetic changes (mismatches, insertions, deletions). We applied MAGE to optimize the 1-deoxy-d-xylulose-5-phosphate (DXP) biosynthesis pathway in Escherichia coli to overproduce the industrially important isoprenoid lycopene. Twenty-four genetic components in the DXP pathway were modified simultaneously using a complex pool of synthetic DNA, creating over 4.3 billion combinatorial genomic variants per day. We isolated variants with more than fivefold increase in lycopene production within 3 days, a significant improvement over existing metabolic engineering techniques. Our multiplex approach embraces engineering in the context of evolution by expediting the design and evolution of organisms with new and improved properties.

Bricks and blueprints: methods and standards for DNA assembly

“…Multiplex Automatable Genome Engineering (MAGE) entails the delivery of synthetic DNA oligonucleotides into growing cells to mutate specific genomic sequences during DNA replication 70 . It works efficiently in E. coli and was used impressively to recode all 321 TAG stop codons in the E. coli genome to TAA stop codons in order to provide a genomically-recoded organism capable of utilising non-standard amino acids 71 .…”

Synthetic RNA devices to expedite the evolution of metabolite-producing microbes

“…Improving product yields or pathway efficiencies, however, requires optimization of various metabolic pathways in the cellular metabolism-a difficult task using rational approaches 5 . Advances in generating diverse cellular phenotypes have made it possible to achieve a desired phenotype through directed evolution [6][7][8][9] . Nevertheless, because a majority of target products of interest are not associated with a conspicuous phenotype, the improved strains are beyond the reach of a general screening tool and cannot be readily obtained 10 .…”

A standardized workflow for surveying recombinases expands bacterial genome‐editing capabilities

“…While Rec2 performs at levels below those of Recβ in E. coli (Wang et al ., 2009; Nyerges et al ., 2016), we note that the frequencies reported in this study reflect a single cycle of recombineering. We expect that optimized protocols involving several cycles would produce results closer to those seen with Recβ in E. coli .…”

“…As mentioned, MMR machinery is known to interfere with the MAGE platform in various bacterial species, dampening final rates of mutagenesis. For this reason, experimental set‐ups aimed to facilitate deep genome engineering usually rely on permanent or transient suppression of MMR (Wang et al ., 2009; Gallagher et al ., 2014; Lennen et al ., 2016; Nyerges et al ., 2016). We expect that implementation of the same approach in P. putida would greatly enhance Rec2 performance, facilitating for projects based on the deployment of high levels of combinatorial genomic diversity.…”

“…(2009) developed the landmark recombineering protocol known as Multiplex Automated Genomic Engineering (MAGE): a library of synthesized mutagenic ss‐oligonucleotides is introduced into β‐expressing E. coli , allowing for rapid, multisite genomic modification that can be cycled to generate vast spaces of genomic diversity. Since its advent, MAGE recombineering has brought about a multitude of advances, including genome‐wide codon replacement, biosynthesis of lycopene and aromatic amino acids, and comprehensive mutant library generation (Wang et al ., 2009; Isaacs et al ., 2011; Gallagher et al ., 2014; Nyerges et al ., 2016). Unfortunately, β‐recombinase (Recβ) recombineering activity is not well‐conserved in bacterial species outside of the Enterobacteriaceae family (Van Kessel and Hatfull, 2008; Binder et al ., 2013; Lennen et al ., 2016); this limited conservation is thought to stem from specificity of host–phage recombinase interactions (Sun et al ., 2015).…”