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 environments. However, genomic diversity is difficult to generate in the laboratory and new phenotypes do not easily arise on practical timescales. Although in vitro and directed evolution methods 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.

Figure 1. Multiplex automated genome engineering enables the rapid and continuous generation of sequence diversity at many targeted chromosomal locations across a large population of cells through the repeated introduction of synthetic DNA

Each cell contains a different set of mutations, producing a heterogeneous population of rich diversity (denoted by distinct chromosomes in different cells). Degenerate oligo pools that target specific genomic positions enable the generation of a diverse set of sequences at each chromosomal location.

Figure 2. Characterization of allelic replacement efficiency as a function of the type and scale of genetic modifications

a, Introducing mismatch mutations of up to 30 bp. b, Inserting exogenous sequences of up to 30 bp. c, Removing up to 45 kbp of chromosomal sequence using a single oligo. d, Correlation of replacement efficiency and two-state hybridization energy ΔG between the oligo and the targeted complement region in the genome. See Supplementary Fig. 1 for an illustration of oligo interaction with genomic targets and Supplementary Table 3 for a list of oligos and corresponding ΔG values. Dashed line is the linear regression correlation (y = −0.288x – 13.7, R² = 0.799). All oligos used were 90 bp with two phosphorothioate bonds at the 3′ and 5′ ends. All error bars indicate ± s.d.; n = 3.

Figure 3. Sequence diversity generated across three separate cell populations as a function of the number of MAGE cycles

Three 90-mer oligo pools were investigated: cN30, iN6 and cN6. cN30 contains oligos with 30 bp of consecutive degeneracy; iN6 contains oligos with 6 bp of degeneracy spaced every 5 bp; cN6 contains oligos with 6 bp of consecutive degeneracy. Frequency of strains in each population that contains 0 to 7+ bp of differences from the wild-type lacZ sequence are colour-coded. The inset shows average number of base pairs changed from wild type across the whole cell population as a function of the number of MAGE cycles using the three oligo pools cN30 (orange line), cN6 (blue line) and iN6 (red line).

Figure 4. MAGE automation

a, Detailed schematic diagram of MAGE prototype including climate-regulated growth chambers with real-time cell density monitors (green), anti-fouling fluidics for transfer of cells between growth chambers and exchange of media and buffers (blue), and real-time generation of competent cells for transformation with synthetic DNA (yellow). Cultures are carried through different chambers at different temperature regimes (30 °C, 42 °C, 4 °C) depending on the necessary MAGE steps (that is, cell growth, heat-shock, cooling). Cells are made electrocompetent by concentration onto a filter membrane and resuspension with wash buffer. Oligos are delivered into cells by electroporation. b, Step-by-step diagram of MAGE cycling steps at a total run time of 2–2.5 h per cycle. Owing to high voltage (18 kV cm⁻¹) electroporation, ~95% of cells are killed at each cycle. Hence, the electroporation event serves to both introduce oligos into cells and to dilute the cell population, cells are then recovered and grown to mid-log phase (7 × 10⁸ cells ml⁻¹) in liquid medium for the subsequent cycle.

Figure 5. Optimization of the DXP biosynthesis pathway for lycopene production

a, Genomic positions of 24 targeted genes with the RBS optimization strategy on the left (red) and gene knockout strategy on the right (blue). The gene knockout strategy involves the introduction of two nonsense mutations. All 90-mer oligos contain two phosphorothioated bases at the 3′ and 5′ termini. b, Black bars represent the growth rate of isolated variants (EcHW2a–f) relative to the ancestral EcHW1 strain. White bars represent lycopene production in p.p.m., which is normalized by dry cell weight in ancestral and mutant strains. Colour-coded labels in each white bar represent genetic modifications found by sequencing. All error bars indicate ± s.d.; n = 3. c, Modifications to the lycopene biosynthesis pathway of isolated variants EcHW2a–f with relevant genes highlighted by rectangular boxes. Blue labels represent knockout targets, red labels represent RBS tuning targets. AcCoA, acetyl-CoA; CDP-ME, 4-diphosphocytidyl-2-C-methyl-d-erythritol; CDP-MEP, 4-diphosphocytidyl-2C-methyl-d-erythritol-2-phosphate; DMAPP, dimethylallyl diphosphate; FPP, farnesyl diphosphate; G3P, glyceraldehyde 3-phosphate; HMBPP, (E)-4-hydroxy-3-methylbut-2-enyl-diphosphate; IPP, isopentenyl diphosphate; MEP, 2-C-methyl-d-erythritol-4-phosphate; MEC, 2C-methyl-d-erythritol-2,4-cyclodiphosphate.