Inducible directed evolution of complex phenotypes in bacteria

Abstract

Directed evolution is a powerful method for engineering biology in the absence of detailed sequence-function relationships. To enable directed evolution of complex phenotypes encoded by multigene pathways, we require large library sizes for DNA sequences >5-10 kb in length, elimination of genomic hitchhiker mutations, and decoupling of diversification and screening steps. To meet these challenges, we developed Inducible Directed Evolution (IDE), which uses a temperate bacteriophage to package large plasmids and transfer them to naive cells after intracellular mutagenesis. To demonstrate IDE, we evolved a 5-gene pathway from Bacillus licheniformis that accelerates tagatose catabolism in Escherichia coli, resulting in clones with 65% shorter lag times during growth on tagatose after only two rounds of evolution. Next, we evolved a 15.4 kb, 10-gene pathway from Bifidobacterium breve UC2003 that aids E. coli's utilization of melezitose. After three rounds of IDE, we isolated evolved pathways that both reduced lag time by more than 2-fold and enabled 150% higher final optical density. Taken together, this work enhances the capacity and utility of a whole pathway directed evolution approach in E. coli.

Figure 1.

Conceptual overview of Inducible Directed Evolution (IDE). The pathway of interest is cloned into a P1 phagemid (PM) and transformed into a diversification strain containing the mutagenesis plasmid (MP) and P1 phage. MP is induced with anhydrotetracycline (+aTc) to create mutations, and then P1 lysis is induced with arabinose (+ara) to produce phage particles containing mutated P1 and PM. Phage lysate is then used to infect a screening strain to select/screen for the desired phenotype. After selection/screening, successful strains can enter another cycle of IDE.

Figure 2.

Optimizing P1 phagemid infection rate. (A) Engineered P1kc (P1kcΔcoi and P1kc::10kb) increases packaging/infection rates of phagemid compared to wild-type P1kc by >10-fold (ns P> 0.05, ***P< 0.002, Student's t test, Bonferroni correction). (B) The effect of insert size on phagemid transfer is negligible. PM-9 (9.7 kb), PM-12 (12.8 kb), PM-24 (24.0 kb) and PM-42 (42.0 kb) phagemids were packaged from the same strain (E. coli C600) containing P1kc::10kb and the same amount of phage lysate was used to infect wild-type E. coli C600 (*P< 0.05, Student's t test, Bonferroni correction).

Figure 3.

IDE allows high, tunable mutation rates. (A) Mutation rate of original MP6 induced with 20 mM arabinose, compared to aTc-MP induced with 200 ng/ml anhydrotetracycline and the base mutation rate of E. coli C600. The aTc-MP mutation rate is 6-fold lower than that of MP6 during induction, and 10-fold lower without induction. (B) Single stop codon reversion in CmR. A single stop codon was introduced to CmR (W16*) and reverted via IDE. Colonies from both induced and uninduced aTc-MP were counted after 8 and 16 hours to test mutation rate tunability with IDE (*P< 0.02, Student's t test). Three replicate cultures were used for both rifampicin assay and CmR stop codon reversion.

Figure 4.

P1 phage's broad infectivity extends IDE screening possibilities to different E. coli strains. (A) Overview of using different E. coli strains in an IDE cycle for diversification and screening steps. (B) Heat map summarizing infection/packaging rates (CFU/mL) of phage lysate produced from different E. coli strains (C600 is a lab strain, MG1655 is an industrial strain, and Nissle 1917 is a probiotic strain) and used to infect the same 3 strains.

Figure 5.

IDE allows for the selection of single variants. Co-infection of lysate produced from strains containing P1kc:10kb::kanR (kanR) with PM-AmpR (AmpR) or PM-CmR (CmR). Phage lysate produced from these two strains was used to infect WT E. coli C600 (1:1 ratio, 0.5 mL from each lysate and 1 mL resuspended cells in ePLM) and plated on LB plates containing Cm, Amp, Kan, Cm/Amp, Cm/Kan or Amp/Kan to select for cells infected with either P1kc:10kb::kanR, PM-Amp, PM-CmR or combinations of two constructs. The rates single infections are >330-fold higher than co-infection (***P< 0.0001, Student's t test). Phage lysate was produced from three biological replicates; each dot represents one biological replicate.

Figure 6.

Directed evolution of a 5.2 kb tagatose pathway. (A) Overview of evolving a tagatose pathway via IDE. (B) Comparison of growth characteristics of variants isolated from IDE and control (mutagenesis uninduced) tagatose selections. Variants from IDE and control selections were grown in tagatose minimal media and optical density was measured over time in a microplate reader. AUC (Area Under the Curve) is calculated by summing OD600 values obtained over the course of the experiment. **** P< 0.0001, Student's t test. (C). Isolated tagatose pathway variants show improved growth on tagatose minimal media after the evolved pathway was re-cloned into the wild-type backbone of the phagemid. The growth curves of the four evolved isolates were compared to the growth curves of E. coli C600 and E. coli C600 + wild-type pathway. (D) Single and double mutations from evolved pathway #3 and #4 were introduced into wild-type pathway and grown in tagatose media. three biological replicates were used for all growth curves.

Figure 7.

Directed evolution of a 15.4 kb melezitose consumption pathway. Top: Layout of a melezitose utilization operon from B. breve UCC2003. Bottom: Isolated melezitose pathway variants show improved growth on melezitose minimal media after the evolved phagemids were transformed to wildtype E. coli C600. The growth curves of the 5 evolved isolates were compared to the growth curves of E. coli C600 and E. coli C600 + wild-type pathway.