Directed evolution can rapidly improve the activity of chimeric assembly-line enzymes

Abstract

Nonribosomal peptides (NRPs) are produced by NRP synthetase (NRPS) enzymes that function as molecular assembly lines. The modular architecture of NRPSs suggests that a domain responsible for activating a building block could be replaced with a domain from a foreign NRPS to create a chimeric assembly line that produces a new variant of a natural NRP. However, such chimeric NRPS modules are often heavily impaired, impeding efforts to create novel NRP variants by swapping domains from different modules or organisms. Here we show that impaired chimeric NRPSs can be functionally restored by directed evolution. Using rounds of mutagenesis coupled with in vivo screens for NRP production, we rapidly isolated variants of two different chimeric NRPSs with approximately 10-fold improvements in enzyme activity and product yield, including one that produces new derivatives of the potent NRP/polyketide antibiotic andrimid. Because functional restoration in these examples required only modest library sizes (10(3) to 10(4) clones) and three or fewer rounds of screening, our approach may be widely applicable even for NRPSs from genetically challenging hosts.

Fig. 1.

Using directed evolution to improve the activity of chimeric NRPSs. A heterologous A domain is swapped into an NRPS, typically resulting in a significant loss of synthetase activity. A library of chimeric synthetase mutants is constructed in which the heterologous A domain has been diversified (for example, by mutagenic PCR). The library is subjected to an in vivo screen for production of the NRP variant. Clones showing improved production are characterized and subjected to further rounds of diversification and screening.

Fig. 2.

The enterobactin and andrimid assembly line enzymes. (a) Chemical structures of enterobactin, andrimid, N-octatrienoyl-β-phenylalanine, and andrimid derivatives. Building blocks incorporated by the wild-type NRPS are shown in red, and building blocks incorporated by evolved chimeric NRPSs are shown in blue. (b) The enterobactin NRPS. The EntF A domain and the serine it incorporates are highlighted in red. (c) The andrimid NRPS–polyketide synthase. The AdmK A domain and the valine it incorporates are shown in red.

Fig. 3.

EntF-SyrE-A1, AdmK-CytC1, and AdmK-BacA-A1 are rapidly improved by directed evolution. (a) Summary of results from the EntF-SyrE-A1 screen showing the colony sizes of biochemical activity of each clone. The colony images come from strains grown on the same plate. (b) Summary of results from the AdmK-CytC1 screen showing the zone-of-inhibition size and relative yield of andrimid for each clone. (c) Summary of results from the AdmK-BacA-A1 screen showing the zone-of-inhibition size and relative yield of andrimid (AdmK) or the isoleucine derivative of andrimid (AdmK-BacA-A1 and 5A-06) for each clone. Relative yield is defined as the yield of andrimid or the isoleucine derivative of andrimid compared with the parental AdmK-CytC1 or AdmK-BacA-A1 chimera, with the yields of the parental chimeras normalized to a value of 1.

Fig. 4.

Structural mapping of the mutations in evolved clones 1B-06, 5A-06, and 6C-06. Two views of the A domain PheA (Protein Data Bank ID code 1AMU) are shown in which a molecule of adenosine-5′-monophosphate (yellow) partially occupies the enzyme's active site. Mutations were mapped to the structure of PheA by amino acid sequence alignment. 1B-06 mutation sites are shown as red spheres, 5A-06 mutation sites are shown as blue spheres, and 6C-06 mutation sites are shown as green spheres.