Overview

We create mutants through error prone PCR, site directed mutation and PACE (Phage-assisted continuous evolution). Among them, mutants produced by error-prone PCR and site directed mutation are screened by fluorescence,and mutants produced by PACE are screened by the device of PACE.

Strain

TyrRS specifically recognizes tyrosine and catalyzes the formation of tyrosyl-tRNA. During the Screening by Fluorescence,to avoid affecting the normal function of the original TyrRS in cells, we applied the modified TyrRS to the orthogonal translation system. This modified TyrRS catalyzes the aminoacylation of an artificial tRNA whose anticodon is UAG. Therefore, E. coli C321 ΔA muts-T7 strain was chosen as the host, in whose genome the UAG codons have been replaced by other stop codon, in order to avoid the incorporation of Tyr in UAG anticodon. The orthogonal TyrRS and modified strain enable the incorporation of noncanonical amino acids without interfering with the normal growth of E. coli.

Screening by Fluorescence

In order to select aaRS variants with potential capability of L-DOPA incorporation, sfGFP Y66-S205C containing the L-DOPA incorporation was applied as a reporter. It can emit green fluorescence (absorption peak at Ex=450 nm/Em=500 nm) with residue Y66 while emitting orange fluorescence (absorption peak at Ex=535 nm/Em=585 nm) with residue DOPA66. The intensity of orange fluorescence is positively proportional to the increase of L-DOPA incorporation. Thereby aaRS variants’ specificity and activity of binding L-DOPA could be quantified.

Through the above design, we can use the fluorescence value to represent the potential capability of L-DOPA incorporation of aaRS. We measure the fluorescence after induced expression of fluorescent protein to screen out mutant strains with high orange MFI. In general, the fluorescence intensity is positively correlated with the reproduction of the strains (the expression amount of sfGFP) and the L-DOPA incorporation efficiency. However, considering the influence of background fluorescence intensity on instrument detection, we usually consider data with low OD600 to be unreliable. Therefore, the variants with high MFI/OD600 but too low OD600 will be eliminated. The variants MFI/OD600 was higher than that of the control group, and the colony reproduction was at a reasonable level (1. 0 < OD600< 1. 4). They are considered benign mutants to bind L-DOPA strongly.

Screening by PACE

PACE is a high-throughput and time-saving strategy^1,2. During PACE, phage infection and target gene activity are coupled. Host cells are infected with phages containing target genes in a flowing lagoons. On account of the induction of MP, the mutations in the whole genome of E. coli could be significantly increased, so as to establish a mutation library for the target gene. Only phages carrying the desired variants could better trigger the expression of gⅢ in host cells, which enabled themselves to propagate normally and exist stably in the lagoon.

Based on this, we designed a specific PACE system for directed evolution of TyrRS. The system evolves TyrRS through positive and negative selections. During the positive selection, the desired TyrRS mutant can bind L-DOPA to the amber suppressor tRNA, thereby allowing the complete translation of the amber terminator-containing T7 RNAP to initiate the expression of gⅢ, which in turn enables the phage to complete its propogation normally. During the negative selection, using tyrosine instead of L-DOPA as the enzyme substrate, the undesired TyrRS mutant can bind tyrosine to tRNA, so that T7 RNAP can be translated normally, and then the gⅢ-neg can be expressed, which fails the propagation of the phages, even if gⅢ expresses normally. Thus, TyrRS mutants that catalyze the binding of tyrosine to tRNAs are washed out from the lagoon. Through the positive and negative screening, we hope to increase the affinity of TyrRS for L-DOPA and decrease the binding capacity of TyrRS for tyrosine.

Reference

1 K. M. Esvelt, J. C. Carlson, D. R. Liu, A system for the continuous directed evolution of biomolecules. Nature 472, 499-U550 (2011). https://10. 1038/nature09929

2 S. M. Miller, T. Wang, D. R. Liu, Phage-assisted continuous and non-continuous evolution. Nature Protocols 15, 4101-4127 (2020). https://10. 1038/s41596-020-00410-3