Enlarging the Amino Acid Set of Escherichia coli by Infiltration of the Valine Coding Pathway Authors V.Döring, H.D. Mootz, L.A. Nangle, T. L. Hendrickson, V. de Crécy-Lagard, P. Schimmel, P. Marlière

Supplementary Material

Directed evolution scheme. To avoid a decrease of cysteine concentration in the medium due to oxidation, the selections were arried out under strictly anaerobic conditions. The biomass of cells in cultures relative to the cysteine concentration (measured as optical density at 600 nm after 24 hours of growth at 30°C) gradually increased fourfold when the thyA strain b5366 [DthyA::erm⁺ pTS13 (bla⁺ thyA:146GUA)] was propagated by serial transfer in mineral glucose medium supplemented with 1.5 mM cysteine. A high concentration of cysteine was required, because the input population could hardly be propagated otherwise. After 16 inoculations, each at a dilution of 1/100 (a total of about 100 generations), single colonies were isolated on mineral glucose plates supplemented with thymidine (a representative of which was designated strain b5520).

One-step selection scheme. For the one-step selections, cysteine oxidation under aerobiosis and subsequent cystine precipitation from the growth medium was avoided by use of the nonoxidizable precursor S-carbamoyl-cysteine (Scc). Scc is a poor precursor of cysteine and sustains growth of a Cys-auxotrophic E. coli strain less efficiently than cysteine. However, at concentrations above 1 mM, Scc gave rise to suppression of the thymidine auxotrophy of strain b5520; in the absence of thymidine, no growth was detectable below 1 mM Scc. When b5419 cells bearing the thyA:Val146GUA allele on a high-copy plasmid were plated on mineral glucose plates supplemented with Scc (3 mM), no colonies grew after prolonged incubation at 30° C. (The experiment was designed to detect a mutant at a frequency of 10^-10.) To increase the mutation frequency, a mutS::spc⁺ (1) mutator allele was introduced in the genetic background of strain b5419 by P1 transduction, yielding strain b5432 [the mutS::spc⁺ disruption disables the mismatch repair system and leads to random transitions and frameshifts (2)]. Colonies then appeared on the same medium with a frequency of about 10^-8. No colonies were found on plates lacking Scc. A comparable frequency of Scc-suppressible clones was obtained after introduction by P1 transduction (1) of a dnaQ mutator allele (1) (to give strain b5435).

Abu misincorporation. The valine auxotroph CU505 (1) was grown in the presence of a limiting supply of valine and increasing concentrations of Abu (Fig. 1). The biomass of cells in cultures relative to the valine concentration in the medium did not change up to a 1 mM concentration of Abu. In contrast, when the valST222P allele was introduced into the chromosome (strain b5498), the yield of cells was diminished in the absence of Abu but increased up to 30% when Abu (0.2 mM) was added.

References and notes

1. mutS::spc⁺ allele (gift of F. Taddei, Hôpital Necker-Enfants, Paris); DnrdD::kan⁺ allele, laboratory collection; DdnaQ::tet allele (gift of J. Shapiro, University of Chicago, IL); CU505 [D(ilvE-ilvC)2049 leu455 galT1 IN(rrnD-rrnE)1] gift of M. Berlyn from the E. coli Genetic Stock Center (New Haven, CT). Transfer of genes and of resistance markers by P1 transduction were carried out using standard protocols (3).

2. P. Modrich, Annu. Rev. Genet. 25, 229 (1991).

3. J. H. Miller, Experiments in Molecular Genetics (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1972).

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Supplemental Figure 1. Overnight cultures of Dilv auxotrophs containing the WT valS (open circles) or the mutant valS:T222P allele (filled circles) and grown in MS glucose medium with limiting valine (0.04 mM Ile-Val. 0.3 mM Ile-Leu) were diluted 1/1, the Val concentration was adjusted to 0.02 mM and the biomass was determined by measuring the optical density at 600 nm after 24 hours of growth at 30°C.

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