Targeted gene evolution in Escherichia coli using a highly error-prone DNA polymerase I

Abstract

We present a system for random mutagenesis in Escherichia coli for the evolution of targeted genes. To increase error rates of DNA polymerase I (Pol I) replication, we introduced point mutations in three structural domains that govern Pol I fidelity. Expression of error-prone Pol I in vivo results in strong mutagenesis of a target sequence encoded in a Pol I-dependent plasmid (8.1 x 10-4 mutations per bp, an 80,000-fold increase), with a preference for plasmid relative to chromosome sequence. Mutagenesis is maximal in cultures maintained at stationary phase. Mutations are evenly distributed and show a variety of base pair substitutions, predominantly transitions. Mutagenesis extends at least 3 kb beyond the 400-500 nt reportedly synthesized by Pol I. We demonstrate that our error-prone Pol I can be used to generate enzymes with distinct properties by generating TEM-1 beta-lactamase mutants able to hydrolyze a third-generation lactam antibiotic, aztreonam. Three different mutations contribute to aztreonam resistance. Two are found in the extended-spectrum beta-lactamases most frequently identified in clinical isolates, and the third (G276R) has not been previously described. Our system of targeted mutagenesis in E. coli should have an impact on enzyme-based applications in areas such as synthetic chemistry, gene therapy, and molecular biology. Given the structural conservation between polymerases, this work should also provide a reference for altering the fidelity of other polymerases.

Fig. 1.

Two-plasmid system for in vivo mutagenesis. JS200 (polA^ts) cells were sequentially transformed with two plasmids. Plasmid I carries the polA gene under control of the tac promoter; it is a low-copy plasmid with a pSC101 origin of replication and carries a cm-resistance marker (25). Plasmid II carries the target gene placed in close proximity downstream of a pUC19 (ColE1-type) origin of replication; it is high-copy and carries a kan-resistance marker. Error-prone Pol I is expressed from plasmid I and initiates replication of plasmid II encoding the targeted gene downstream from ori; it has been reported that Pol I synthesizes the first 400–500 nt before a switch to the more accurate and processive Pol III (12). Pol III is the main replicative polymerase and is responsible for replicating the majority of chromosomal DNA. Pol I catalyzes Okazaki fragment joining and plays a role in DNA repair (2). Sequences synthesized by Pol I and Pol III are indicated in light blue and green, respectively.

Fig. 2.

D424A I709N A759R Pol I achieves efficient and preferential mutagenesis, and mutagenesis is sensitive to culture conditions. JS200 cells transformed with a plasmid expressing Pol I and a plasmid encoding the β-lactamase reporter were grown under “initial” or “optimized” conditions as described in Materials and Methods. The plot represents carbenicillin (as an indicator of plasmid mutagenesis, solid columns) or rifampin resistance (as a readout for chromosomal mutagenesis, white columns) as a function of different Pol I low-fidelity mutants. Each point was plated in triplicate, and error bars represent standard error of the mean (P < 0.05).

Fig. 4.

Distribution of mutagenesis in the target plasmid. (A) Location of secondary mutations within 650 bp of target sequence. JS200 cells expressing the D424A I709N A759R polymerase and carrying the reporter plasmid pLA230 were grown under optimized conditions for mutagenesis and plated in 50 μg/ml carbenicillin. Single carbenicillin-resistant colonies were grown as described in Materials and Methods.(B) Mutagenesis is distance-sensitive but remains significant for at least 3.7 kb. JS200 cells were transformed with a plasmid containing D424A I709N A759R Pol I and with plasmids pLA230, pLA700, pLA1600, pLA2200, pLA2800, or pLA3700 as reporters. Cultures were grown under optimized conditions for mutagenesis and plated in the presence or absence of carbenicillin. Each point represents the average of two independent cultures, each plated in duplicate.

Fig. 3.

Reporter plasmid is less abundant in cells expressing D424A I709N A759R Pol I. JS200 cells transformed with the Pol I and reporter plasmids were grown under “optimized” conditions as described in Materials and Methods. These plasmids were restricted with HindIII (with a unique site on both plasmids) and run on a 1% agarose gel. In cells expressing wild-type Pol I, pLA230 plasmid signal is on average 18.6-fold greater than that of the Pol I plasmid. In cells expressing D424A I709N A759R Pol I, the signal of the reporter plasmid is only 2.0-fold that of the Pol I plasmid.