Lessons from diversity of directed evolution experiments by an analysis of 3,000 mutations

Abstract

Diversity generation by random mutagenesis is often the first key step in directed evolution experiments and screening of 1,000-2,000 clones is in most directed evolution campaigns sufficient to identify improved variants. For experimentalists important questions such as how many positions are mutated in the targeted gene and what amino acid substitutions can be expected after screening of 1,000-2,000 clones are surprisingly not answered by a statistical analysis of mutant libraries. Therefore three random mutagenesis experiments (epPCR with a low- and a high-mutation frequency and a transversion-enriched sequence saturation mutagenesis method named SeSaM-Tv P/P) were performed on the lipase BSLA and in total 3,000 mutations were analyzed to determine the diversity in random mutagenesis libraries employed in directed evolution experiments. The active fraction of the population ranged from 15% (epPCR-high), to 52% (SeSaM-Tv P/P), and 55% (epPCR-low) which correlates well with the average number of amino acid substitutions per protein (4.1, 1.6 and 1.1). In the epPCR libraries transitions were the predominant mutations (>72%), and >82% of all mutations occurred at A- or T-nts. Consecutive nucleotide (nt) mutations were obtained only with a low fraction (2.8%) under highly error-prone conditions. SeSaM-Tv P/P was enriched in transversions (43%; >1.7-fold more than epPCR libraries), and consecutive nt mutations (30.5%; 11-fold more than epPCR-high). A high fraction of wild-type BSLA protein (33%) was found in the epPCR-low mutant library compared to 2% in epPCR-high and 13% in SeSaM-Tv P/P. An average of 1.8-1.9 amino acid substitutions per residue was obtained with epPCR-low and -high compared to 2.1 via SeSaM-Tv P/P. The chemical composition of the amino acid substitutions differed, however, significantly from the two epPCR methods to SeSaM-Tv P/P.

Towards Understanding Directed Evolution: More than Half of All Amino Acid Positions Contribute to Ionic Liquid Resistance of Bacillus subtilis Lipase A

“…These results teach us a further lesson for directed enzyme evolution: random mutagenesis methods that generate diverse amino acid substitution patterns (e.g., from aliphatic to charged or to polar) increase the likelihood of finding BSLA variants with improved IL resistance, thereby minimize screening efforts. Chemically diverse mutant libraries are generated with a transversion bias or by subsequent mutations in a codon [49,56,57] and therefore transversion enriched methods should be preferred. [58] Notably most commonly used epPCR random mutagenesis methods have a strong transition bias which preserves the chemical properties.…”

Are Directed Evolution Approaches Efficient in Exploring Nature’s Potential to Stabilize a Lipase in Organic Cosolvents?

“…The investigated random mutagenesis libraries (epPCR-low, -high, and SeSaM-Tv P/P) allow only the identification of a small fraction of all obtainable beneficial substitutions. Despite the fact that these shortcomings of random mutagenesis methods are commonly known [1,27] and accepted, this study is the first to quantify this phenomenon in the example of organic cosolvents. A more suitable approach to overcome the limitation of a single random mutagenesis method could be the combination of two different methods as shown in Table 2.…”

“…A more suitable approach to overcome the limitation of a single random mutagenesis method could be the combination of two different methods as shown in Table 2. The SeSaM-Tv P/P method was optimized to generate a complementary bias to the often transition-biased epPCR-based methods and to introduce subsequent mutations (>30%), which cannot be achieved by conventional epPCR approaches [27,34]. By performing different rounds of directed evolution utilizing epPCR and SeSaM-Tv P/P in alternating order, a larger pool of beneficial substitutions (up to 16% using the epPCR/SeSaM-Tv P/P combination vs. just up to 12% using epPCR only; Table 2) could be accessed.…”

“…The generation of the three random mutagenesis libraries was described before [27]. Error-prone PCR (epPCR) was performed using two different concentrations of MnCl 2 (0.1 mM and 0.3 mM) to obtain a library with high mutagenesis frequency (epPCR-high,~4 aa per sequence) and low mutagenesis frequency (epPCR-low,~1 aa per sequence).…”

“…Error-prone PCR (epPCR) was performed using two different concentrations of MnCl 2 (0.1 mM and 0.3 mM) to obtain a library with high mutagenesis frequency (epPCR-high,~4 aa per sequence) and low mutagenesis frequency (epPCR-low,~1 aa per sequence). Additionally, a transversion-enriched Sequence Saturation Mutagenesis (SeSaM-Tv P/P) experiment was performed in accordance with a protocol described before [27,34] in order to obtain a third random mutagenesis library. Single clones of each library were randomly picked and sent for sequencing.…”