For convenience, all variants of gus-wt and gus-tr3337 produced by mutagenesis will be designated gus-wt* and gus-tr3337*, respectively. During the directed evolution process, selected gus-wt* and gus-tr3337* were sequenced to ensure that they were unique. A summary of the observed changes in the 10 variants from each generation is presented in Table 4.2 while a full list of all changes present in the 50 variants from each library is presented in Table 4.3. The sequences are grouped according their generation so that it is easy to observe the evolutionary process. The variants 1P17G2 implies that it is a first generation (1P) and was found in tray 17 and well G2. In any generation, some new changes appear – a few maybe functional, but most are likely not to be beneficial, but simply occur in the gene in addition to a beneficial mutation (Table 4.3). Changes that enhance activity to a significant (i.e. detectable) degree are retailed while the DNA shuffling tends to result in the elimination of neutral mutation. DNA shuffling also accelerated the accumulation of positive mutations. Thus, in generation 5, a number of positive changes were found in all variants – that is, the evolutionary process has converged for these mutations. This extensive analysis of the sequences has provided an interesting example of the progression of directed evolution.
In GUS-WT library, only one of the eight changes that arose in the first generation was maintained in the variant population through to generation 5. This mutation, T509A, reached saturation by generation 3. Another mutation that reached saturation in the finalist population is from generation 2, K568Q. K568Q must be highly beneficial for activity in the
61 selection conditions since it became saturating within one generation whereas T509A required two generations to achieve saturation.
Similarly, in the GUS-TR3337 library, only one of the eight mutations, S550N, appeared in the first generation and was maintained through to generation 5. Another three mutations that reached saturation in a final population were Q498, S566N and K568E that all appeared in generation 2. Among these four mutations, positions 550 and 566 were reversions to wild- type residues. The reason S550N took three generations to achieve saturation was probably due to the competition between this change and the combination of F551L, S566N and K568E changes. The S550N and F551L arose from changes that were relatively close in the nucleotide sequence so that a recombination event to bring them together would be rare.
Table 4.2 Comparison of GUS-WT and GUS-TR3337 variants over 5 generations
The 10 selected parents from each generation are summarized with the mutations in primary sequence indicated. The asterisks (*) indicate residues that were mutated back to their native residues. The hash (#) symbol is associated to mutated residues that were found in both libraries. Residues that confer enhanced thermostability are bordered in black. Shading has been used to highlight the significant differences. The number of occurrences of the mutation is presented by the numbers in superscript.
* * # # # # # # Generation 64 279 350 469 474 480 493 498 509 516 532 550 557 559 561 566 568 597 GUS-WT A E D Y Q T Q Q T M M N S G L N K P WT1 A E D Y Q T Q Q A6 M M N P2 G L N K Q WT2 A E D Y Q T Q L2 A7 M K2/ V N S G L N Q2 Q3/ L WT3 A E D Y Q T Q Q A10 V2 K N P3 G L N Q10 Q8 WT4 A E D N Q T Q Q A10 V5 M N P3 G L N Q10 Q6 WT5 E3 D3 G5 N6 Q T Q Q A10 M M N P6 G L N Q10 Q9 GUS-TR3337 A E D Y Q T R Q A M T S S S L S K P THERMO1 A E D Y K M R Q A R T G/ N2 P4 S L N K P THERMO2 A E D H3 K3 A R K A M M N3 P5 S S N2 E2 P THERMO3 A E D H2 H M R K A M M2 N5 P3 S S N7 E7 P THERMO4 A E D Y Q M8 R K10 A M T N10 S S L N10 E10 P THERMO5 A E D Y Q M8 R K10 A M T N10 S S L N10 E10 P Reversion Reversion
62
Table 4.3 Changes in GUS-WT and GUS-TR3337 sequences over 5 generations
Residues that are different to the native amino acid are shaded grey and bordered in black at the point where they first appeared. Only the unique variants selected to be parents for future generation are presented. The asterisks (*) indicate residues that were mutated back to their native residues. The hash (#) symbol is associated to mutated residues that were found in both libraries. A schematic representation of the protein domains of β-GUS is illustrated below the table. Domain I, II, III comprising amino acid residue 1-180, 181-274, 275- 603 correspond to sugar-binding domain, immunoglobulin domain and TIM barrel, respectively. A schematic representation of the actual domain size is presented at the bottom. The scale bar corresponds to the length in amino acids of β-GUS. (a) GUS-WT library (b) GUS- TR3337 library (a) # # # # # # # # # # # I II III 1 100 200 300 400 500 600 I II III
63 (b) * * * # # # # # # # # # # # I II III 1 100 200 300 400 500 600 I II III
64