  Protein variant libraries produced by site-directed mutagenesis are a useful
tool utilized by protein engineers to explore variants with potentially
improved properties, such as activity and stability. These libraries are
commonly built by selecting residue positions and alternative beneficial
mutations for each position. All possible combinations are then constructed and
screened, by incorporating degenerate codons at mutation sites. These
degenerate codons often encode additional unwanted amino acids or even STOP
codons. Our study aims to take advantage of annealing based recombination of
oligonucleotides during synthesis and utilize multiple degenerate codons per
mutation site to produce targeted protein libraries devoid of unwanted
variants. Toward this goal we created an algorithm to calculate the minimum
number of degenerate codons necessary to specify any given amino acid set, and
a dynamic programming method that uses this algorithm to optimally partition a
DNA target sequence with degeneracies into overlapping oligonucleotides, such
that the total cost of synthesis of the target mutant protein library is
minimized. Computational experiments show that, for a modest increase in DNA
synthesis costs, beneficial variant yields in produced mutant libraries are
increased by orders of magnitude, an effect particularly pronounced in large
combinatorial libraries.
