Hemanshu Mundhada

• Elucidating structure-function relationships in proteins and adapting biocatalysts to non-conventional substrates and production conditions are of high academic and economical interest. Independent from any structural information, directed evolution can steer protein properties in iterative cycles of diversity generation on the gene level and screening for improved variants on the protein level. Homogeneous distribution of mutations, consecutive nucleotide exchanges and transversion mutations are the keys to achieve chemically diverse amino acid substitutions. However, commonly used random mutagenesis methods can only generate point mutations and achieve chemically conserved amino acid substitutions due to transition biased mutational spectra or cannot give homogeneous distribution of mutations thus severely hampering the diversity. Sequence Saturation Mutagenesis (SeSaM-Tv+) is a novel random mutagenesis method which can construct transversion biased mutant library and consecutive mutations. Four steps involved for construction of mutant libraries of the gene are (i) Generation of DNA fragments by random incorporation of cleavable dNTPαS in the gene by PCR followed by cleavage and purification of the fragments, (ii) Enzymatic addition of universal base/s, (iii) Elongation of fragments (iv) Replacement of universal bases by standard nucleotides. Choice of dNTPαS used to generate pool of DNA fragments and bias of replacing universal base is responsible for mutational flexibility in SeSaM and consecutive mutations can be created by addition of more than one universal base. SeSaM-Tv+ was reported to achieve enrichment of desired transversions.