Jeremy Minshull

Protein evolution by molecular breeding.

Natural evolution has guided the development of molecular breeding processes used in the laboratory for the rapid modification of subgenomic sequences including single genes. The most significant recent development has been the in vitro permutation of natural diversity. Homologous recombination of multiple related sequences produced high-quality libraries of chimeric sequences encoding proteins with functions that differ dramatically from any of the parents. Increasingly powerful screening methods are also being developed, allowing these libraries to be screened for novel biocatalysts.

Synthetic shuffling expands functional protein diversity by allowing amino acids to recombine independently.

We describe synthetic shuffling, an evolutionary protein engineering technology in which every amino acid from a set of parents is allowed to recombine independently of every other amino acid. With the use of degenerate oligonucleotides, synthetic shuffling provides a direct route from database sequence information to functional libraries. Physical starting genes are unnecessary, and additional design criteria such as optimal codon usage or known beneficial mutations can also be incorporated. We performed synthetic shuffling of 15 subtilisin genes and obtained active and highly chimeric enzymes with desirable combinations of properties that we did not obtain by other directed-evolution methods.

Novel enzyme activities and functional plasticity revealed by recombining highly homologous enzymes

BACKGROUNDnDirected evolution by DNA shuffling has been used to modify physical and catalytic properties of biological systems. We have shuffled two highly homologous triazine hydrolases and conducted an exploration of the substrate specificities of the resulting enzymes to acquire a better understanding of the possible distributions of novel functions in sequence space.nnnRESULTSnBoth parental enzymes and a library of 1600 variant triazine hydrolases were screened against a synthetic library of 15 triazines. The shuffled library contained enzymes with up to 150-fold greater transformation rates than either parent. It also contained enzymes that hydrolyzed five of eight triazines that were not substrates for either starting enzyme.nnnCONCLUSIONSnPermutation of nine amino acid differences resulted in a set of enzymes with surprisingly diverse patterns of reactions catalyzed. The functional richness of this small area of sequence space may aid our understanding of both natural and artificial evolution.

Molecular breeding: the natural approach to protein design.

Publisher Summary This chapter reveals the advances in protein design termed “molecular breeding,” allows protein engineers to homologously recombine multiple related genes by a process that closely mimics sexual recombination to generate functionally diverse libraries of chimeric proteins from which improved variants can be selected. Molecular breeding effects the permutation of diversity within a pool of related sequences and has proven to be an extraordinarily effective method to evolve proteins and pathways for better function. The most widely used format for molecular breeding is in vitro fragmentation and reassembly of DNA. The highly active, functionally diverse gene libraries generated by molecular breeding have extended directed evolution to a plethora of proteins for which only limited throughput screens are feasible. The chapter discusses the method for molecular breeding involves recombination of homologous genes obtained from nature, in order to permutate the proven diversity. Molecular breeding (also called DNA shuffling) was developed to mimic this essential feature of natural evolution.

Systematic Variation of Amino Acid Substitutions for Stringent Assessment of Pairwise Covariation

During protein evolution, amino acids change due to a combination of functional constraints and genetic drift. Proteins frequently contain pairs of amino acids that appear to change together (covariation). Analysis of covariation from naturally occurring sets of orthologs cannot distinguish between residue pairs retained by functional requirements of the protein and those pairs existing due to changes along a common evolutionary path. Here, we have separated the two types of covariation by independently recombining every naturally occurring amino acid variant within a set of 15 subtilisin orthologs. Our analysis shows that in this family of subtilisin orthologs, almost all possible pairwise combinations of amino acids can coexist. This suggests that amino acid covariation found in the subtilisin orthologs is almost entirely due to common ancestral origin of the changes rather than functional constraints. We conclude that naturally occurring sequence diversity can be used to identify positions that can vary independently without destroying protein function.