Systematic approaches to directed evolution of proteins have been documented since the 1970s. The ability to recruit new protein functions arises from the considerable substrate ambiguity of many proteins. The substrate ambiguity of a protein can be interpreted as the evolutionary potential that allows a protein to acquire new specificities through mutation or to regain function via mutations that differ from the original protein sequence. All organisms have evolutionarily exploited this substrate ambiguity. When exploited in a laboratory under controlled mutagenesis and selection, it enables a protein to "evolve" in desired directions. One of the most effective strategies in directed protein evolution is to gradually accumulate mutations, either sequentially or by recombination, while applying selective pressure. This is typically achieved by the generation of libraries of mutants followed by efficient screening of these libraries for targeted functions and subsequent repetition of the process using improved mutants from the previous screening. Here we review some of the successful strategies in creating protein diversity and the more recent progress in directed protein evolution in a wide range of scientific disciplines and its impacts in chemical, pharmaceutical, and agricultural sciences.

https://mmbr.asm.org/content/mmbr/69/3/373.full.pdf

Laboratory-Directed Protein Evolution

Directed evolution: selecting today's biocatalysts

https://www.cell.com/trends/biotechnology/pdf/S0167-7799(07)00186-2.pdf

Engineering microbial cell factories for biosynthesis of isoprenoid molecules: beyond lycopene

Copyright: 2012. Taylor & Francis Online. Due to copyright restrictions, only the abstract is available. For access to the full text item, please consult the publisher's website. The definitive version of the work is published in Cricital reviews in Biotechnology, Vol 33, No.4 365-378; http://dx.doi.org/10.3109/07388551.2012.716810

Directed evolution: tailoring biocatalysts for industrial applications

Methods and devices are provided for manipulating droplets on a support using surface tension properties, moving the droplets along a predetermined path and merging two droplets together enabling a number of chemical reactions. Disclosed are methods for controlling the droplets volumes. Disclosed are methods and devices for synthesizing at least one oligonucleotide having a predefined sequence. Disclosed are methods and devices for synthesizing and/or assembling at least one polynucleotide product having a predefined sequence from a plurality of different oligonucleotides having a predefined sequence. In exemplary embodiments, the methods involve synthesis and/or amplification of different oligonucleotides immobilized on a solid support, release of synthesized/amplified oligonucleotides in solution to form droplets, recognition and removal of error-containing oligonucleotides, moving or combining two droplets to allow hybridization and/or ligation between two different oligonucleotides, and further chain extension reaction following hybridization and/or ligation to hierarchically generate desired length of polynucleotide products.

Methods and Microfluidic Devices for the Manipulation of Droplets in High Fidelity Polynucleotide Assembly

Evolutionary protein engineering is now proceeding to a new stage in which novel technologies, besides the conventional point mutations, to generate a library of proteins, are required. In this context, a novel method forshuffling and rearranging DNA blocks (leading to protein libraries) is reported. A cycle of processes for producing combinatorial diversity was devised and designated Y-ligation-based block shuffling (YLBS). Methodological refinement was made by applying it to the shuffling of module-sized and amino acid-sized blocks. Running three cycles of YLBS with module-sized GFP blocks resulted in a high diversity of an eight-block shuffled library. Partial shuffling of the central four blocks of GFP was performed to obtain in-effect shuffled protein, resulting in an intact arrangement. Shuffling of amino acid monomer-sized blocks by YLBS was also performed and a diversity of more than 10 1 0 shuffled molecules was attained. The deletion problems encountered during these experiments were shown to he solved by additional measures which tame type IIS restriction enzymes. The frequency of appearance of each block was skewed but was within a permissible range. Therefore, YLBS is the first general method for generating a huge diversity of shuffled proteins, recombining domains, exons and modules with ease.

/pdf/construction-of-block-shuffled-libraries-of-dna-for-1busy1z4ej.pdf

Shinsuke Narasaki

Papers

Construction of block-shuffled libraries of DNA for evolutionary protein engineering: Y-ligation-based block shuffling