Methods for enzyme library creation: Which one will you choose?

Abstract

Enzyme engineering allows to explore sequence diversity in search for new properties. The scientific literature is populated with methods to create enzyme libraries for engineering purposes, however, choosing a suitable method for the creation of mutant libraries can be daunting, in particular for the novices. Here, we address both novices and experts: how can one enter the arena of enzyme library design and what guidelines can advanced users apply to select strategies best suited to their purpose? Section I is dedicated to the novices and presents an overview of established and standard methods for library creation, as well as available commercial solutions. The expert will discover an up-to-date tool to freshen up their repertoire (Section I) and learn of the newest methods that are likely to become a mainstay (Section II). We focus primarily on in vitro methods, presenting the advantages of each method. Our ultimate aim is to offer a selection of methods/strategies that we believe to be most useful to the enzyme engineer, whether a first-timer or a seasoned user.

piggyBac-Mediated Genomic Integration of Linear dsDNA-Based Library for Deep Mutational Scanning in Mammalian Cells

“…Second, the screening capacity of libraries constructed is considerable. As an example, the 3NNK-EGFP in-vitro ligation-PCR library constructed in two 10-cm dish-seeded HEK293T cells theoretically contains more than 8×10 5 variant members (20×20×20×100), since the 1:100 diluted library covers all eight X65-Y66-G67-pattern green fluorescence-chromophores (Figure 4), This library size is relatively large from the perspective of screening capacity [7,30,31] , especially considering its further growth potential with the expansion of transfection scale. Third, piggyBac transposon system offers a large cargocarrying capacity (over 200 kb) [22] , comparing to <200 bp insertion mediated by ssODNs in existing CRISPR/Cas9-based strategies.…”

“…Further reduction of the redundancy can rely on the other randomization schemes, e.g. the SILM strategy or 22c-trick, which use the mixtures of ordinary primers to achieve zero or near-zero redundancy [30,[36][37][38] . However, the economic cost of library construction will increase due to the larger number of primers, which should be traded off by investigators based on their own situations [38] .…”

uPIC–M: efficient and scalable preparation of clonal single mutant libraries for high-throughput protein biochemistry

“…Conversely, existing techniques for generating mutant libraries, while powerful, are typically not suited for generating large-scale user-defined clonal mutant libraries. 13 For example, error-prone PCR [14][15][16] and mutagenic oligos containing degenerate codons 17,18 allow generation of extremely large mutant libraries (10 7 -10 9 ) at relatively low cost; such libraries are ideal for selecting constructs with desired characteristics, but these mutagenesis strategies do not allow generation of a desired set of defined sequences.…”

Convergence in Determining Enzyme Functional Descriptors across Kemp Eliminase Variants

“…[16][17][18] Wild-type enzymes usually exhibit low specificity for converting non-native substrate and feeble activity for catalyzing new-to-nature reactions. Experimental strategies of enzyme engineering, such as random mutagenesis, [19][20][21] gene shuffling/recombination, 22,23 CASTing, 24,25 and directed evolution, [26][27][28][29] have been leveraged to optimize enzymes' capability for accommodating non-native substrates or catalyzing new-to-nature reactions. These strategies require extensive efforts for screening and selecting mutants to achieve desired functions.…”