Abstract
The Golden Gate strategy entails the use of type IIS restriction enzymes, which cut outside of their recognition sequence. It enables unrestricted design of unique DNA fragments that can be readily and seamlessly recombined. Successfully employed in other synthetic biology applications, we demonstrate its advantageous use to engineer a biocatalyst. Hot-spots for mutations were individuated in three distinct regions of Candida antarctica lipase A (Cal-A), the biocatalyst chosen as a target to demonstrate the versatility of this recombination method. The three corresponding gene segments were subjected to the most appropriate method of mutagenesis (targeted or random). Their straightforward reassembly allowed combining products of different mutagenesis methods in a single round for rapid production of a series of diverse libraries, thus facilitating directed evolution. Screening to improve discrimination of short-chain versus long-chain fatty acid substrates was aided by development of a general, automated method for visual discrimination of the hydrolysis of varied substrates by whole cells.
Highlights
Effective mutagenesis strategies in enzyme engineering are often dependent on the generation of small and targeted, high-quality libraries of mutants[1]
The long chain of a C18 fatty acid substrate has been hypothesized to bind in a tunnel where PEG crystalized, and targeted NDT mutagenesis of that region had previously shown some effect on cis-trans substrate selectivity[25]
For the randomized Part 2 library, 66% of the variants retained activity: the ratio between variants active on short-chain vs. long-chain fatty acids was 39:1. These results demonstrate the suitability of the Golden Gate strategy to achieve rapid generation of readily recombined functional diversity
Summary
Effective mutagenesis strategies in enzyme engineering are often dependent on the generation of small and targeted, high-quality libraries of mutants[1]. Such ‘smart’ libraries are consistent with practical constraints imposed by the screening effort: while point-mutant libraries are readily screened, we need creative solutions to improve our capacity to explore the combinatorial complexity of sequence space. Simultaneous amino acid substitutions may have non-additive or epistatic effects (cooperative or antagonistic) on protein function[1,2,3,4]. To sample complex mutational patterns, efforts are increasingly made to maximize protein sequence diversity while keeping the library size manageable. Strategies include controlling mutational bias through the use of a reduced genetic alphabet
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