Enzyme engineering: A synthetic biology approach for more effective library generation and automated high-throughput screening

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.

Highly efficient one-step scarless protein tagging by type IIS restriction endonuclease-mediated precision cloning

“…Once these custom sticky ends join in the new vector (such as the tag clone in pBSII-KS-4B in Figure 4), the type IIS restriction enzymes can no longer separate them away, which allows digestion and ligation occur in the same tube and simplifies cloning procedures. This unique property has been exploited in plant synthetic biology for large DNA assembly or effective library generation [16,17]. In our trial on the transmembrane protein THSD1 (Figure 7), we had a satisfying cloning result (100% success rate for getting the template clone and each tag clone).…”

Darwin Assembly: fast, efficient, multi-site bespoke mutagenesis

“…Consequently, expression and functional optimization, or more radical engineering is often required to generate the desired enzymatic activity, whether boosting an existing activity or changing enzyme function altogether. Those needs have led to the flourishing of directed evolution and protein engineering and it has repeatedly proven possible to enhance a number of protein properties, including expression, folding (2), thermostability (3), substrate specificity (4)(5)(6) and catalytic efficiency (7,8). While single amino acid mutations can have profound effects, they are rarely sufficient to generate the desired function and often multiple, distal mutations are required in the protein of intereste.g.…”

“…Some methods are not effective at mutating the whole population (45,46) and/or incorporating all mutations (47,48). Methods that can introduce mutations effectively and efficiently tend to require complex and time-consuming steps, such as phagemid propagation to generate dU-containing DNA (49,50), chemical degradation steps(51), multiple PCR reactions followed by overlap extension PCR reactions to assemble the mutated gene (46,52,53) or modularization of the library into PCR-tractable libraries that can be later re-assembled via Golden Gate assembly (6). Other methods, such as TAMS (54) and OD SPM (55), are simple and effective.…”

Golden Mutagenesis: An efficient multi-sitesaturation mutagenesis approach by Golden Gate cloning with automated primer design

“…However, so far only very few examples were reported employing Golden Gate cloning for mutagenesis in the context of directed evolution approaches. 16,24,25 We have developed and optimized a hands-on protocol for the implementation of Golden Gate-based Mutagenesis (coined Golden Mutagenesis) in any laboratory focusing on rational or random protein engineering. The entire Golden Mutagenesis protocol requires solely three enzymes (BsaI, BbsI and a DNA ligase) and one to two different plasmids (one cloning and one E. coli expression plasmid)-a Golden Gate compatible pET28b based expression vector was constructed and deposited at Addgene.…”

“…This offers an efficient approach for a controlled, hierarchical mutagenesis approach that was employed in previous studies in a similar method. 24 The final gene assembly step into the compatible E. coli expression vector (pAGM22082_CRed)-either directly from the obtained PCR fragments or via intermediate subcloning of individual fragments-is performed using an E. coli BL21(DE3) expression strain, enabling the direct assessment of the protein phenotypes of the respective clones. The DE3 genotype is crucial since it includes a T7 RNA polymerase gene thus enabling T7 promoter-dependent target gene expression.…”