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Split inteins are biological mechanisms for the operation of the spatiotemporal control of protein activities. They function through protein trans -splicing, in which their N- and C-terminal fragments are expressed contiguously with two protein halves. The subsequent self-excision upon recognition of the complimentary fragment yields a mature, complete, and functional protein. The conditional regulation of protein splicing through environmental factors or the attachment of regulatory modules can be used to determine when and where a protein will operate, providing potential novel approaches for engineering biology applications. This review will discuss current split intein applications and the mechanistic basis for novel species classification. These considerations can provide guidance in intein and extein engineering through activation strategies, in the design of spatial arrangements, and in taking advantage of unique reaction environments. This can pave the way for the future implementation of novel split intein discoveries and the selection of appropriate intein species and aid in designing novel protein engineering strategies.

Background: Large serine integrases (LSIs, derived from temperate phages) have been adapted for use in a multipart DNA assembly process in vitro, called serine integrase recombinational assembly (SIRA). The versatility, efficiency, and fidelity of SIRA is limited by lack of a sufficient number of LSIs whose activities have been characterized in vitro., Methods and Major Results: In this report, we compared the activities in vitro of 10 orthogonal LSIs to explore their suitability for multiplex SIRA reactions. We found that Bxb1, ϕR4, and TG1 integrases were the most active among the set we studied, but several others were also usable. As proof of principle, we demonstrated high-efficiency one-pot assembly of six DNA fragments (made by PCR) into a 7.5 kb plasmid that expresses the enzymes of the β-carotenoid pathway in Escherichia coli, using six different LSIs. We further showed that a combined approach using a few highly active LSIs, each acting on multiple pairs of att sites with distinct central dinucleotides, can be used to scale up "poly-part" gene assembly and editing., Conclusions and Implications: We conclude that use of multiple orthogonal integrases may be the most predictable, efficient, and programmable approach for SIRA and other in vitro applications., (© 2022 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.)

Serine integrases are emerging as core tools in synthetic biology and have applications in biotechnology and genome engineering. We have designed a split-intein serine integrase-based system with potential for regulation of site-specific recombination events at the protein level in vivo. The ϕC31 integrase was split into two extein domains, and intein sequences (Npu DnaEN and Ssp DnaEC) were attached to the two termini to be fused. Expression of these two components followed by post-translational protein trans-splicing in Escherichia coli generated a fully functional ϕC31 integrase. We showed that protein splicing is necessary for recombination activity; deletion of intein domains or mutation of key intein residues inactivated recombination. We used an invertible promoter reporter system to demonstrate a potential application of the split intein-regulated site-specific recombination system in building reversible genetic switches. We used the same split inteins to control the reconstitution of a split Integrase-Recombination Directionality Factor fusion (Integrase-RDF) that efficiently catalysed the reverse attR x attL recombination. This demonstrates the potential for split-intein regulation of the forward and reverse reactions using the integrase and the integrase-RDF fusion, respectively. The split-intein integrase is a potentially versatile, regulatable component for building synthetic genetic circuits and devices., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)