microorganisms Review Inteins in Science: Evolution to Application Ananya Nanda , Sourya Subhra Nasker, Ashwaria Mehra, Sunita Panda and Sasmita Nayak * School of Biotechnology, Kalinga Institute of Industrial Technology, Bhubaneswar 754021, Odisha, India; [email protected] (A.N.); [email protected] (S.S.N.); [email protected] (A.M.); [email protected] (S.P.) * Correspondence: [email protected] Received: 12 November 2020; Accepted: 9 December 2020; Published: 16 December 2020 Abstract: Inteins are mobile genetic elements that apply standard enzymatic strategies to excise themselves post-translationally from the precursor protein via protein splicing. Since their discovery in the 1990s, recent advances in intein technology allow for them to be implemented as a modern biotechnological contrivance. Radical improvement in the structure and catalytic framework of cis- and trans-splicing inteins devised the development of engineered inteins that contribute to various efficient downstream techniques. Previous literature indicates that implementation of intein-mediated splicing has been extended to in vivo systems. Besides, the homing endonuclease domain also acts as a versatile biotechnological tool involving genetic manipulation and control of monogenic diseases. This review orients the understanding of inteins by sequentially studying the distribution and evolution pattern of intein, thereby highlighting a role in genetic mobility. Further, we include an in-depth summary of specific applications branching from protein purification using self-cleaving tags to protein modification, post-translational processing and labelling, followed by the development of intein-based biosensors. These engineered inteins offer a disruptive approach towards research avenues like biomaterial construction, metabolic engineering and synthetic biology. Therefore, this linear perspective allows for a more comprehensive understanding of intein function and its diverse applications. Keywords: inteins; splicing; evolution; application; protein engineering 1. Introduction Splicing mechanism can be broadly categorized as RNA splicing and protein splicing, two mechanisms responsible for the flow of information from a gene to its protein product to yield a functional protein whose sequence is strictly noncolinear with the gene. While group I introns self-splice at a precursor RNA level, intein splicing involves the removal of an intervening sequence at a precursor polypeptide level [1]. Initially, this intervening polypeptide sequence was termed as spacer or protein introns, currently termed as inteins (INTervening protEINS) [2]. In a radical post-translational event, inteins excise themselves precisely from a larger precursor protein by sequential cleavage of peptide bonds and concomitant ligation by peptide bond formation between the flanking amino-terminal (N-) and carboxy-terminal (C-) residues termed as exteins, resulting in the formation of an active protein product [3,4]. The intein-mediated splicing mechanism lacks the use of any exogenous cofactors or high-energy molecules [3,5]. The embodiment of intein-mediated protein splicing in the “central dogma” of molecular biology puts in an additional level of complexity to the mechanism of gene expression [6]. Most of the inteins are interrupted by a homing endonuclease domain (HED) [7–9]. However, the HED can be removed from within the intein, without entirely compromising the splicing activity [10]. Thus the presence of HEDs increases the allele frequency at a rate higher than that of Mendelian Microorganisms 2020, 8, 2004; doi:10.3390/microorganisms8122004 www.mdpi.com/journal/microorganisms Microorganisms 2020, 8, 2004 2 of 30 Microorganisms 2020, 8, x FOR PEER REVIEW 2 of 30 rates [2,11]. Homing endonucleases encoded within an intein contains the prefix “PI-” in terms of ratesintein [2,11]. nomenclature Homing endonucleases [12,13]. Conventionally, encoded within intein nomenclaturean intein contains comprises the prefix abbreviated “PI-” in namesterms of of inteinboth genus nomenclature and species [12,13]. followed Conventionally, by the name intein of the protein;nomenclature an intein comprises residing abbreviated in the GyrA proteinnames of of bothMycobacterium genus and xenopi speciesis designatedfollowed by as theMxe nameGyrA, of forthe instance protein; [ 13an, 14intein]. Mxe residingGyrA, coincidentally,in the GyrA protein is also ofthe Mycobacterium smallest known xenopi naturally is designated occurring as inteinMxe GyrA, [15]. for instance [13,14]. Mxe GyrA, coincidentally, is also theInteins smallest naturally known exist natu inrally three occurring different intein configurations [15]. (Figure1): (1) full-length inteins, where a sequence-specificInteins naturally homing exist in endonuclease three different domain configurat is embeddedions (Figure in 1): between (1) full-length the splicing inteins, (catalytic) where a sequence-specificdomains; (2) mini-inteins, homing endonuclease lacking the homing domain endonuclease is embedded domain in between and containing the splicing a contiguous(catalytic) domains;protein splicing (2) mini-inteins, domain; and lacking (3) split the inteins, homing transcribed endonuclease and translateddomain and as two containing separate a polypeptides contiguous proteineach joined splicing with domain; an extein and [4,8 (3),16 split,17]. inteins, The study transcribed of intein distribution, and translated dissemination as two separate and theirpolypeptides potential eachbiological joined functions with an are extein particularly [4,8,16,17]. fascinating The study in the of field intein of translational distribution, research. dissemination Inteins distribution and their potentialis sporadic biological in the genomes functions of are organisms particularly spanning fascinating from in archaea, the field bacteria of translational and eukaryotes research. to Inteins several distributionviral genomes is sporadic [18–20]. in The the reasongenomes for of such organisms anomalous spanning distribution from archaea, has spurred bacteria the and proposal eukaryotes for tonumerous several viral evolutionary genomes scenarios, [18–20]. includingThe reason the fo roler such of inteinsanomalous in genetic distribution mobility has and spurred as a selfish the proposalDNA [19 –for21 ].numerous Still, the evolutionary question remains scenarios, as to whyincluding inteins the persisted role of inteins for millions in genetic of years? mobility Do theyand asperform a selfish a beneficialDNA [19–21]. role inStill, the the host question or are they remains just aas selfish to why gene? inteins This persisted phenomenon for millions is puzzling of years? and Doneeds they to perform be explored a beneficial further. role in the host or are they just a selfish gene? This phenomenon is puzzling and needs to be explored further. a) N-Extein N-Intein HED C-Intein C-Extein 2 b) N-Extein Mini intein C-Extein 2 c) N-ExteinN-Intein C-Intein C-Extein 2 FigureFigure 1. InteinIntein configurations. configurations. Schematic Schematic representation representation of of various various types types of of intein: intein: ( (aa)) full full length length inteinintein with with Homing Homing endonuclease endonuclease domain domain (HED), (HED), ( (b)) mini-intein mini-intein and and ( (c)) split split intein. intein. TheThe potential potential to to exploit exploit inteins inteins for for a a practical practical purpose purpose has has led led to to the the development development of of a a diverse diverse arrayarray of of applications applications in in modern biotechnology. Inteins Inteins can can be be engineered engineered to to undergo undergo conditional proteinprotein splicing (CPS) whichwhich requiresrequires environmentalenvironmental or or molecular molecular triggers triggers like like light, light, changes changes in in pH pH or ortemperature, temperature, change change in redoxin redox state state or addition or addition of small of small molecules molecules [22–27 [22–27].]. The bias The nature bias nature of inteins of inteinstoward toward plant and plant human and human pathogens pathogens makes itmakes an attractive it an attractive tool for tool novel for drug novel development drug development [28–30]. [28–30].Development Development of engineered of engineered inteins orinteins synthetic or sy inteinnthetic systems intein systems has encouraged has encouraged efficient efficient protein proteinpurification, purification, ligation ligation and cyclization and cyclization strategies strategies [9,31,32 [9,31,32].]. Recent Recent advances advances in intein in intein research research have haveextended extended these thesein vitro in applicationvitro application to whole to organismswhole organisms [9]. Such [9]. developing Such developing applications applications suggest suggestthat inteins that areinteins becoming are becoming a mature a andmature critical and biological critical biological tool, capable tool, ofcapable widening of widening the aperture the apertureto new avenues to new ofavenues scientific of research,scientific includingresearch, enhancedincluding transgenicenhanced transgenic plants and plants novel therapeuticand novel therapeuticstrategies [9 strategies,28,33]. [9,28,33]. 2.2. Intein Intein Distribution Distribution and and Evolution Evolution TheThe first first intein intein sequence sequence was was discovered discovered 32 32 years years ago ago in in the the SaccharomycesSaccharomyces cerevisiae cerevisiae VMA1 VMA1 genegene