Engineered lasso cyclases

Abstract

The present application is related to novel engineered lasso cyclases having altered properties, including tolerance of amino acid sequences of a lasso peptide that are not tolerated by a wild¬ type or parent lasso cyclase during enzymatically catalyzed cyclization of a lasso core peptide substrate leading to formation of a properly folded lasso peptide. In certain embodiments, the novel engineered lasso cyclases have one or more of improved catalytic rates, thermal stability, non-natural amino acid tolerance, and folding of lasso peptides from peptide precursors without leader sequences. The present application also covers the use of engineered lasso cyclases for the production of novel lasso peptide variants that are difficult or impossible to produce using wild-type cyclases, compositions and methods of producing engineered lasso cyclases, and compositions and uses of novel lasso peptide variants produced by engineered lasso cyclases.

Description

ENGINEERED LASSO CYCLASES1. CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of priority from U.S. Provisional Application No. 63 / 564,911, filed March 13, 2024. The entire contents of which is hereby incorporated by reference.2. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing, which has been submitted via Patent Center. The Sequence Listing titled 200301-012002_PCT_SL.xml, which was created on March 6, 2025 and is 71,102 bytes in size, is hereby incorporated by reference in its entirety.3. FIELD

[0003] The field of invention covers novel engineered lasso cyclases having altered properties, including improved catalytic rates, thermal stability, acceptance of amino acid substitutions in lasso peptide substrates and products, non-natural amino acid tolerance, and folding of lasso peptides from peptide precursors without leader sequences. The field of invention also covers the use of engineered lasso cyclases for the production of novel lasso peptide variants that are difficult or impossible to produce using wild-type cyclases, compositions and methods of producing engineered lasso cyclases, and compositions and uses of novel lasso peptide variants produced by engineered lasso cyclases.4. BACKGROUND

[0004] Lasso peptides are kinetically trapped rotaxanes (Montalban-Lopez, M. etal. Nat. Prod. Rep. 2021, 38, 130-239) produced by bacteria and comprise highly stable, mutable structures that consist of a ring, loop, and tail and hold great promise for biomedical applications (FIG. 1) (Wang et al., Front. Bioeng. Biotechnol., 2021 9:741364. doi: 10.3389 / fbioe.2021.741364). The biosynthetic pathway involves transformation of a linear precursor peptide into a lasso peptide via two main enzymes, a lasso peptidase and a lasso cyclase (FIG. 2A and FIG. 2B). Lasso peptide biosynthesis begins with translation of a precursor peptide composed of an N- terminal leader region and a C-terminal core region (Duquesne, S., et al., Chem. BioP 2007, 14, 793-803). A lasso peptidase enzyme first cleaves the leader peptide from a precursor peptide to liberate a core peptide. The class-defining slipknot structure of lasso peptides is createdby an ATP-dependent lasso cyclase enzyme that catalyzes reaction between the N-terminus of a core peptide and an adenylated acceptor residue (Asp or Glu), thus forming an isopeptide bond and closing the macrocycle with the C-terminus trapped inside (Yan, K-P., et al., Chem- BioChem, 2012, 13, 1046-1052).

[0005] Lasso cyclases have evolved from asparagine synthetases to fold specific linear peptide substrates into the characteristic knotted configuration of a lasso peptide (Hegemann, J. D., et al., Acc. Chem. Res., 2015, 48, 1909-1919). Natural lasso cyclases have been shown to tolerate sequence variation in the ring (Hegemann, J. D., J. Am. Chem. Soc., 2013, 135, 210-222), the loop (Ducasse, R. et al. ChemBioChem, 2012, 13, 371-380), and the tail (Zong, C., et al., ACS Chem. BioL, 2016, 11, 61-68) regions of the core peptide. For example, wild-type lasso cyclases catalyzing the formation of klebsidin (KleC; Hills, E., et al., ACS Chem. BioL, 2022, 17(4), 998-1010), microcin J25 (McjC; Pavlova, O., et al., J. Biol. Chem., 2008, 283, 25589- 2559), ubonodin (UboC; Thokkadam, A. et al., ACS Cent. Sci., 2023, 9, 540-550), lariatin A (LarB; Inokoshi, J., et al., Sci. Report, 2016, 6:30375), capistruin (CapC; Knappe, T. A., et al., Chemistry & Biology, 2009, 16, 1290-1298), and stlassin (StlaC; Liu, T. et al., Chem Sci., 2021, 12(37), 12353-12364), were found to accept single-site mutations throughout the core peptide. Also, the cyclase associated with fusilassin (FusC) was shown to tolerate multi-site mutational variation in the ring region, but loop mutations were not readily accepted (Si, Y., et al., J. Am. Chem. Soc. 2021, 143, 5917-5927).

[0006] Despite the examples outlined above, the sequence space accessible using these wildtype lasso cyclases remains limited, especially when attempting to introduce multiple mutations simultaneously, or when attempting to introduce non-natural amino acids, thus hindering the development of lasso peptides as a new therapeutic modality. Numerous lasso cyclases have been shown to properly process certain substrates that deviate from the wild-type sequence, yet wild-type cyclases have evolved naturally for their substrates and they encounter challenges when attempting to fold a wide range of potentially useful non-natural substrates. Lasso cyclases dictate the types of amino acids and the amino acid sequences that will be tolerated for proper folding of a substrate into a lasso peptide product. The lack of lasso cyclase substrate tolerance and / or the inability of a lasso cyclase to accept variations in the amino acid sequence of a core peptide or lasso peptide greatly limits the diversity and utility of lasso peptides as a source of novel products. Also, lasso cyclases can exhibit slow rates, which greatly limits the ability to produce or manufacture a given lasso peptide product. Lasso cyclases are sensitive enzymes that are difficult to isolate and characterize - no structure of a lasso cyclasehas been reported, and thermostable lasso cyclases would facilitate structure elucidation and ease the use of lasso cyclases. Finally, lasso peptide biosynthesis generally requires a long precursor peptide (>40 amino acids) and the involvement of both peptidase and cyclase enzymes, thus obviating the ability to chemically synthesize and enzymatically cyclize the smaller core peptides into lasso peptide products. While one example of a lasso cyclase that properly folds a core peptide without a leader sequence or peptidase has been reported (Zyubko, T., et al., Chem. Set., 2019, 10, 9699-9707), this is atypical and evolving lasso cyclases that generally can fold just the core peptide could greatly facilitate the production of many novel lasso peptide variants. There is an important need for methods to improve the properties and functional performance of lasso cyclases, including an improvement in lasso cyclase catalytic rates, thermal stability, substrate tolerance, ability to properly fold linear core peptides, and the acceptance of a wider range of amino acid substitutions, including non-natural amino acids, in order to generate diverse lasso peptide products for use as therapeutics, biomaterials, and diagnostic agents. The present disclosure involves the advancement of novel engineered lasso cyclases with improved properties and functional performance to meet this need and provides related advantages. The present disclosure also involves novel methods for engineering lasso cyclases with such improved properties and functional performance.5. SUMMARY

[0007] Provided herein are methods of engineering lasso cyclases to improve their properties and functional performance are provided. Methods for using engineered lasso cyclases to improve the production of lasso peptide products or provide access to lasso peptide sequences that are inaccessible through the wild-type lasso cyclase are provided. Compositions of novel engineered lasso cyclases and novel lasso peptide products are provided.

[0008] Particularly, in a first aspect of the present disclosure, provided herein is an engineered lasso cyclase that tolerates amino acid sequences of a lasso peptide that are not tolerated by a wild-type or parent lasso cyclase during enzymatically catalyzed cyclization of a lasso core peptide substrate leading to formation of a properly folded lasso peptide. In certain embodiments, the engineered lasso cyclase forms a properly folded lasso peptide with at least 20% higher titer, rate, and / or yield relative to the wild-type or parent lasso cyclase. In an embodiment, the engineered lasso cyclase comprises a variant amino acid sequence of the parent lasso cyclase, wherein the parent lasso cyclase is McjC (SEQ ID NO: 18). In an embodiment, the engineered lasso cyclase comprises a mutation at amino acid position K388. In anembodiment, the engineered lasso cyclase comprises a K388A mutation. In an embodiment, the engineered lasso cyclase comprises a mutation at amino acid position K252. the engineered lasso cyclase comprises a K252A mutation. In an embodiment, the engineered lasso cyclase comprises a mutation at amino acid positions K252 and K388. In certain embodiments, the engineered lasso cyclase comprises one or more features in comparison to the wild-type or parent lasso cyclase, wherein the features comprise one or more of i) higher cyclase stability to heat; ii) increased titer, rate, and / or yield of a lasso peptide product; and iii) increased acceptance of non-natural amino acids.

[0009] In certain embodiments, the engineered lasso cyclase produces a lasso peptide comprising an amino acid sequence corresponding to any one of SEQ ID NOs: 1-8. In certain embodiments, the engineered lasso cyclase produces a lasso peptide comprising the amino acid sequence corresponding to any one of SEQ ID NOs: 1-8 at a greater titer than the wild-type or parent lasso cyclase. In certain embodiments, the engineered lasso cyclase produces a lasso peptide comprising the amino acid sequence corresponding to any one of SEQ ID NOs: 4-8 at a 2-fold to 20-fold greater titer than the wild-type or parent lasso cyclase. In certain embodiments, the engineered lasso cyclase produces a lasso peptide that is an inhibitor of an integrin or an integrin receptor. In certain embodiments, the lasso cyclase produces a lasso peptide that is an inhibitor of integrin avP6 or avP8.

[0010] In certain aspects, provided herein is a method of engineering a lasso cyclase with improved properties or functional performance in comparison to the wild-type or parent lasso cyclase, the method comprising one or more of:(i) building a computational model based on a known lasso cyclase structure or predicting a lasso cyclase structure,(ii) predicting a lasso cyclase substrate binding site and catalytic pocket,(iii) docking an ensemble of pre-folded lasso core substrates in different conformational states that simulate the transformation of substrate to lasso peptide product in the modeled cyclase substrate binding site and catalytic pocket,(iv) identifying and selecting cyclase residues that are predicted by computational modeling and conformational dynamics simulations to extend into the substrate binding and catalytic pocket,(v) identifying and selecting cyclase residues that are predicted to engage in atomic interactions (attractive, neutral, or repulsive) with the docked pre-folded lasso core substrates and / or lasso peptide product, whereby an interaction is defined as atoms of the cyclase and lasso substrate that approach within < 6Angstroms of each other, an attractive interaction is one that lowers binding free energy (e.g., hydrophobic, H-bonding, salt bridge) and a repulsive interaction is one that increases binding free energy (e.g., steric, same charge interactions),(vi) identifying and selecting cyclase residues that reside < 6 Angstroms from a cyclase ATP binding site,(vii) identifying and selecting cyclase residues with atoms that are predicted to approach atoms in residues of the predicted pre-folded or folded loop of the lasso peptide product within a distance of < 6 Angstroms,(viii) identifying and selecting cyclase residues with atoms that are predicted to approach atoms in residues of the predicted pre-folded or folded ring of the lasso peptide product within a distance of < 6 Angstroms,(ix) performing mutagenesis of the lasso cyclase, including alanine scanning, site- directed or random multi-site mutagenesis of the cyclase amino acid residues selected in steps (iv)-(viii),(x) screening of mutated cyclase variants and selecting evolved cyclases with improved properties or functional performance relative to the wild-type or parent lasso cyclase.

[0011] In certain embodiments of the methods provided herein, the mutagenesis comprises one or more of alanine scanning, site-directed and random multi-site mutagenesis.

[0012] In some embodiments, for a method provided herein, 3D computational models of a target lasso cyclase are built based on the atomic coordinates of the lasso cyclase obtained from a protein structure database or scientific literature. In some embodiments, for a method provided herein, the atomic coordinates of the lasso peptides are obtained from a protein structure database that archives structure data of biological macromolecules or from the scientific literature. In some embodiments, the protein structure database is selected from worldwide Protein Data Bank (PDB; http: / / www.rcsb.org / pdb / '), Cambridge Structure Database, Molecular Model Database of National Center for Biotechnology Information (NCBI), and Biological Magnetic Resonance Data Bank (BMRB) database. In some embodiments, the atomic coordinates of the lasso peptide are obtained by subjecting the lasso peptide to nuclear magnetic resonance (NMR) analysis, X-ray crystallography, neutron diffraction, or 3-dimensional electron microscopy (3D-EM). In some embodiments, the X-ray crystallography is serial femtosecond crystallography. In some embodiments, the 3D-EM is cryogenic electron microscopy (cryo-EM).In some embodiments, for a method provided herein, step (i) further comprises computationally modeling the 3D structure of the lasso peptide based on X-ray diffraction data, and / or cryo- EM data and / or nuclear magnetic resonance (NMR) data of the lasso cyclase and atomic coordinates of a reference protein, and wherein the lasso cyclase has at least 30% amino acid sequence similarity to the reference lasso peptide. In some embodiments, the X-ray diffraction or cryo-EM data of the lasso cyclase are obtained by subjecting a crystal of the lasso cyclase to X-ray crystallography or cryogenic electron microscopy analysis. In some embodiments, step (i) comprises: (a) obtaining atomic coordinates of the lasso peptide based on the X-ray diffraction or cryo-EM data; and (b) refining the atomic coordinates of the lasso cyclase based on the atomic coordinates of the reference protein. In certain embodiments, the method comprises building the computational model based on atomic coordinates of a lasso cyclase obtained from nuclear magnetic resonance (NMR) analysis, X-ray crystallography, neutron diffraction, or 3-dimensional electron microscopy (3D-EM). In certain embodiments, the method comprises predicting a structure of target lasso cyclase using a protein structure prediction algorithm. In certain embodiments, the protein structure prediction algorithm comprises an algorithm selected from AlphaFold, AlphaFold2, RosettaFold, and I-TASSER.

[0013] In certain embodiments, for the method provided herein, 3D structures of a target lasso cyclase are created and computationally modeled based on the amino acid sequence of the target cyclase and atomic coordinates of one or more structurally characterized reference polypeptide; and wherein the amino acid sequence of the target lasso cyclase is at least about 25- 30% identical to the amino acid sequence of the reference polypeptide. In some embodiments, for a method provided herein, computational modeling the 3D structure of the target lasso cyclase is performed using homology modeling. In some embodiments, for a method provided herein, homology modeling is performed in combination with a protein structure prediction algorithm.

[0014] In some embodiments, lasso substrate binding site and catalytic pocket prediction is performed using an algorithms, a machine learning algorithms, or deep learning algorithm.

[0015] In some embodiments, ligand (e.g., lasso core peptide substrate) binding to a protein (e.g., lasso cyclase) is defined to occur if the distance between any one of the atoms in the ligand molecule and at least one of the atoms in the amino acid residue of the protein does not exceed the sum of the radii of these two atoms plus 0.5 A, then the amino acid residue is regarded as a ligand binding residue. Atomic interactions between atoms of amino acid residuesof a ligand and a protein can be modeled and predicted at interatomic distances of < 6 Angstroms, and such atomic interactions can be attractive, neutral, or repulsive.

[0016] In some embodiments, an ensemble of conformations of pre-folded lasso core peptide substrates and / or lasso peptide products are docked into the predicted lasso substrate binding site and catalytic pockets of the lasso cyclase using a computational modeling system.

[0017] In some embodiments, an ensemble of conformations of pre-folded lasso core substrates and / or lasso peptide products are modeled and atomic interactions (attractive, neutral, or repulsive) between residues of pre-folded lasso core substrates and / or lasso peptide products and lasso cyclases are identified, using in silico docking algorithms including, but not limited to, CABS-dock, Macromodel, FlexPepDock, DynaDock, Autodock, MOE-Dock, Surfl ex-dock, Glide, AutoDock Vina, or ICM. In some embodiments, pre-folded lasso core substrates and / or lasso peptide products having improved atomic interactions (attractive, neutral, or repulsive) with a target cyclase are identified and selected using in silico docking algorithms together with conformational analysis on 3D model structures of lasso cyclases and lasso peptides using molecular dynamics simulation algorithms, including but not limited to the publicly available or commercial programs, GROMACS, AMBER, CHARMM, and Schroedinger’s MAESTRO.

[0018] In some embodiments, an ensemble of conformations of pre-folded lasso core substrates and / or lasso peptide products are modeled and atomic interactions (attractive, neutral, or repulsive) between residues of pre-folded lasso core substrates and / or lasso peptide products and lasso cyclases are analyzed and identified by using artificial intelligence, deep learning, or machine learning algorithms for in silico virtual screening and / or de novo design to define the overall lasso peptide structural topologies encompassing the loop, ring, and tail size, as well as amino acid residues that suitably fit inside and favorably interact in the lasso-binding site of a target cyclase in order to improve functional performance, which may be refined further through iterative in silico docking and molecular dynamics simulations approaches. In some embodiments, one or more different artificial intelligence, deep learning, or machine learning algorithms are used, including but not limited to, support vector machines, random forest, k- nearest neighbors, as well as neural networks with or without autoencoders, such as convolutional neural network, recurrent neural network, deep neural network, generative neural network, and generative adversarial neural network.

[0019] In some embodiments, docked models enable the identification and selection of residues in the docked ensemble of conformations of pre-folded lasso core peptide substratesand / or lasso peptide products that are predicted to be in proximity (< 6 Angstroms) to cyclase residues that extend into the catalytic pocket. In other embodiments, docked models enable the identification and selection of residues in the ensemble of conformations of docked prefolded lasso core peptide substrates and / or lasso peptide products that are predicted to be in proximity (< 6 Angstroms) to cyclase residues that surround the cyclase ATP -binding site. In another embodiment, docked models enable the identification and selection of residues in the loop of a docked ensemble of an ensemble of pre-folded lasso core peptide substrates and / or lasso peptide products that are predicted to be in proximity (< 6 Angstroms) to cyclase residues during the macrolactam ring closing process. In another embodiment, docked models enable the identification and selection of residues in the ring of a docked ensemble of pre-folded lasso core peptide substrates and / or lasso peptide products that are predicted to be in proximity (< 6 Angstroms) to cyclase residues during the macrolactam ring closing process.

[0020] In some embodiments, selected residues of lasso cyclases are evolved using alanine scanning or site directed mutagenesis guided by structure predictions and modeling. In other embodiments, lasso cyclases are evolved using random mutagenesis methods, such as phage display, mRNA display, or DNA-display, at sites selected using structure predictions and modeling. In other embodiments, libraries of mutated cyclase variants are screened for improved properties or functional performance relative to the wild-type or parent lasso cyclase. For example, improved properties could be higher cyclase stability to heat, and improved functional performance could be better cyclase tolerance to amino acid substitutions in the pre-folded lasso substrate or lasso peptide product, including the acceptance of non-natural amino acids.

[0021] In another aspect of the present disclosure, provided herein are engineered cyclases with improved properties and functional performance. In one embodiment, lasso cyclases are engineered to tolerate amino acid sequences that are not accepted by the wild-type or parent lasso cyclase. In another embodiment, the lasso cyclase is McjC (SEQ ID NO: 18). In another embodiment, McjC is engineered to produce lasso peptide sequences that serve as integrin inhibitors. In another embodiment, McjC mutants allow production of integrin inhibitors that are difficult to produce using the wild-type McjC. In another embodiment, mutant McjC cyclases are used to generate potent inhibitors of integrin receptors. In another embodiment, mutant McjC cyclases are used to generate potent inhibitors of the integrin avP8. In another embodiment, an improved McjC variant has a K252A mutation. In another embodiment, an improved McjC variant has a K388A mutation. In another embodiment, an improved McjC variants has K252A and K388A mutations.

[0022] In another aspect of the present invention, certain lasso peptide sequences are produced, which are otherwise difficult or impossible to produce and characterize by using the wild-type cyclase. In one embodiment, the lasso peptide sequence GGRGSIPEYFQRGDLQSFYG (SEQ ID NO: 6) is produced and characterized using the engineered cyclases with K252A, K338A, and K252A / K388A mutations. In another embodiment, the lasso peptide sequence GGRG- SIIEYFQRGDLQSFYG (SEQ ID NO: 8) is produced and characterized using the engineered cyclases with K252A, K338A, and K252A / K388A mutations.

[0023] In certain aspects, described herein is a recombinant nucleic acid encoding an engineered lasso cyclase of the present disclosure. In an embodiment, the recombinant nucleic acid comprises a nucleotide sequence encoding the engineered lasso cyclase operatively linked to a promoter. In certain aspects, described herein is a vector or plasmid comprising the recombinant nucleic acid. In an aspect, described herein is a non-naturally occurring microbial organism comprising the recombinant nucleic acid or the vector or plasmid.6. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The details of one or more embodiments of the subject application are set forth in the accompanying drawings and the description below. Other features, objects, and benefits of the embodiments described herein will be apparent from the description and drawings, and from the claims. All publications, patents and patent applications cited herein are hereby expressly incorporated by reference for all purposes.

[0025] The embodiments of the description described herein are not intended to be exhaustive or to limit the disclosure to the precise forms disclosed in the following drawings or detailed description. Rather, the embodiments are chosen and described so that others skilled in the art can appreciate and understand the principles and practices of the description.

[0026] FIG. 1 A schematic illustration of a lasso peptide showing the characteristic lasso (lariat) topology.

[0027] FIG. 2A. A schematic illustration of the lasso peptide biosynthesis pathway involving peptidase- and cyclase-catalyzed enzymatic reactions resulting in transformation of precursor peptide into a lasso peptide. The cyclase reaction requires ATP to fold the macrocycle and this enzyme dictates the amino acid tolerance and type of lasso peptide variants that can be formed.

[0028] FIG. 2B. A schematic illustration of the lasso peptide biosynthesis pathway involving peptidase- and cyclase-catalyzed enzymatic steps showing pre-folded core peptide substrateand ATP-dependent activation of the core peptide as an AMP ester on the carboxyl side chain of Asp or Glu residues. B and C are the lasso peptidase and lasso cyclase enzymes, respectively.

[0029] FIG. 3. A schematic illustration of a method to evolve a cyclase by randomly mutating four amino acids in proximity to the ATP -binding site of a lasso cyclase.

[0030] FIG. 4. Structure of starting scaffold MccJ25 (Lasso 1) with amino acid sequence and numbering. Amino acids are labeled via standard single letter nomenclature. Figure discloses SEQ ID NO: 1.

[0031] FIG. 5. A schematic illustration of the strategy used to generate potent lasso peptide inhibitors of integrin avP8.

[0032] FIG. 6. A model illustrating the AlphaFold2 predicted structure of McjC, the wild-type cyclase that leads to the formation of MccJ25. MccJ25 is shown docked into the cyclase active site for illustration purposes only.

[0033] FIG. 7A. AlphaFold2 predicted structure of McjC and CASTp predicted catalytic pocket circled in white. ATP and two prominent residues, K252 and K388, in the pocket are highlighted with arrows.

[0034] FIG. 7B. An illustration showing a different perspective and an expanded view of the predicted location of K252 and K388 relative to docked model of the incipient MccJ25 loop being formed.

[0035] FIG. 8. Representative ICso curves for lasso peptides (SEQ ID NOS: 1-8) inhibition of avP8.7. DETAILED DESCRIPTION

[0036] The novel features of the subject matter described herein are set forth specifically in the appended claims. A better understanding of the features and benefits of the present disclose will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the claimed embodiments are utilized. To facilitate a full understanding of the disclosure set forth herein, a number of terms are defined below.7.1. General Techniques

[0037] Techniques and procedures described or referenced herein include those that are generally well understood and / or commonly employed using conventional methodology by thoseskilled in the art, such as, for example, the widely utilized methodologies described in Sam- brook et al.. Molecular Cloning: A Laboratory Manual (4th ed. 2012); Current Protocols in Molecular Biology (Ausubel et al. eds., 2003); Therapeutic Monoclonal Antibodies: From Bench to Clinic (An ed. 2009); Monoclonal Antibodies: Methods and Protocols (Albitar ed. 2010); Antibody Engineering Vols 1 and 2 (Kontermann and Diibel eds., 2nd ed. 2010); Molecular Biology of the Cell (6th Ed., 2014); March's Advanced Organic Chemistry (6thed. 2007); Lasso Peptides, (Li, Y.; Zirah, S.; Rebuffet, S., Springer; New York, 2015); Natural Products in Medicinal Chemistry, Methods and Principles in Medicinal Chemistry (Hanessian, S., ed., Wiley-VCH; 1st edition, 2014); and Basic Principles of Drug Discovery and Development (Blass, B. Academic Press; 2ndedition, 2021).7.2. Terminology

[0038] Unless described otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. For purposes of interpreting this specification, the following description of terms will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. All patents, applications, published applications, and other publications are incorporated by reference in their entirety. In the event that any description of terms set forth conflicts with any document incorporated herein by reference, the description of term set forth below shall control.Conventions and Abbreviations

[0039] The singular terms “a,” “an,” and “the” as used herein include the plural reference unless the context clearly indicates otherwise.

[0040] The term “alpha V beta 8” or “avP8” or similar term refers to an integrin having an a chain V subunit and an P chain 8 subunit from any vertebrate source, including mammals such as primates (e.g., humans, cynomolgus monkey (cyno)), dogs, and rodents (e.g., mice and rats), unless otherwise indicated. The term avP8 encompasses “full-length,” unprocessed a chain V subunit and P chain 8 subunit, as well as any form of a chain V subunit and P chain 8 subunit or any fragment thereof that results from processing in the cell. The term avP8 also encompasses naturally occurring variants of avP8, such as isoforms, SNP variants, splice variants and allelic variants. The full-length amino acid sequence of human a chain V subunit is provided above as SEQ ID NO: 66. The full-length amino acid sequence of human P chain 8 subunit can be found at GenBank Accession No. P26012.1 and is provided below:MCGSALAFFTAAF VCLQNDRRGP ASFLWAAWVF SLVLGLGQGEDNRC AS SNAAS- CAR-CLALGPECGWCVQEDFISGGSRSERCDIVSNLISKGCSVDSIEYPSVHVIIPTENEINTQ VTPGEVSIQLRPGAEANFMLKVHPLKKYPVDLYYLVDVSASMHN-NIEKLNSVGNDLSRK-MAFFSRDFRLGFGSYVDKTVSPYISIHPERIHNQCSDYNLDCMPPHGYIHVLSLTENIT EFEKAVHRQKISGNIDTPEGGFDAMLQAAVCESHIG-WRKEAKRLLLVMTDQTSHLALDSKLAGIVVPNDGNCHLKNNVYVKSTTMEHPSLG QLSEKLIDNNINVIFAVQGKQFHWYKDLLPLLPGTIAGEIESKAANLNNLVVEAYQK- LISEV-KVQVENQVQGIYFNITAICPDGSRKPGMEGCRNVTSNDEVLFNVTVTMKKCDVTGG KNYAIIKPIGFNETAKIHIHRNCSCQCEDNRGPKGKCVDET-FLDSKCFQCDENKCHFDEDQFSSESCKSHKDQPVCSGRGVCVCGKCSCHKIKLGKV YGKYCEKDDFSCPYHHGNLCAGH-GECEAGRCQCFSGWEGDRCQCPSAAAQHCVNSKGQVCSGRGTCVCGRCECTDPRSI GRFCEHCPTCYTACKENWNCMQCLHPHNLS-QAILDQCKTSCALMEQQHYVDQTSECFSSPSYLRIFFIIFIVTFLIGLLKVLIIRQVILQW NSNKIKSSSDYRVSASKKDKLILQSVCTRAVTYRREKPEEIKMDISKLNAHETFRCNF (SEQ ID NO: 24).

[0041] Other related avP8 integrins that are also encompassed by the term avP8 include fragments, derivatives (e.g., substitution, deletion, truncations, and insertion variants), fusion polypeptides, and interspecies homologs that retain avP8 activity and / or are sufficient to generate avP8 signaling. As those skilled in the art will appreciate, an engineered lasso peptide described herein can bind to an avP8 integrin, a fragment of avP8 integrin, and / or an epitope on the avP8 integrin. An epitope may be part of a larger polypeptide, which may be part of a larger avP8 protein, which, in turn, may be part of a larger avP8 complex. An avP8 integrin may exist in a native or denatured form. An avP8 integrin described herein may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods. An avP8 integrin may include a polypeptide having the same amino acid sequence as a corresponding avP8 integrin derived from nature.

[0042] The terms “amino acid”, “amino acid residue”, and “residue” refer to naturally occurring and non-naturally occurring alpha-amino acids, as well as alpha-amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring alpha-amino acids. Naturally encoded amino acids are the 22 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, pyrrolysine and selenocysteine). Amino acid analogs or derivatives refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, z.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and a side chain R group, such as, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (such as, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. The terms “nonnatural amino acid” or “non-proteinogenic amino acid” or “unnatural amino acid” or “non- canonical” refer to alpha-amino acids that contain different side chains (different R groups) relative to those that appear in the twenty-two common or naturally occurring amino acids listed above. In addition, these terms also can refer to amino acids that are described as having D-stereochemistry, rather than L-stereochemistry of natural amino acids, despite the fact that some amino acids do occur in the D-stereochemical form in nature (e.g., D-alanine and D- serine).

[0043] The terms “binding” and “binds” refer to an interaction between molecules (e.g. , nucleic acids, oligonucleotides, proteins, polypeptides, or peptides) including, for example, the formation of a complex. Interactions can be, for example, non-covalent interactions including hydrogen bonds, ionic bonds, hydrophobic interactions, and / or van der Waals interactions. A complex can also include the binding of two or more molecules held together by covalent or non-covalent bonds, interactions, or forces. The strength of the total non-covalent interactions between a single target-binding site of a binding molecule (e.g., protein, polypeptide, or peptide) and a single target site of a target molecule is the affinity of the binding molecule for that target site. For example, the ratio of dissociation rate (koir) to association rate (kon) of a binding protein (e.g., an engineered lasso peptide) to a monovalent target site (k0ff / k0n) is the equilibrium dissociation constant KD, which is inversely related to affinity. The KD is commonly used to describe how tightly a ligand binds to a particular protein, and is the inverse of the association constant. The lower the KD value, the higher the affinity of the binding protein. The value of KD varies for different complexes of binding molecules and target molecules and depends on both kon and koff. When the binding of a binding protein results in inhibition of the target molecule, such binding can be described by an inhibition constant (Ki), which is the equilibrium dissociation constant for an enzyme inhibitor, and provides an indication of the potency of an inhibitor. In other words, the Ki also represents a KD, but more narrowly for the binding of an inhibitor to a target molecule; a binding protein whose binding reduces the activity of the target molecule. The binding equilibrium described by the Ki value depends on the kinetic mechanism of inhibition. The KD or Ki for a binding protein (e.g., an engineered lasso peptide) provided herein can be determined using any method provided herein or any other method well known to those skilled in the art. In general, the Ki value is used whenever the binding constant is measured through inhibition kinetics, while the KD value is preferred when the binding is measured more directly (e.g., by fluorescence quenching, isothermal titration calorimetry, or surface plasmon resonance). The affinity at one binding site does not always reflect the true strength of the interaction between a binding protein and the target molecule. When complex target molecule containing multiple, repeating target sites, such as a polyvalent target protein, come in contact with lasso peptides containing multiple target binding sites, the interaction of the lasso peptide with the target protein at one site will increase the probability of a reaction at a second site.

[0044] The term “binding affinity” generally refers to the strength of the sum total of noncova- lent interactions between a single binding site of a molecule (e.g., a binding protein such as anengineered lasso peptide) and its binding partner (e.g., a target protein). Binding affinity may be the intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair (e.g., an engineered lasso peptide and target protein). The affinity of a binding molecule X for its binding partner Y can generally be represented by the KD. Similarly, the affinity of an inhibiting molecule X for its binding partner Y can generally be represented by the Ki. Affinity can be measured by common methods known in the art, including those described herein. Low-affinity lasso peptides generally bind target proteins slowly and tend to dissociate readily, whereas high-affinity lasso peptides generally bind target proteins faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure. Specific illustrative embodiments include the following: the “KD” or “KD value” can be measured by assays known in the art, for example by a binding assay, including in a radioimmunoassay (RIA), a surface plasmon resonance assay as provided by Biacore®, using, for example, a Biacore®TM-2000 or a Biacore®TM-3000, or by biolayer interferometry using, for example, the Octet® QK384 system, performed with the engineered lasso peptide described herein and its target protein (e.g., integ- rin); an “on-rate” or “rate of association” or “association rate” or “kon” can also be determined with the same surface plasmon resonance or biolayer interferometry techniques described above using, for example, a Biacore®TM-2000 or a Biacore®TM-3000, or the Octet®QK384 system; and the “Ki” or “Ki value” can be measured by assays known in the art, for example, direct estimation of Ki and rate of enzyme inactivation (kinact) from time-dependent ICso values as described in Krippendorff et al., J. Biomolecular Screening, 2009, 14(8): 913-923, or an endpoint competition assay as described in Miyahisa et al., Angew Chem Int Ed Engl. 2015, 54(47): 14099-14102.

[0045] The terms “computational modeling,” “computational design,” “in silica modeling,” and “in silica design” may be used interchangeably and refer to methods of applying computational algorithms or programs to predict, simulate, analyze, and assess the structure and properties of a molecule, especially with respect to the interaction between one molecule and another, such as a lasso peptide interacting with a target protein.

[0046] The term “docking” refers to the use of computer algorithms to fit a model ligand compound structure, such as a model three-dimensional structure of a lasso peptide, into the space of a binding site of a model target protein, and adjusting the models according to chemical and physical principles to simulate and predict the energy of their interactions. In some embodiments, amino acid residue interactions between the lasso peptide and the target protein areanalyzed and energy is minimized to predict the lowest energy or highest-affinity binding. In some embodiments, the three-dimensional structure of the target protein is known or can be computationally created so that a plurality of model three-dimensional structures of the lasso peptide can be docked onto a known or predicted lasso-binding site of the target protein. In certain embodiments, interactions between amino acid residues of a plurality of lasso peptides and the target protein are analyzed and energy is minimized to predict and rank the lowest- energy or highest-affinity binding lasso peptide using scoring functions. In some embodiments, rigid docking methods are used to predict and rank the binding affinities of docked lasso peptides. In some embodiments, flexible docking methods are used to predict and rank the binding affinities of docked lasso peptides.

[0047] The term “encoding” or grammatical equivalents thereof as it is used in reference to nucleic acid molecule refers to a nucleic acid molecule in its native state or when manipulated by methods well known to those skilled in the art that can be transcribed to produce mRNA, which is then translated into a polypeptide and / or a fragment thereof. The antisense strand is the complement of such a nucleic acid molecule, and the encoding sequence can be deduced therefrom.

[0048] The terms “engineered” and “variant” as used here in when used in reference to any peptide, polypeptide, protein, nucleic acid or polynucleotide described herein refer to a sequence of amino acids or nucleic acids having at least one alteration (e.g., substitution) at an amino acid residue or nucleic acid base as compared to a parent sequence. The parent sequence of amino acids or nucleic acids can be, for example, a wild-type sequence or a homolog thereof, or a variant of a wild-type sequence or homolog thereof. Such an engineered or variant sequence of amino acids or nucleic acids is not naturally occurring. Accordingly, an engineered lasso peptide or engineered lasso refers to a non-naturally occurring analog, derivative, or variant of a naturally occurring lasso peptide, which analog, derivative or variant is also a lasso peptide itself.

[0049] The term “ICso” refers an amount, concentration, or dosage of a compound that results in 50% inhibition of a maximal response in an assay that measures such response.

[0050] The term “grafting” or a grammatical equivalent thereof as used herein means inserting a sequence (e.g., in case of a linear motif) or multiple sequences (e.g., in case of a conformational motif), such as an integrin binding motif described herein, into the sequence of a different peptide or polypeptide, such that the grafted peptide or polypeptide gains the activity of thegrafted motif (e.g., binding capability and specificity). Accordingly, a motif of a peptide or polypeptide can be “grafted” into another peptide or polypeptide, for example, by replacing a segment (e.g., in case of a linear motif) or segments (e.g., in case of a conformation motif) of the original sequence of the peptide or peptide with the sequence or sequences of the motif.

[0051] The terms “binding epitope” or “epitope” can be used interchangeably and refer to a sequence or collection of amino acids in a protein or polypeptide, such as a lasso peptide, that interacts with specific amino acids of a target biomolecule (e.g., a receptor or enzyme) leading to a binding event.

[0052] The term “evolution” or “molecular evolution” or “evolve” as used herein refers to varying the sequence of a parent protein or peptide by introducing one or more mutations in the oligonucleotide sequence encoding the amino acid sequence of the protein or peptide. In some embodiments, a parent lasso peptide is evolved by introducing one or more mutations within the oligonucleotide sequence encoding a parent lasso peptide. In another embodiment, a parent lasso peptide is evolved by introducing one or more mutations within the amino acid sequence of the parent lasso peptide. In another embodiment, a parent lasso peptide is evolved by introducing one or more mutations within the amino acid sequence of the parent lasso peptide, including the introduction of natural or non-natural amino acids.

[0053] The term “homology modeling” is a term of art and refers to a procedure that generates a previously unknown protein structure by “fitting” its sequence (target) into a known structure (template), given a certain level of sequence(at least 30%) between target and template. Homology modeling thus predicts the 3D structure of a query protein through the sequence and structure alignment with one or more template proteins of known 3D structure. Generally, the process of homology modeling involves four steps: template identification, sequence alignment, model building, and model refinement (Meier and Soding, PLoS Comput Biol. 2015; 11(10): el004343. doi: 10.1371 / journal.pcbi.1004343).

[0054] The terms “inhibition” or “inhibitor” as used herein refers to the act of or a molecule which is capable of inhibiting or reducing (including partially inhibiting or allosteric inhibition) one or more of the normal biological activities of a target molecule (e.g., an integrin). Inhibitors, for example, act by reducing or suppressing the activity of a target molecule and / or reducing or suppressing signal transduction that normally is induced by the binding of a natural ligand to the target protein. In some instances, an engineered lasso peptide described herein can be characterized an integrin inhibitor because it causes substantially complete inhibition ofone or more of the 24 human integrins described herein. In particular, an engineered lasso peptide described herein causes substantially complete inhibition of one or more integrins selected from avP6, avP8, and avpi. A molecule may also be referred to as a “partial inhibitor,” which refers to a molecule which can induce a partial response for example, by partially reducing or suppressing the activity of a target molecule and / or partially reducing or suppressing signal transduction. In some instances, a partial inhibitor mimics the spatial arrangement, electronic properties, or some other physicochemical and / or biological property of the inhibitor. In some instances, in the presence of elevated levels of an inhibitor, a partial inhibitor competes with the inhibitor for occupancy of the target molecule and provides a reduction in efficacy, relative to the inhibitor alone. Inhibition may be partial (such as, 1%, 2%, 5%, 10%, 20%, 25%, 50%, 75%, 90%, 95%, 99%) or complete (z.e., 100%) inhibition.

[0055] The terms “isolated,” “isolate,” and “isolating,” or grammatical equivalent thereof, when used in reference to a nucleic acid, protein, polypeptide, peptide, or cell, refer to a nucleic acid, protein, polypeptide, peptide, or cell that is substantially free of at least one component relative to the referenced nucleic acid, protein, polypeptide, peptide, or cell is found in nature or in its current environment. The term includes a nucleic acid, protein, polypeptide, peptide or cell that is removed from some or all components as it is found in its natural environment. Therefore, an isolated nucleic acid, protein, polypeptide, peptide, or cell is partly or completely separated from other substances as it is found in nature or as it is grown, stored or subsisted in non-naturally occurring environments (e.g., laboratories). Specific examples of isolated nucleic acid, protein, polypeptide, peptide, or cell include a partially pure nucleic acid, protein, polypeptide, peptide, or cell, a substantially pure nucleic acid, protein, polypeptide, peptide, or cell, a cell cultured in a medium that is non-naturally occurring, a protein, polypeptide, or peptide purified from other components and substances present their natural environment, including other proteins, polypeptides, or peptides, or an isolated nucleic acid that is substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence. As another example, an isolated nucleic acid can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. A substantially pure molecule can include isolated forms of the molecule.

[0056] The terms “lasso core peptide” and “core peptide” refer to the peptide or the peptide segment of the precursor peptide that is processed into or otherwise forms an engineered lassopeptide having the lariat-like topology. As used herein, a core peptide can have the same amino acid sequence as an engineered lasso peptide, but it has not matured to have the lariat-like topology of an engineered lasso peptide. Core peptides can have different lengths of amino acid sequences. For example, the core peptide of the engineered lasso peptides described herein are typically 15 to about 24 amino acids long, but other core peptides can be, for example, about 12 amino acids long to as many as about 65 amino acids long. The lasso core peptide serves as the “substrate” that is converted into a lasso peptide by a lasso cyclase.

[0057] The terms “lasso cyclase” or “cyclase” are used interchangeably herein and refer to the enzyme capable of catalyzing cyclization of the ring portion of a lasso core peptide leading to formation of a lasso peptide (e.g., a lasso peptide variant).

[0058] The term “lasso peptidase” as used herein refers to the enzyme capable of catalyzing the removal of the leader sequence from a lasso precursor peptide to produce a lasso core peptide.

[0059] The terms “lasso peptide” and “lasso” and “lasso peptide product” are used interchangeably herein, and are used to refer to a class of peptide or polypeptide having the general lariatlike topology as exemplified in FIG. 1. As shown in the figure, the lariat-like topology can be generally divided into a ring portion, a loop portion, and a tail portion. Particularly, a region on one end of the peptide forms the ring around the tail on the other end of the peptide, the tail is threaded through the ring, and a middle loop portion connects the ring and the tail, together forming the lariat-like topology. Particularly, the amino acid residues that are joined together to form the ring are herein referred to as the “ring-forming amino acids.” Ring-forming amino acids can be located at the N-terminus or C-terminus of the lasso peptide (“terminal ring-forming amino acid”) and in the middle (but not necessarily the center) of a lasso peptide (“internal ring-forming amino acid”). Internal ring-forming amino acids are typically an aspartic acid (D) or glutamic acid (E) residue located in the middle the lasso peptide. An engineered lasso peptide can be referred to here as being “cyclized” when such a lariat-like topology is formed by the engineered lasso peptide. For example, a lasso peptide can be described as being Gl- D9 cyclized when referring to a lasso peptide, which means that the lasso peptide has a N- terminal ring-forming amino acid of a glycine residue (Gl) and an internal ring-forming amino acid of an aspartate residue at position 9 (D9), where the amino group of Gl and the carboxyl group of D9 form an isopeptide bond, thus forming the ring portion of the lasso peptide. The fragment of a lasso peptide between and including the two ring-forming amino acid residues is the ring portion. The fragment of a lasso peptide between the internal ring-forming amino acidand where the peptide threaded through the plane of the ring is the loop portion. The remaining fragment of a lasso peptide starting from where the peptide is threaded through the plane of the ring is the tail portion. Determining the position of a given residue within a lasso peptide is based on counting from the N-terminal residue of the linear core peptide as the first amino acid (position 1). The boundary between the ring and the loop portion of lasso peptide is determined by the formation of an isopeptide bond between the ring-forming amino acids. The boundary between the loop and tail of a lasso peptide can vary, but is generally dependent upon the location of the locking residues and is typically determined based on the 3D structure of the lasso peptide. In addition to the lariat-like topology, additional topological features of a lasso peptide can further include intra-peptide disulfide bonding, such as disulfide bond(s) between the tail and the ring, between the ring and the loop, and / or between different locations within the loop or tail. A lasso peptide can include both naturally-existing peptides and engineered peptides that have the lariat-like topology as described herein.

[0060] The term “lasso peptide biosynthesis component” as used herein refers to a protein including one or more of (i) a lasso peptidase, (ii) a lasso cyclase, and (iii) RRE. In some instances, the lasso peptidase and RRE proteins are fused, while in others, they operate as separate proteins. An exemplary process of lasso peptide production using lasso peptide biosynthesis components from a lasso precursor peptide is depicted in FIG. 2A and FIG. 2B. An additional lasso peptide biosynthesis component can be a transporter that is specific for secreting lasso peptides outside the cell.

[0061] The terms “lasso peptide variant” or “engineered lasso peptide” are used herein interchangeably and refer to a derivative of a natural lasso peptide that has been modified or changed relative to its original structure or atomic composition. The engineered lasso peptide can (i) have at least one amino acid alteration (e.g., substitution(s), insertion(s) or deletion(s)) as compared to the sequence of a lasso peptide; (ii) have at least one modification to the amino acids as compared to a reference lasso peptide, wherein such modifications include, but are not limited to, acylation, biotinylation, O-methylation, N-methylation, amidation, glycosylation, pegylation, esterification, halogenation, amination, hydroxylation, dehydrogenation, prenylation, lipidoylation, heterocyclization, phosphorylation; (iii) have at least one unnatural amino acid(s) as compared to the sequence of a lasso peptide; (iv) have at least one different isotope(s) as compared to the lasso peptide molecule; or any combination of (i) to (iv). An engineered lasso peptide can be a conjugate or fusion made of a lasso peptide or an engineered lasso peptide and one or more additional molecule(s). In some instances, the additional molecule canbe another peptide or protein, including but not limited a lasso peptide and a cell surface receptor or an antibody or an antibody fragment. In some instances, the additional molecule can be a non-peptidic molecule, such as a drug molecule, a fatty acid or lipid molecule, an isoprenoid molecule, or an oligonucleotide molecule. In some instances, the engineered lasso peptide retain the same general lasso topology as shown in FIG. 1. In some instances, production of an engineered lasso peptide may occur by introducing a modification into the gene of a lasso precursor or core peptide, followed by transcription and translation and cyclization using cell- free or cell-based methods, as described herein, leading to an engineered lasso peptide containing that modification. In an alternative aspect, production of an engineered lasso peptide may occur by introducing a modification into a lasso precursor or core peptide, followed by cyclization of each using cell-free or cell-based methods, as described herein, leading to an engineered lasso peptide containing that modification. In another aspect, production of an engineered lasso peptide may occur by introducing a modification into a pre-formed lasso peptide, leading to a lasso peptide containing that modification. In another aspect, engineered lasso peptides are designed using structural information and in silico modeling algorithms, including docking and molecular dynamics algorithms, and such engineered lasso peptide may be produced by cell- free or cell-based methods. In another aspect, engineered lasso peptide are designed de novo using computer algorithms, including artificial intelligence, machine learning, deep learning, neural nets, etc., and such engineered lasso peptides may be produced by cell-free or cell-based methods.

[0062] An engineered lasso peptide that specifically or preferentially binds to a target protein (e.g., an integrin, or a specific integrin like avP8) can be identified, for example, by immunoassays (e.g., ELISA, fluorescent immunosorbent assay, chemiluminescence immune assay, radioimmunoassay (RIA), enzyme multiplied immunoassay, solid phase radioimmunoassay (SPRIA), a surface plasmon resonance (SPR) assay (e.g., Biacore®), a fluorescence polarization assay, a fluorescence resonance energy transfer (FRET) assay, Dot-blot assay, fluorescence activated cell sorting (FACS) assay, or other techniques known to those of skill in the art. Typically, a specific or selective reaction will be at least twice background signal or noise and can be more than 10 times background.

[0063] The terms “lasso core peptide substrate” or “lasso core substrate” or “lasso substrate” can be used interchangeably and refer to the peptide or the peptide segment of the precursor peptide that is processed by a lasso cyclase into a lasso peptide having the lariat-like topology. As used herein, the enzyme capable of catalyzing cyclization of the ring portion of a lasso corepeptide is referred to as the “lasso cyclase.” As used herein, a lasso core peptide may have the same amino acid sequence as a lasso peptide, but has not matured to have the lariat-like topology of a lasso peptide. The terms “pre-folded lasso substrate” or “pre-folded lasso core substrate” or “pre-folded core peptide” or “pre-folded core peptide intermediate” can be used interchangeably and refer to the predicted intermediate folded structure of the core peptide that is transformed into the lasso peptide product through the action of the lasso cyclase (FIG. 2B). The terms “activated core peptide” or “activated core peptide intermediate” refer to a prefolded core peptide that is adenylated on the carboxyl side chain of a Glu or Asp reside at one of the positions 7-10 of the core peptide In various embodiments, core peptides, lasso substrates, pre-folded lasso substrates, and activated core peptides can have different lengths of amino acid sequences. In various embodiments, lasso substrates, pre-folded lasso substrates, and activated core peptides may have the same amino acid sequence as a lasso peptide, but have not matured to have the lariat-like topology of a lasso peptide.

[0064] The terms “lasso precursor peptide” or “precursor peptide” as used herein refer to a precursor that is processed into or otherwise forms a lasso peptide. A lasso precursor peptide can include at least one engineered lasso core peptide portion. A lasso precursor peptide can also include one or more amino acid residues or amino acid fragments that do not belong to an engineered lasso core peptide, such as a leader sequence that facilitates recognition of the lasso precursor peptide by one or more lasso processing enzymes.

[0065] The terms “lasso peptide analog” or “lasso peptide variant” are used herein interchangeably and refer to a derivative of a natural lasso peptide that has been modified or changed relative to its original structure or atomic composition. In various embodiments, the lasso peptide analog can (i) have at least one amino acid substitution(s), insertion(s) or deletion(s) as compared to the sequence of a lasso peptide; (ii) have at least one different modification(s) to the amino acids as compared to a lasso peptide, such modifications include but are not limited to acylation, biotinylation, O-methylation, N-methylation, amidation, glycosylation, pegyla- tion, esterification, halogenation, amination, hydroxylation, dehydrogenation, prenylation, lip- idoylation, heterocyclization, phosphorylation; (iii) have at least one unnatural amino acid(s) as compared to the sequence of a lasso peptide; (iv) have at least one different isotope(s) as compared to the lasso peptide molecule; or any combination of (i) to (iv). As used herein, the term of “lasso peptide analog” also includes a conjugate or fusion made of a lasso peptide or a lasso peptide analog and one or more additional molecule(s). In some embodiments, the additional molecule can be another peptide or protein, including but not limited a lasso peptide anda cell surface receptor or an antibody or an antibody fragment. In some embodiments, the additional molecule can be a non-peptidic molecule, such as a drug molecule, a fatty acid or lipid molecule, an isoprenoid molecule, or an oligonucleotide molecule. In some embodiments, the lasso peptide analogs retain the same general lasso topology as shown in FIG. 1. In some embodiments, production of a lasso peptide analog may occur by introducing a modification into the gene of a lasso precursor or core peptide, followed by transcription and translation and cyclization using cell-free or cell -based methods, as described herein, leading to a lasso peptide containing that modification. In an alternative embodiment, production of a lasso peptide analog may occur by introducing a modification into a lasso precursor or core peptide, followed by cyclization of each using cell-free or cell-based methods, as described herein, leading to a lasso peptide containing that modification. In another embodiment, production of a lasso peptide analog may occur by introducing a modification into a pre-formed lasso peptide, leading to a lasso peptide containing that modification. In another embodiment, lasso peptide analogs are designed using structural information and in silico modeling algorithms, including docking and performing conformational analysis on 3D model structures using molecular dynamics simulation algorithms to obtain conformational states of 3D model structure of lasso peptides, and such “designed” lasso peptides may be produced by cell-free or cell-based methods. In another embodiment, lasso peptide analogs are designed de novo using computer algorithms, including artificial intelligence, machine learning, deep learning, neural nets, etc., and such “designed” lasso peptides may be produced by cell-free or cell-based methods.

[0066] The term “lasso peptide library” as used herein refers to a collection of at least two lasso peptides or lasso peptide analogs, or combinations thereof, which may be pooled together as a mixture or kept separated from one another. In some embodiments, the lasso peptide library is kept in vitro, such as in tubes or wells. In some embodiments, the lasso peptide library may be created by biosynthesis of at least two lasso peptides or lasso peptide variants using a cell-free system. In some embodiments, the lasso peptide library may be created by biosynthesis of at least two lasso peptides or lasso peptide variants using a cell -based system. In some embodiments, the lasso peptides or lasso peptide variants of the library may be mixed with one or more component of the cell-free or cell-based systems. In other embodiments, the lasso peptides or lasso peptide variants may be purified from the cell-free or cell-based systems. In some embodiments, the lasso peptides or lasso peptide variants may be partially purified. In some embodiments, the lasso peptides or lasso peptide variants may be substantially purified. In some embodiments, the lasso peptides may be isolated. In some embodiments, the lasso peptidelibrary may be created by isolating at least two lasso peptides from their natural environment. In some embodiments, the lasso peptides may be partially isolated. In some embodiments, the lasso peptides may be substantially isolated.

[0067] The term “leader sequence” as used in reference to a lasso peptide, such as a lasso precursor peptide, refers to an amino acid sequence that facilitates recognition and processing by the lasso peptide processing enzymes described herein to form a cyclized lasso peptide. The leader sequence may determine substrate specificity of the processing enzymes. Accordingly, a lasso core peptide (e.g., an engineered lasso peptide) having a leader sequence can be referred to as a lasso precursor peptide.

[0068] The term “maximal percent inhibition” as used herein refers to the maximal level of inhibition achievable based a dose-response curve in an assay that measures such a response.

[0069] The term “maximal percent reduction” as used herein refers to the maximal level of reduction achievable based a dose-response curve in an assay that measures such a response.

[0070] The terms “microbial,” “microbial organism” and “microorganism” as used herein refer to any organism that exists as a microscopic cell that is included within the domains of archaea, bacteria or eukarya. Therefore, the terms encompass prokaryotic or eukaryotic cells or organisms having a microscopic size and includes bacteria, archaea and eubacteria of all species as well as eukaryotic microorganisms such as yeast and fungi. The terms also include cells of any species that can be cultured for the production of a biochemical (e.g., a lasso peptide, including an engineered lasso peptide). A cell, such as a microbial organism, that produces an engineered lasso peptide of the present disclosure can include a bacterial and archaea host cells into which nucleic acids encoding the lasso peptide component have been introduced. Suitable host cells are disclosed below.

[0071] The terms “modulating” and “modulate” as used herein refer to an effect of altering a biological activity (i.e. increasing or decreasing the activity), especially a biological activity associated with a particular biomolecule such as a, enzyme or cell surface receptor. For example, an inhibitor of a particular biomolecule modulates the activity of that biomolecule, e.g., an enzyme, by decreasing the activity of the biomolecule, such as an enzyme. Such activity is typically indicated in terms of an inhibitory concentration (ICso) of the compound for an inhibitor with respect to, for example, an enzyme or a cell surface receptor.

[0072] As used herein the general terms “molecular modeling” and “computational modeling” are used interchangeably and are terms of art referring to the use of a computer algorithm togenerate a predicted structural model of a molecule, including a protein or peptide, or a set of interacting molecules, such as a lasso peptide and a target protein, including a lasso cyclase. A structural model generated as such is herein referred to as a “model structure,” such as a three- dimensional (3D) model structure. Molecul r modeling can be performed with a collection of computer-based techniques for deriving, representing, simulating, predicting, and manipulating the structures and behaviors of molecules, as well as reactions and interactions between molecules, and those properties that are dependent on these three-dimensional atomic structures. Molecular modeling implements molecular mechanics calculations based on equilibrium bond lengths, bond angles, partial charge values, force constants, and van der Waals parameters, which collectively are referred to as force fields. Deviations from these equilibrium force field functions, along with non-bonded van der Waals and electrostatic interactions, will increase or decrease the energy of a system. Energy minimization and optimization algorithms are used to find the lowest energy arrangements of interacting atoms or molecules that are defined by force fields.

[0073] The terms “motif’ and “epitope” and “binding motif’ and “binding epitope” are used interchangeably herein to mean the set of distinct amino acid residues on a binding molecule (e.g., a lasso peptide) that allows a binding interaction to occur between the binding molecule and a target molecule (e.g., a target protein). A binding motif can be linear or conformational, based on the structure and interaction with the binding target. A conformational motif is formed by the 3D conformation adopted by the interaction of discontinuous segments of amino acid residue(s). In contrast, a linear motif is formed by the interaction of contiguous amino acid residues. The ability of a linear motif to bind a target molecule may not be determined solely by the primary structure of the involved amino acids. Residues that flank such amino acid residues, as well as more distant amino acid residues of the binding molecule can affect the ability of the linear motif to adopt the motif s 3D conformation required for activity. For example, other adjacent or proximal residues can be used to enhance the binding interactions of a binding motif. Accordingly, “expanded motifs” can be created by adding amino acids to an originally defined minimal motif like RGD, such as RGDX1X2, which is an example of an expanded motif of RGD, wherein Xi and X2 are amino acid residues that have been shown to enhance the binding affinity and / or selectivity of molecules to certain target integrins, relative to RGD alone.

[0074] The terms “naturally occurring,” “natural,” and “native” when used in connection with naturally occurring biological materials, such as nucleic acid molecules, oligonucleotides,amino acids, polypeptides, peptides, metabolites, small molecule natural products, host cells, and the like, refer to materials that are found in or isolated directly from nature and are not changed or manipulated by humans.

[0075] The terms “non-naturally occurring,” “non-natural,” “unnatural” and “non-native” as used herein refer to a material, substance, molecule, cell, nucleic acid, oligonucleotide, nucleotide, enzyme, protein, polypeptide, peptide, or amino acid that is not known to exist or is not found in Nature or that has been structurally modified and / or synthesized by humans. Such terms when used in reference to a microbial organism, cell extract, or nucleic acid of the disclosure mean that the microbial organism, cell extract, or nucleic acid has at least one genetic alteration not normally found in a naturally occurring strain or a naturally occurring nucleic acid of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, for example, introduction of expressible oligonucleotides or nucleic acids encoding polypeptides (e.g., an engineer lasso peptide), nucleic acid additions, substitutions, or deletions and / or other functional disruption of the microbial organism’s genetic material. Such alterations include, for example, nucleotide changes, additions, substitutions or deletions in the genomic coding regions and functional fragments thereof, used for heterologous, homologous or both heterologous and homologous expression of polypeptides. Additional alterations include, for example, nucleotide changes, additions, substitutions or deletions in the genomic non-coding and / or regulatory regions in which the modifications alter expression of a gene or operon. Such terms when used in reference to a protein, polypeptide, or peptide are used to refer to a protein, polypeptide, or peptide having amino acids that are introduced into the amino acid sequence of the protein, polypeptide, or peptide to modify the properties of the polypeptide.

[0076] The terms “oligonucleotide” and “nucleic acid” refer to oligomers of deoxy ribonucleotides (e.g., DNA) or ribonucleotides (e.g., RNA) and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless specifically limited otherwise, the term also refers to oligonucleotide analogs including PNA (peptidonucleic acid), analogs of DNA used in antisense technology (phosphorothioates, phos- phoroamidates, and the like). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including but not limited to, degenerate codon substitutions) and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer, M.A., et aL, Nucleic Acid Res., 1991, 19, 5081-1585; Ohtsuka, E. et al., J. Biol. Chem., 1985, 260, 2605-2608; and Rossolini, G.M., et al., Mol. Cell. Probes, 1994, 8, 91-98). “Oligonucleotide,” as used herein, refers to short, generally single-stranded, synthetic polynucleotides that are generally, but not necessarily, fewer than about 200 nucleotides in length. The terms “oligonucleotide” and “polynucleotide” are not mutually exclusive. The description above for oligonucleotides is equally and fully applicable to polynucleotides. Unless specified otherwise, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5’ end; the left-hand direction of doublestranded polynucleotide sequences is referred to as the 5’ direction. The direction of 5’ to 3’ addition of nascent RNA transcripts is referred to as the transcription direction; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 5’ to the 5’ end of the RNA transcript are referred to as “upstream sequences”; sequence regions on the DNA strand having the same sequence as the RNA transcript that are 3 ’ to the 3 ’ end of the RNA transcript are referred to as “downstream sequences.”

[0077] The terms “parent scaffold peptide,” “parent scaffold,” and “scaffold” as used herein are used interchangeably and mean the lasso peptide from which an engineered lasso peptide was designed from as a starting peptide into which the integrin binding motif was introduced to and, in some instances, further engineered as described herein.

[0078] The term “peptide” as used herein refers to a polymer chain containing between two and fifty (2-50) amino acid residues. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-natu- rally occurring amino acid, e.g., an amino acid analog or non-natural amino acid.

[0079] The terms “polypeptide” and “protein” are used interchangeably herein to refer to a polymer of greater than about fifty (50) amino acid residues. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-naturally occurring amino acid, e.g., an amino acid analog.

[0080] The terms “post-translationally modified peptide (RiPP) recognition element” or “RRE” refer to a facilitator protein element encoded in some lasso peptide biosynthetic gene clusters that facilitates the recognition of and binding to the leader sequence by the lasso peptidase during biosynthesis of the lasso peptide.

[0081] The terms “preferential binding” and “preferentially binds to” when used in reference to a particular polypeptide or peptide (an engineered lasso peptide) on a particular target molecule (e.g., integrin avP8) with respect to a reference molecule (e.g., integrin avP3) refer to binding of the target molecule that is measurably higher than binding of the reference molecule, while the reference molecule may or may not also bind to the engineered lasso peptide. For example, an engineered lasso peptide described herein can preferentially binds to avP8 over avP3. Preferential binding can be determined, for example, by determining the binding affinity for the target molecule and the reference molecule. For example, an engineered lasso peptide that preferentially binds to a target molecule over a reference molecule can bind to the target molecule with a KD less than the KD exhibited relative to the reference molecule.

[0082] The term “promoter” as used herein in reference to a nucleic acid encoding a protein, polypeptide or peptide refers to a nucleotide sequence where transcription of a linked open reading frame (e.g., a nucleotide sequence encoding an engineered lasso peptide) by an RNA polymerase begins. A promoter sequence can be located directly upstream or at the 5' end of the transcription initiation site. RNA polymerase and the necessary transcription factors bind to a promoter sequence and initiate transcription. Promoter sequences define the direction of transcription and indicate which DNA strand will be transcribed, i.e. the sense strand.

[0083] The term “protein structure prediction” refers to the use of algorithms for the prediction of tertiary protein structure based on primary sequence data, conformational modeling, and energy minimization. Numerous advanced algorithms have been developed for accurate protein structure prediction, including trRosetta (Yang, J., et al., Improved protein structure prediction using predicted inter-residue orientations. Proc. Nat. Acad. Sci., USA. 2020, 117(3), 1496-1503), AlphaFold (Senior, A.W., et al., Improved protein structure prediction using potentials from deep learning. Nature, 2020, 577, 706-710) and AlphaFold 2 (Skolnick et al., AlphaFold 2: Why It Works and Its Implications for Understanding the Relationships of Protein Sequence, Structure, and Function. J Chem Inf Model., 2021 Oct 25;6I(10):4827-4831). Protein structure prediction algorithms may be used alone or in combination with homology modeling to create accurate 3D computational models of target proteins.

[0084] The term “recombinant” as used herein with respect to a nucleic acid, such as a nucleic acid having a gene that encodes a protein or polypeptide (e.g., an engineered lasso peptide described herein), refers to: a nucleic acid that has been artificially supplied to a biological system; a nucleic acid that has been modified within a biological system, or a nucleic acid whose expression or regulation has been manipulated within a biological system. Therecombinant nucleic acid can be supplied to the biological system, for example, by introduction of the nucleic acid into genetic material of a microbial organism, such as by integration into a microbial organism chromosome, or as non-chromosomal genetic material such as a plasmid. A recombinant nucleic acid that is introduced into or expressed in a microbial organism can be a nucleic acid that comes from a different organism or species from the microbial organism, or can be a synthetic nucleic acid, or can be a nucleic acid that is also endogenously expressed in the same organism or species as the microbial organism. A recombinant nucleic acid that is also endogenously expressed in the same organism or species as the microbial organism can be considered heterologous if: the sequence of the recombinant nucleic acid is modified relative to the endogenously expressed sequence, the sequence of a regulatory region such as a promoter that controls expression of the nucleic acid is modified relative to the regulatory region of the endogenously expressed sequence, the nucleic acid is expressed in an alternate location in the genome of the microbial organism relative to the endogenously expressed sequence, the nucleic acid is expressed in a different copy number in the microbial organism relative to the endogenously expressed sequence, and / or the nucleic acid is expressed as non-chromosomal genetic material such as a plasmid in the microbial organism.

[0085] The terms “selective inhibition of’ and “selectively inhibits” as used herein with regard to inhibition of a target molecule by an engineered lasso peptide refer to inhibition of the target molecule activity is measurably stronger than inhibition of a reference molecule activity. For example, in some instances, an engineered lasso peptide selectively inhibits integrin avP8 over avP3 and avP5. Selective inhibition can be determined, for example, by determining the IC50 value. For example, an engineered lasso peptide that selectively inhibits a target protein can exhibit an IC50 value for the target protein that is less than the IC50 exhibited for a second target protein. In some instances, the engineered lasso peptide selectively inhibits a target protein with an IC50 for the target protein that is less than half of the IC50 exhibited for a second target protein. In some instances, the lasso peptide selectively inhibits a target protein with an IC50 for the target protein that is about 75%, about 50%, about 25%, about 10%, about 5%, about 2.5%, or about 1% of the IC50 exhibited for a second target protein. In some instances, the ratio between the IC50 exhibited by the engineered lasso peptide with respect to the second target protein and the IC50 exhibited with respect to the target protein is at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 100 fold, at least 500 fold, at least 103fold, at least 104fold, or at least 105fold.

[0086] The terms “specific binding,” “specifically binds to,” and “is specific for” when used in reference to a particular molecule (e.g., protein, polypeptide, or peptide, such as an engineered lasso peptide) refer to binding that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of nonlabeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target. The terms also include binding where a molecule (e.g., protein, polypeptide, or peptide) binds to a particular protein or fragment of a particular protein without substantially binding to any other protein or protein fragment. Accordingly, an engineered lasso peptide described herein can be described as specifically binding to a target protein when it binds to the target protein with higher affinity than to any cross-reactive target molecule as determined using experimental techniques described herein.

[0087] The term “substantially” means that something takes place, as a function or activity, to provide the expected outcome or result to a large degree and to a great extent, but still not to the fullest extent. For example, if an engineered lasso peptide is substantially purified, the engineered lasso peptide is isolated and purification steps afford the engineered lasso peptide at purity level above 80%, preferably above 90%, and as high as 99.99%.

[0088] The term “substantially all” refers to at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100%.

[0089] The term “therapeutic agent” refers to any agent that can be used in treating or preventing a disease, disorder, or condition, including in the treatment, prevention, or alleviation of one or more symptoms of an integrin receptor-mediated proliferative disease (e.g., cancer or fibrosis), disorder, or condition and / or a symptom related thereto. In some instances, a therapeutic agent refers to an engineered lasso peptide as described herein.

[0090] The term “substitution” when used in reference to a peptide, polypeptide, protein refers to an amino acid residue that has been substituted for a structurally different amino acid residue. Such substitutions can be a conservative substitution, a non-conservative substitution, a substitution to a specific sub-class of amino acids, or a combination thereof as described herein.

[0091] The term “therapeutically effective amount” as used herein refers to the amount of an agent (e.g., an engineered lasso peptide provided herein or any other agent described herein) that is sufficient to manage, prevent, or treat a given disease, disorder, or condition as described herein, and / or a symptom related thereto. A therapeutically effective amount of an agent of the present disclosure (e.g., an engineered lasso peptide) can vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the agent to elicit a desired response in the individual. A therapeutically effective amount encompasses an amount in which any toxic or detrimental effects of the agent are outweighed by the therapeutically beneficial effects. A therapeutically effective amount also encompasses an amount of an engineered lasso peptide or other agent (e.g., drug) effective to treat a disease, disorder, or condition described herein, in a subject or mammal.

[0092] The term “therapy” refers to any protocol, method, and / or agent that can be used in the management, prevention or treatment of an integrin receptor-mediated proliferative disease, disorder, or condition (e.g., cancer or fibrosis). In some instances, the terms “therapies” and “therapy” refer to a biological therapy, supportive therapy, and / or other therapies useful in the management, prevention, and / or treatment of an integrin receptor-mediated proliferative disease, disorder, or condition (e.g, cancer or fibrosis) known to one of skill in the art such as medical personnel.

[0093] The term “vector” refers to a substance that is used to carry or include a nucleic acid sequence, including, for example, a nucleic acid sequence encoding an engineered lasso peptide, a lasso precursor peptide (e.g, an engineered lasso peptide having a leader sequence), or lasso processing enzymes as described herein, in order to introduce the nucleic acid sequence into a host cell. Vectors applicable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which can include selection sequences or markers operable for stable integration into a host cell’s chromosome. Additionally, the vectors can include one or more selectable marker genes and appropriate expression control sequences. Selectable marker genes that can be included, for example, provide resistance to antibiotics or toxins, complement auxotrophic deficiencies, or supply critical nutrients not in the culture media. Expression control sequences can include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, which are well known in the art. When two or more nucleic acid molecules are to be co-expressed (e.g., both an engineered lasso core peptide and a lasso cyclase), both nucleic acid molecules can be inserted, for example, into a single expression vector or in separate expression vectors. Forsingle vector expression, the encoding nucleic acids can be operationally linked to one common expression control sequence or linked to different expression control sequences, such as one inducible promoter and one constitutive promoter. The introduction of nucleic acid molecules into a host cell can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis such as Northern blots or polymerase chain reaction (PCR) amplification of mRNA, immunoblotting for expression of gene products, or other suitable analytical methods to test the expression of an introduced nucleic acid sequence or its corresponding gene product. It is understood by those skilled in the art that the nucleic acid molecules are expressed in a sufficient amount to produce a desired product (e.g., an engineered lasso peptide as described herein), and it is further understood that expression levels can be optimized to obtain sufficient expression using methods well known in the art.

[0094] The terms “wild-type” or “WT” refers to organisms, cells, genes, lasso peptide biosynthetic gene clusters, enzymes, proteins, oligonucleotides, and the like that are found in Nature and are unchanged relative to these components found in Nature (in the wild).Yang, K.K., et al., Nature Methods, 2019, 16(8), 687-694.7.3. Method for Lasso Cyclase Engineering

[0095] A method for the engineering of lasso cyclases to improve their properties or functional activity in comparison to the wild-type or parent lasso cyclase is disclosed herein. The method comprises one or more of the following steps:(i) building a computational model based on a known lasso cyclase structure or predicting a lasso cyclase structure,(ii) predicting a lasso cyclase substrate binding site and catalytic pocket,(iii) docking an ensemble of pre-folded lasso core substrates in different conformational states that simulate the transformation of substrate to lasso peptide product in the modeled cyclase substrate binding site and catalytic pocket,(iv) identifying and selecting cyclase residues that are predicted by computational modeling and conformational dynamics simulations to extend into the substrate binding and catalytic pocket,(v) identifying and selecting cyclase residues that are predicted to engage in atomic interactions (attractive, neutral, or repulsive) with the docked pre-folded lasso core substrates and / or lasso peptide product, whereby an atomic interaction is defined as atoms of the cyclase and lasso substrate that approach within < 6Angstroms of each other, an attractive interaction is one that lowers binding free energy (e.g., hydrophobic, H-bonding, salt bridge) and a repulsive interaction is one that increases binding free energy (e.g., steric, same charge interactions),(vi) identifying and selecting cyclase residues that reside < 6 Angstroms from a cyclase ATP binding site,(vii) identifying and selecting cyclase residues with atoms that are predicted to approach atoms in residues of the predicted pre-folded or folded loop of the lasso peptide product within a distance of < 6 Angstroms,(viii) identifying and selecting cyclase residues with atoms that are predicted to approach atoms in residues of the predicted pre-folded or folded ring of the lasso peptide product within a distance of < 6 Angstroms,(ix) performing mutagenesis of the lasso cyclase, including alanine scanning, site- directed or random multi-site mutagenesis of the cyclase amino acid residues selected in steps (iv)-(viii),(x) screening of mutated cyclase variants and selecting evolved cyclases with improved properties or functional performance relative to the wild-type or parent lasso cyclase.

[0096] The present disclosure involves the use of computational models to identify and select residues of a lasso cyclase that are predicted to interact with an incipient lasso peptide as it is being formed in the pocket of the cyclase enzyme. The interacting residues are selected based on the predicted distance between atoms of a cyclase residue and atoms of a docked pre-folded lasso core substrate in different conformations that simulate the trajectory toward and inclusive of lasso peptide formation. Various computational methods are implemented to identify and select cyclase amino acid residues to mutate. Cyclases may be mutated randomly by various evolution methods, including phage display or error-prone PCR, which can involve difficult screening of very large cyclase mutational libraries. The method disclosed herein allows streamlining of cyclase engineering via focused computationally-guided mutagenesis and screening of smaller cyclase variant libraries, which greatly facilitates the mutation of residues that are expected to impact lasso cyclase properties and function. The present disclosure involves the advancement of novel methods for engineering novel lasso cyclases with improved properties and functional performance in comparison to the wild-type or parent lasso cyclase. In certain embodiments, the improved properties or functional performance in comparison tothe wild-type or parent lasso cyclase comprises one or more of: higher cyclase stability to heat; increased tolerance to amino acid substitutions by a wider range of amino acids in a lasso core peptide substrate or lasso peptide product; increased titer, rate, and / or yield of a lasso peptide product; and increased acceptance of non-natural amino acids.7.4. Lasso Cyclases

[0097] As shown in FIGs. 2A and 2B, lasso peptides are constructed biosynthetically through the action of two main enzymes, a lasso peptidase that cleaves the linear precursor peptide to form the lasso core peptide substrate, and a lasso cyclase, which folds the core substrate into the lasso peptide product. Lasso cyclases are ATP-dependent enzymes that are distantly related to class B asparagine synthetases (protein family PF00733, SF52402) and P-lactam synthetases with sequence identities in the range of 20-23% overall, although sequence similarity to the C- terminal region of asparagine synthetase is significantly higher (38%), suggesting that the N- terminal glutaminase region of Asn synthetase was eliminated during the evolution of lasso cyclases (Severinov, K., et al., Mol. Microbiol., 2007, 65(6), 1380-1394; Duquesne, S., et al., Chem. BioL, 2007, 14, 793-803). The mechanism is not fully elucidated, but all evidence suggests that the cyclase containing ATP adenylates the side chain of an Asp or Glu at position 7-10 of the core peptide substrate, which are typically 15-25 residues long and which are proposed to be configured in a pre-folded conformation in the substrate binding and catalytic pocket of the cyclase enzyme. The pre-folded adenylyl-activated core peptide intermediate then undergoes cyclase-catalyzed macrolactamization whereby the N-terminal amino group reacts with the activated Glu or Asp side chain to form an isopeptide bond and trapping the tail inside the newly formed ring, thus providing the distinctive 3D lariat structure of lasso peptides.

[0098] Lasso cyclases are responsible for interacting with the core substrates, pre-folding them into a constrained, entropically-demanding conformation, and then catalyzing formation of the lasso-defining structure. To force the folding of a linear core peptide into a knotted structure, lasso cyclases must create strong interactions with the lasso intermediates along the reaction trajectory. Accordingly, certain lasso core sequences are likely to be more energetically preferred for folding and lasso cyclases will therefore dictate which amino acid residues in a core lasso are accepted. It does not appear that lasso peptidases are involved in determining amino acid sequence tolerance in lasso peptide products, although this aspect requires further confirmation. Natural lasso cyclases have been shown to tolerate sequence variation in the ring (Hegemann, J. D., J. Am. Chem. Soc., 2013, 135, 210-222), the loop (Ducasse, R. et al.ChemBioChem, 2012, 13, 371-380), and the tail (Zong, C., et al., ACS Chem. BioL, 2016, 11, 61-68) regions of the core peptide. For example, wild-type lasso cyclases catalyzing the formation of klebsidin (KleC; Hills, E., et al., ACS Chem. Biol., 2022, 17(4), 998-1010), microcin J25 (McjC; Pavlova, O., et al., J. Biol. Chem., 2008, 283, 25589-2559), ubonodin (UboC; Thokkadam, A. et al., ACS Cent. Sci., 2023, 9, 540-550), lariatin A (LarB; Inokoshi, J., et al., Sci. Report, 2016, 6:30375), capistruin (CapC; Knappe, T. A., et al., Chemistry & Biology, 2009, 16, 1290-1298), and stlassin (StlaC; Liu, T. et al., Chem Sci., 2021, 12(37), 12353- 12364), were found to accept single-site mutations throughout the core peptide. Also, the cyclase associated with fusilassin (FusC) was shown to tolerate multi-site mutational variation in the ring region, but loop mutations were not readily accepted (Si, Y., et al., J. Am. Chem. Soc. 2021, 143, 5917-5927).

[0099] Despite the examples outlined above, the sequence space accessible using wild-type lasso cyclases remains limited, especially when attempting to introduce multiple mutations simultaneously, or when attempting to introduce non-natural amino acids, thus hindering the development of lasso peptides as a new therapeutic modality. Numerous lasso cyclases have been shown to properly process certain substrates that deviate from the wild-type sequence, yet wild-type cyclases have evolved naturally for their substrates and they encounter challenges when attempting to fold a wide range of potentially useful non-natural substrates. Lasso cyclases dictate the types of amino acids and the amino acid sequences that will be tolerated for proper folding of a lasso core substrate into a lasso peptide product. The lack of lasso cyclase substrate tolerance and / or the inability of a lasso cyclase to accept variations in the amino acid sequence of a core peptide or lasso peptide greatly limits the diversity and utility of lasso peptides as a source of novel products. Also, lasso cyclases can exhibit slow rates, which greatly limits the ability to produce or manufacture a given lasso peptide product. Lasso cyclases are sensitive enzymes that are difficult to isolate and characterize - no structure of a lasso cyclase has been reported, and thermostable lasso cyclases would facilitate structure elucidation and ease the use of lasso cyclases. Finally, lasso peptide biosynthesis generally requires a long precursor peptide (>40 amino acids) and the involvement of both peptidase and cyclase enzymes, thus obviating the ability to chemically synthesize and enzymatically cyclize the smaller core peptides into lasso peptide products. While one example of a lasso cyclase that properly folds a core peptide apparently without a leader sequence or peptidase has been reported (Zyubko, T., et al., Chem. Sci., 2019, 10, 9699-9707), this is atypical and evolving lasso cyclases that generally can fold just the core peptide could greatly facilitate the productionof many novel lasso peptide variants. There is an important need for lasso cyclases with improved properties and functional performance, including an improvement in lasso cyclase catalytic rates, thermal stability, substrate tolerance, ability to properly fold linear core peptides, and the acceptance of a wider range of amino acid substitutions, including non-natural amino acids, in order to generate diverse lasso peptide products for use as therapeutics, biomaterials, and diagnostic agents. The present disclosure involves the advancement of novel lasso cyclases with improved properties and functional performance and provides related advantages to meet this need.

[0100] In certain aspects, provided herein is an engineered lasso cyclase that tolerates amino acid sequences of a lasso peptide that are not tolerated by a wild-type or parent lasso cyclase during enzymatically catalyzed cyclization of a lasso core peptide substrate leading to formation of a properly folded lasso peptide. In certain embodiments, the engineered lasso cyclase forms a properly folded lasso peptide with at least 20% higher titer, rate, and / or yield relative to the wild-type or parent lasso cyclase. In an embodiment, the engineered lasso cyclase comprises a variant amino acid sequence of the parent lasso cyclase, wherein the parent lasso cyclase is McjC (SEQ ID NO: 18). In an embodiment, the engineered lasso cyclase comprises a mutation at amino acid position K388. In an embodiment, the engineered lasso cyclase comprises a K388A mutation. In an embodiment, the engineered lasso cyclase comprises a mutation at amino acid position K252. the engineered lasso cyclase comprises a K252A mutation. In an embodiment, the engineered lasso cyclase comprises a mutation at amino acid positions K252 and K388. In certain embodiments, the engineered lasso cyclase comprises one or more features in comparison to the wild-type or parent lasso cyclase, wherein the features comprise one or more of: i) higher cyclase stability to heat; ii) increased titer, rate, and / or yield of a lasso peptide product; and iii) increased acceptance of non-natural amino acids.

[0101] In certain embodiments, the engineered lasso cyclase produces a lasso peptide comprising an amino acid sequence corresponding to any one of SEQ ID NOs: 1-8. In certain embodiments, the engineered lasso cyclase produces a lasso peptide comprising the amino acid sequence corresponding to any one of SEQ ID NOs: 1-8 at a greater titer than the wild-type or parent lasso cyclase. In certain embodiments, the engineered lasso cyclase produces a lasso peptide comprising the amino acid sequence corresponding to any one of SEQ ID NOs: 4-8 at a 2-fold to 20-fold greater titer than the wild-type or parent lasso cyclase. In certain embodiments, the engineered lasso cyclase produces a lasso peptide that is an inhibitor of anintegrin or an integrin receptor. In certain embodiments, the lasso cyclase produces a lasso peptide that is an inhibitor of integrin avP6 or avP8.

[0102] In certain aspects, described herein is a recombinant nucleic acid encoding an engineered lasso cyclase of the present disclosure. In an embodiment, the recombinant nucleic acid comprises a nucleotide sequence encoding the engineered lasso cyclase operatively linked to a promoter. In certain aspects, described herein is a vector or plasmid comprising the recombinant nucleic acid. In an aspect, described herein is a non-naturally occurring microbial organism comprising the recombinant nucleic acid or the vector or plasmid.7.5. Lasso Cyclase Computational Modeling

[0103] In some embodiments, for a method provided herein, 3D computational models of a target lasso cyclase are built based on the atomic coordinates of the lasso cyclase obtained from a protein structure database or scientific literature. In some embodiments, for a method provided herein, the atomic coordinates of the lasso peptides are obtained from a protein structure database that archives structure data of biological macromolecules or from the scientific literature. In some embodiments, the protein structure database is selected from worldwide Protein Data Bank (PDB; http: / / www.rcsb.org / pdb / ), Cambridge Structure Database, Molecular Model Database of National Center for Biotechnology Information (NCBI), and Biological Magnetic Resonance Data Bank (BMRB) database. In some embodiments, the atomic coordinates of the lasso peptide are obtained by subjecting the lasso peptide to nuclear magnetic resonance (NMR) analysis, X-ray crystallography, neutron diffraction, or 3-dimensional electron microscopy (3D-EM). In some embodiments, the X-ray crystallography is serial femtosecond crystallography. In some embodiments, the 3D-EM is cryogenic electron microscopy (cryo-EM). In some embodiments, for a method provided herein, step (i) further comprises computationally modeling the 3D structure of the lasso peptide based on X-ray diffraction data, and / or cryo- EM data and / or nuclear magnetic resonance (NMR) data of the lasso cyclase and atomic coordinates of a reference protein, and wherein the lasso cyclase has at least 30% amino acid sequence similarity to the reference lasso peptide. In some embodiments, the X-ray diffraction or cryo-EM data of the lasso cyclase are obtained by subjecting a crystal of the lasso cyclase to X-ray crystallography or cryogenic electron microscopy analysis. In some embodiments, step (i) comprises: (a) obtaining atomic coordinates of the lasso peptide based on the X-ray diffraction or cryo-EM data; and (b) refining the atomic coordinates of the lasso cyclase based on the atomic coordinates of the reference protein.

[0104] Currently, no lasso cyclase structures have been reported or deposited in the PDB. In other embodiments, for a method provided herein, 3D structures of a target lasso cyclase are created and computationally modeled based on the amino acid sequence of the target cyclase and atomic coordinates of one or more structurally characterized reference polypeptide; and wherein the amino acid sequence of the target lasso cyclase is at least about 25-30% identical to the amino acid sequence of the reference polypeptide. In some embodiments, for a method provided herein, computational modeling the 3D structure of the target lasso cyclase is performed using homology modeling. In some embodiments, for a method provided herein, homology modeling is performed in combination with a protein structure prediction algorithm, wherein optionally the protein structure prediction algorithm is trRosetta (Du, Z., et al., Nature Protoc., 2021, 16(12), 5634-5651), AlphaFold (Jumper, J. et al. Nature, 2021, 596, 583-589), AlphaFold 2 (Yang, Z , etal. Sig. Transduct. Target. Ther. , 2023, 8, 115. Doi: 10.1038 / s41392- 023-01381-z.), and / or AlphaFold-multimer (Johansson-Akhe, I., and Wallner, B. Front. Bioinform., 2022, 2:959160. Doi: 10.3389 / fbinf.2022.959160).

[0105] In some embodiments, lasso substrate binding site and catalytic pocket prediction is performed using algorithms such as CASTp 3.0 (Tian, W ., et al., Nucleic Acids Res., 2018, 46 (Web Server issue) W363-W367), PatchSurfer2.0 (Zhu X., et 3^., Bioinfor alics, 2015, 31, 707-713), TM-Site (Yang J., et al., Bioinformatics, 2013, 29, 2588-2595), machine learning algorithms P2Rank (KrivakR., HokszaD. J Cheminf, 2018, 10, 39. Doi: 10.1186 / sl 3321 -018- 0285-8), COACH-D (Wu Q., et al., Nucleic Acids Res., 2018, 46, W438-W442), or deep learning algorithm DeepSite (Jimenez J., et al., Bioinformatics, 2017, 33, 3036-3042).

[0106] Li gaud (e.g., lasso core peptide substrate) binding to a protein (e.g., lasso cyclase) is defined to occur if the distance between any one of the atoms in the ligand molecule and at least one of the atoms in the amino acid residue of the protein does not exceed the sum of the radii of these two atoms plus 0.5 A, then the amino acid residue is regarded as a ligand binding residue. Atomic interactions between atoms of amino acid residues of a ligand and a protein can be modeled and predicted at interatomic distances of < 6 Angstroms, and such atomic interactions can be attractive, neutral, or repulsive.

[0107] In some embodiments, an ensemble of conformations of pre-folded lasso core peptide substrates and / or lasso peptide products are docked into the predicted lasso substrate binding site and catalytic pockets of the lasso cyclase using commercially available computational modeling systems such as Orion Molecular Design Platform (OpenEye Scientific), MolecularOperating Environment (Chemical Computing Company), MacroModel (Schroedinger), or Discover Studio (Dessault Systems).

[0108] In some embodiments, an ensemble of conformations of pre-folded lasso core substrates and / or lasso peptide products are modeled and atomic interactions (attractive, neutral, or repulsive) between residues of pre-folded lasso core substrates and / or lasso peptide products and lasso cyclases are identified, using in silico docking algorithms (Pagadala, N.S., et al., Biophys Rev, 2017, 9, 91-102) including, but not limited to, CABS-dock (Kurcinski, M., et al., Protein Science, 2020, 29, 211-222), Macromodel (Mohamadi, F., et al., J. Comput. Chem, 1990, 11, 440-467), FlexPepDock (London, N. et al., Nucleic Acids Res. 2010 39, W249- W253), DynaDock (Antes, I., Proteins, 2010, 78, 1084-1104), Autodock (Morris, G.M., et al., J Comput. Chem., 2009, 30, 2785-2791), MOE-Dock (Corbell, C.R., et al., J Comput Aided Mol Des., 2012, 26, 775-786), Surflex-dock (Jain, A.N., J Med Chem, 2003, 46, 499- 511), Glide (Friesner, R.A., et al., J. Med. Chem., 2004, 47, 1739-1749) AutoDock Vina (Trott, O.; Olson, A. J., J. Comput. Chem., 2010, 31 (2), 455-461), or ICM (Neves, M.A.C., et al., J Comput Aided Mol Des., 2012, 26, 675-686). In some embodiments, pre-folded lasso core substrates and / or lasso peptide products having improved atomic interactions (attractive, neutral, or repulsive) with a target cyclase are identified and selected using in silico docking algorithms together with conformational analysis on 3D model structures of lasso cyclases and lasso peptides using molecular dynamics simulation algorithms, including but not limited to the publicly available or commercial programs, GROMACS, AMBER, CHARMM, and Schroedinger’ s MAESTRO.

[0109] In some embodiments, an ensemble of conformations of pre-folded lasso core substrates and / or lasso peptide products are modeled and atomic interactions (attractive, neutral, or repulsive) between residues of pre-folded lasso core substrates and / or lasso peptide products and lasso cyclases are analyzed and identified by using artificial intelligence, deep learning, or machine learning algorithms for in silico virtual screening and / or de novo design to define the overall lasso peptide structural topologies encompassing the loop, ring, and tail size, as well as amino acid residues that suitably fit inside and favorably interact in the lasso-binding site of a target cyclase in order to improve functional performance, which may be refined further through iterative in silico docking and molecular dynamics simulations approaches. In some embodiments, one or more different artificial intelligence, deep learning, or machine learning algorithms are used, including but not limited to, support vector machines (Warmuth, M.K., et al., J. Chem. Inf. Comput. Sci., 2003, 43, 667-673), random forest (Deshmukh A.L., et al., Mol.Biosyst., 2017, 13, 1630-1639), ^-nearest neighbors (Luo, M., et al., Mol. Inf., 2016, 35, 36 - 41), as well as neural networks with or without autoencoders, such as convolutional neural network ( Jimenez, J., et al., J. Chem. Inf. Model., 2018, 58, 287-296), recurrent neural network (Sattarov, B., et al., J. Chem. Inf. Model. 2019, 59, 1182-1196; Muller, A.T., et al., J. Chem. Inf. Model., 2018, 58, 2, 472-479), deep neural network (Ma, J., et al., J. Chem. Inf. Model., 2015, 55, 263-274), generative neural network (Gupta, A., et al., Mol. Inf, 2018, 37, 1700111), and generative adversarial neural network (Prykhodko, O, et al., J Cheminform., 2019, 11:74; doi.org / 10.1186 / sl3321-019-0397).

[0110] In some embodiments, docked models enable the identification and selection of residues in the docked ensemble of conformations of pre-folded lasso core peptide substrates and / or lasso peptide products that are predicted to be in proximity (< 6 Angstroms) to cyclase residues that extend into the catalytic pocket. In other embodiments, docked models enable the identification and selection of residues in the ensemble of conformations of docked prefolded lasso core peptide substrates and / or lasso peptide products that are predicted to be in proximity (< 6 Angstroms) to cyclase residues that surround the cyclase ATP -binding site. In another embodiment, docked models enable the identification and selection of residues in the loop of a docked ensemble of an ensemble of pre-folded lasso core peptide substrates and / or lasso peptide products that are predicted to be in proximity (< 6 Angstroms) to cyclase residues during the macrolactam ring closing process (FIG. 2A and FIG. 2B). In another embodiment, docked models enable the identification and selection of residues in the ring of a docked ensemble of pre-folded lasso core peptide substrates and / or lasso peptide products that are predicted to be in proximity (< 6 Angstroms) to cyclase residues during the macrolactam ring closing process.7.6. Lasso Cyclase Engineering

[0111] Enzyme engineering is an approach that involves mutating the amino acids of and / or evolving an enzyme to improve its properties (Yang, K.K., et al., Nature Methods, 2019, 16(8), 687-694). Engineering lasso cyclases could allow these enzymes to expand beyond their natural lasso substrate tolerance and provide access to previously unattainable lasso peptide variants with much broader sequence diversity. It is estimated that cyclase engineering could generate cyclase variants that properly fold lasso peptide variants with >95% of theoretical mutational efficiency at each lasso peptide residue, thus allowing the creation of highly diverse lasso peptide libraries. Thus, an important aim of cyclase engineering could be to enable theformation of lasso peptides with maximum sequence diversity, which is likely instrumental for optimizing properties such as target binding affinity, selectivity, and pharmacokinetics of lasso peptides in biopharmaceutical applications.

[0112] In some embodiments, selected residues of lasso cyclases are evolved using alanine scanning or site directed mutagenesis guided by structure predictions and modeling. In other embodiments, lasso cyclases are evolved using random mutagenesis methods, such as phage display, mRNA display, or DNA-display, at sites selected using structure predictions and modeling. In other embodiments, libraries of mutated cyclase variants are screened for improved properties or functional performance relative to the wild-type or parent lasso cyclase. For example, improved properties could be higher cyclase stability to heat, and improved functional performance could be better cyclase tolerance to amino acid substitutions in the pre-folded lasso substrate or lasso peptide product, including the acceptance of non-natural amino acids.

[0113] As disclosed herein, a set of nucleic acids encoding the desired activities of a lasso peptide biosynthesis pathway can be introduced into a host organism to produce a lasso peptide or can be introduced into a cell-free biosynthesis reaction mixture containing a cell extract or other suitable medium to produce a lasso peptide. In some cases, it can be desirable to modify the properties or biological activities of a lasso peptide to improve its therapeutic potential. In other cases, it can be desirable to modify the activity or specificity of lasso peptide biosynthesis pathway enzymes or proteins to improve the production of lasso peptides. For example, mutations can be introduced into an encoding nucleic acid molecule (e.g., a gene), which ultimately leads to a change in the amino acid sequence of a protein, enzyme, or peptide, and such mutated proteins, enzymes, or peptides can be screened for improved properties. Such optimization methods can be applied, for example, to increase or improve the activity or substrate scope of an enzyme, protein, or peptide and / or to decrease an inhibitory activity. Lasso peptides are derived from precursor peptides that are ribosomally produced by transcription and translation of a gene encoding the linear precursor peptide. Ribosomally produced peptides, such as lasso precursor peptides, are well-known to be readily evolved and optimized through variation of nucleotide sequences within genes that encode for the amino acid residues that comprise the peptide. Similarly, biosynthetic enzymes, such as lasso cyclases, are ribosomally produced and can be evolved in a similar fashion. Large libraries of peptide or enzyme mutational variants have been produced by methods well known in the art, and some of these methods are referred to as directed or random evolution.

[0114] Directed evolution is a powerful approach that involves the introduction of mutations targeted to a specific gene, specific oligonucleotide sequences within a gene (e.g., oligonucleotides corresponding to specific amino acid residues in the peptide product), or an oligonucleotide sequence containing a gene, in order to improve and / or alter the properties or production of an enzyme (e.g., lasso cyclase), protein or peptide (e.g., a lasso peptide). Improved and / or altered enzymes, proteins or peptides can be identified through the development and implementation of sensitive high-throughput assays that allow automated screening of many enzyme or peptide variants (for example, >104). Iterative rounds of mutagenesis and screening typically are performed to afford an enzyme or peptide with optimized properties. Computational algorithms that can help to identify areas of the gene for mutagenesis also have been developed and can significantly reduce the number of enzyme or peptide variants that need to be generated and screened (See: Fox, R.J., et al., Trends Biotechnol., 2008, 26, 132-138; Fox, R.J., et al., Nature Biotechnol., 2007, 25, 338-344). Numerous directed and random evolution technologies have been developed and shown to be effective at creating diverse variant libraries, and these methods have been successfully applied to the improvement of a wide range of properties across many enzyme and protein classes (for reviews, see: Hibbert et al., Biomol. Eng., 2005, 22,11-19; Huisman and Lalonde, In Biocatalysis in the pharmaceutical and biotechnology industries, pgs. 717-742 (2007), Patel (ed.), CRC Press; Otten and Quax, Biomol. Eng., 2005, 22, 1-9; and Sen et al., AppL Biochem.Biotechnol., 2007, 143, 212-223). Enzyme and protein characteristics that have been improved and / or altered by directed and random evolution technologies include, for example: selectivity / specificity, for acceptance of non-native sequences or conversion of non-natural substrates; temperature stability, for robust high temperature processing; pH stability, for bioprocessing under lower or higher pH conditions; substrate or product tolerance, so that high product titers can be achieved; binding (Km), including broadening of ligand or substrate binding to include non-natural substrates; inhibition (Ki), to remove inhibition by products, substrates, or key intermediates; activity (kcat), to increase enzymatic reaction rates to achieve desired flux; isoelectric point (pl) to improve protein or peptide solubility; acid dissociation (pKa) to vary the ionization state of the protein or peptide with repect to pH; expression levels, to increase protein or peptide yields and overall pathway flux; oxygen stability, for operation of air-sensitive enzymes or peptides under aerobic conditions; and anaerobic activity, for operation of an aerobic enzyme or peptide in the absence of oxygen.

[0115] A number of exemplary methods have been developed for the mutagenesis and diversification of genes and oligonucleotides to introduce desired properties into specific enzymes,proteins and peptides. Such methods are well-known to those skilled in the art and allow either single-site or multi-site mutagenesis of a gene. Any of these can be used to alter and / or optimize the activity of a lasso peptide biosynthetic pathway enzyme, protein, or peptide, including a lasso precursor peptide, a lasso core peptide, or a lasso peptide. Such methods include, but are not limited to error-prone polymerase chain reaction (EpPCR), which introduces random point mutations by reducing the fidelity of DNA polymerase in PCR reactions (See: Pritchard et al., J. Theor.BioL, 2005, 234:497-509); Error-prone Rolling Circle Amplification (epRCA), which is similar to epPCR except a whole circular plasmid is used as the template and random 6-mers with exonuclease resistant thiophosphate linkages on the last 2 nucleotides are used to amplify the plasmid followed by transformation into cells in which the plasmid is re-circularized at tandem repeats (Fujii et al., Nucleic Acids Res., 2004, 32:el45; and Fujii et al., Nat. Protoc., 2006, 1, 2493-2497); DNA, Gene, or Family Shuffling, which typically involves digestion of two or more variant genes with nucleases such as Dnase I or EndoV to generate a pool of random fragments that are reassembled by cycles of annealing and extension in the presence of DNA polymerase to create a library of chimeric genes (Stemmer, Proc. Natl. Acad. Sci. U.S.A., 1994, 91, 10747-10751; and Stemmer, Nature, 1994, 370, 389-391); Staggered Extension Process (StEP) in vitro recombination, which entails template priming followed by repeated cycles of 2-step PCR with denaturation and very short duration of annealing / extension (as short as 5 sec) (Zhao et al., Nat. BiotechnoL, 1998,16, 258-261); Random Priming Recombination (RPR), in which random sequence primers are used to generate many short DNA fragments complementary to different segments of the template (Shao et al., Nucleic Acids Ae .,1998, 26, 681-683).

[0116] Additional methods include Heteroduplex Recombination, in which linearized plasmid DNA is used to form heteroduplexes that are repaired by mismatch repair (See: Volkov et al, Nucleic Acids Res., 1999, 27:el8; Volkov et al., Methods EnzymoL, 2000, 328, 456-463); Random Chimeragenesis on Transient Templates (RACHITT), which employs Dnase I fragmentation and size fractionation of single-stranded DNA (ssDNA) (See: Coco, W.M., et al., Nat. BiotechnoL, 2001, 19, 354-359); Recombined Extension on Truncated Templates (RETT), which entails template switching of unidirectionally growing strands from primers in the presence of unidirectional ssDNA fragments used as a pool of templates (See: Lee et al., J. Mol. Cat., 2003, 26, 119-129); Degenerate Oligonucleotide Gene Shuffling (DOGS), in which degenerate primers are used to control recombination between molecules; (Bergquist, P.L., Meth- odsMol. BioL, 2007, 352, 191-204; Bergquist, P.L., et al., Biomol. Eng., 2005, 22, 63-72; Gibbset al., Gene, 2001, 271, 13-20); Incremental Truncation for the Creation of Hybrid Enzymes (ITCHY), which creates a combinatorial library with 1 base pair deletions of a gene or gene fragment of interest (See: Ostermeier, M., et al., Proc. Natl. Acad. Sci. U.S.A., 1999, 96, 3562- 3567; and Ostermeier, M., et al., Nat. BiotechnoL, 1999, 17, 1205-1209); Thio-Incremental Truncation for the Creation of Hybrid Enzymes (THIO-ITCHY), which is similar to ITCHY except that phosphothioate dNTPs are used to generate truncations (See: Lutz, S., et al., Nucleic Acids Res., 2001, 29, El 6); SCRATCHY, which combines two methods for recombining genes, ITCHY and DNA Shuffling (See: Lutz, S., et al., Proc. Natl. Acad. Sci. U.S.A., 2001, 98, 11248-11253); Random Drift Mutagenesis (RNDM), in which mutations made via epPCR are followed by screening / selection for those retaining usable activity (See: Bergquist, P.L., et al., Biomol. Eng., 2005, 22, 63-72); Sequence Saturation Mutagenesis (SeSaM), a random mutagenesis method that generates a pool of random length fragments using random incorporation of a phosphothiolate nucleotide and cleavage, which is used as a template to extend in the presence of “universal” bases such as inosine, and replication of an inosine-containing complement gives random base incorporation and, consequently, mutagenesis (See: Wong, T.S., et al., BiotechnoL J., 2008, 3, 74-82; Wong, T.S., et al., Nucleic Acids Res. , 2004, 32, e26; Wong, T.S., et al., Anal. Biochem., 2005, 341, 187-189); Synthetic Shuffling, which uses overlapping oligonucleotides designed to encode “all genetic diversity in targets” and allows a very high diversity for the shuffled progeny (See: Ness, J.E., et al., Nat. BiotechnoL, 2002, 20, 1251- 1255); Nucleotide Exchange and Excision Technology (NexT), which exploits a combination of dUTP incorporation followed by treatment with uracil DNA glycosylase and then piperidine to perform endpoint DNA fragmentation (See: Muller, K., et al., Nucleic Acids Res. , 2005, 33 : el l7. Doi: 10.1093 / nar / gnil l6).

[0117] Further methods include Sequence Homology-Independent Protein Recombination (SHIPREC), in which a linker is used to facilitate fusion between two distantly related or unrelated genes, and a range of chimeras is generated between the two genes, resulting in libraries of single-crossover hybrids (See: Sieber et al., Nat. BiotechnoL, 2001, 19, 456-460); Gene Site Saturation Mutagenesis™ (GSSM™), in which the starting materials include a supercoiled double stranded DNA (dsDNA) plasmid containing an insert and two primers which are degenerate at the desired site of mutations, enabling all amino acid variations to be introduced individually at each position of a protein or peptide (See: Kretz, K., et al., Methods EnzymoL, 2004, 388, 3-11); Combinatorial Cassette Mutagenesis (CCM), which involves the use of short oligonucleotide cassettes to replace limited regions with a large number of possible amino acidsequence alterations (See: Reidhaar-Olson, J.F., et al. Methods EnzymoL , 1991, 208, 564-586; Reidhaar-Olson, J.F., et al. Science, 1988, 241, 53-57); Combinatorial Multiple Cassette Mutagenesis (CMCM), which is essentially similar to CCM and uses epPCR at high mutation rate to identify hot spots and hot regions and then extension by CMCM to cover a defined region of protein sequence space (See: Reetz, M., et al., Angew. Chem. Int. EdEngL, 2001, 40, 3589- 3591); the Mutator Strains technique, in which conditional is mutator plasmids, utilizing the mutD5 gene, which encodes a mutant subunit of DNA polymerase III, to allow increases of 20 to 4000x in random and natural mutation frequency during selection and block accumulation of deleterious mutations when selection is not required (See: Selifonova et al., AppL Environ. Microbiol., 2001, 67, 3645-3649); Low et al., J. Mol. Biol., 1996, 260, 3659-3680).

[0118] Additional exemplary methods include Look-Through Mutagenesis (LTM), which is a multidimensional mutagenesis method that assesses and optimizes combinatorial mutations of a selected set of amino acids (See: Rajpal et al., Proc. Natl. Acad. Sci. U.S.A., 2005, 102, 8466-8471); Gene Reassembly, which is a homology-independent DNA shuffling method that can be applied to multiple genes at one time or to create a large library of chimeras (multiple mutations) of a single gene (See: Short, J.M., US Patent 5,965,408, Tunable GeneReassem- bly™); in silico Protein Design Automation (PDA), which is an optimization algorithm that anchors the structurally defined protein backbone possessing a particular fold, and searches sequence space for amino acid substitutions that can stabilize the fold and overall protein energetics, and generally works most effectively on proteins with known three-dimensional structures (See: Hayes et al., Proc. Natl. Acad. Sci. U.S.A., 2002, 99, 15926-15931); and Iterative Saturation Mutagenesis (ISM), which involves using knowledge of structure / function to choose a likely site for improvement, performing saturation mutagenesis at chosen site using a mutagenesis method such as Stratagene QuikChange (Stratagene; San Diego CA), screen- ing / selecting for desired properties, and, using improved clone(s), starting over at another site and continue repeating until a desired activity is achieved (See: Reetz, M., et al., Nat. Protoc., 2007, 2, 891-903; Reetz, M., et al., Angew. Chem. Int. EdEngL, 2006, 45, 7745-7751). Saturation mutagenesis at multiple sites within a protein or peptide simultaneously (e.g., generate all possible variants using, for example, NNK codons at 2-sites, 3 -sites, 4-sites, etc. within a given parent enzyme or peptide) allows for the production of random mutagenesis libraries with the potential to identify synergistic combinations of mutations to improve performance and / or properties.

[0119] A rapid way to create and screen very large random libraries of diverse peptides involves the use of display technologies (For a review, see: Ullman, C.G., et al., Briefings Functional Genomics, 2011, 10, 125-134). Peptide display technologies offer the benefit that specific peptide encoding information (e.g., RNA or DNA sequence information) is linked to, or otherwise associated with, each corresponding enzyme or peptide in a library, and this information is accessible and readable (e.g., by amplifying and sequencing the attached DNA oligonucleotide) after a screening event, thus enabling identification of the individual peptides within a large library that exhibit desirable properties (e.g., high amino acid tolerance leading to high lasso peptide sequence diversity). Lasso peptide cell-free biosynthesis (CFB) methods are known (Si, Y., et al., J. Am. Chem. Soc., 2021, 143, 5917-5927. Doi: 10.1021 / jacs. lc01452) and can facilitate and enable the creation of large lasso cyclase libraries containing analogs that can be screened for favorable properties. Lasso cyclase mutants that exhibit the desired improved properties (hits) may be subjected to additional rounds of mutagenesis to allow creation of highly optimized lasso peptide variants. The CFB methods and systems can be used in combination with enzyme display technologies to establish a platform to rapidly produce high density libraries of lasso cyclase variants and to identify promising lasso cyclase analogs with desirable properties.

[0120] Any of the aforementioned methods for mutagenesis and / or display can be used alone or in any combination to evolve and improve the performance of lasso peptide biosynthesis pathway enzymes, proteins, and peptides. Similarly, any of the aforementioned methods for mutagenesis and / or display can be used alone or in any combination to enable the creation of engineered lasso cyclases which may be selected for improved properties.

[0121] In one embodiment of the invention, lasso cyclases are evolved whereby mutants of a parent lasso cyclase are created and the lasso cyclase variants are screened for improved properties or functional activity. In another embodiment of the invention, lasso cyclases are evolved whereby mutants of a parent lasso cyclase are created, as guided by computational modeling to identify key sites for mutagenesis, and the lasso cyclase variants are screened for improved properties or functional activity (FIG. 3). In some embodiments, multiple sites of a parent lasso cyclase are varied individually or simultaneously to generate a directed or random library of cyclase mutants that are screened for improved properties. In some embodiments, multiple sites of a parent lasso cyclase are varied individually or simultaneously to generate a directed or random library of cyclase mutants that are screened for improved production of a desired lasso peptide variant. In some embodiments, multiple sites of a parent lasso cyclase inproximity to the ATP -binding site are varied individually or simultaneously to generate a directed or random library of cyclase mutants that are screened for improved production of a desired lasso peptide variant (FIG. 3). In some embodiments, multiple sites of a parent lasso cyclase that are predicted to interact with the lasso loop region are varied individually or simultaneously to generate a directed or random library of cyclase mutants that are screened for improved production of a desired lasso peptide variant. In another embodiment, multiple sites of a parent lasso cyclase that are predicted to interact with the lasso ring region are varied individually or simultaneously to generate a directed or random library of cyclase mutants that are screened for improved production of a desired lasso peptide variant. In another embodiment, multiple sites of a parent lasso cyclase that are predicted to interact with the lasso loop and ring regions are varied individually or simultaneously to generate a directed or random library of cyclase mutants that are screened for improved production of a desired lasso peptide variant. In another embodiment, mutational variants or a mutational library of lasso core peptides are created and converted by an engineered lasso cyclase into a library of lasso peptide variants that are screened for improved properties.

[0122] Using the methods described herein, certain modifications are computationally predicted, identified, and selected for enhancing the properties and / or activity of an engineered lasso cyclase. Numerous exemplary alterations or modifications that are computationally predicted to impact the properties and / or functional activity of a lasso cyclase are shown in Table 1. In some embodiments, the engineered lasso cyclase further includes one, two, three, or four amino acid alterations relative to a parent lasso cyclase. In some embodiments, the engineered lasso cyclase further includes one amino acid alteration relative to a parent lasso cyclase. In some embodiments, the engineered lasso cyclase further includes two amino acid alterations relative to a parent lasso cyclase. In some embodiments, the engineered lasso cyclase further includes two amino acid alterations relative to a parent lasso cyclase, and that amino acid alteration involves changing two lysine residues to alanine. In some embodiments, the engineered lasso cyclase further includes three amino acid alterations relative to a parent lasso cyclase. In some embodiments, the engineered lasso cyclase further includes four amino acid alterations relative to a parent lasso cyclase. Such alterations include changes to the amino acid sequence of the parent lasso cyclase. These alterations include an amino acid substitution, deletion, insertion, or combination thereof. In some embodiments, the alterations include one or more amino acid substitutions. In some embodiments, the alterations include one or more amino acid deletions. In some embodiments, the alterations include one or more amino acidinsertions. The amino acid deletions or insertions include insertion or deletion of one or more amino acid residues. Accordingly, in some embodiments, the amino acid insertion includes the insertion of one amino acid residue, the insertion of two amino acid residues, the insertion of three amino acid residues, or the insertion of four amino acid residues. In some embodiments, the amino acid deletion includes the deletion of one amino acid residue, the deletion of two amino acid residues, the deletion of three amino acid residues, or the deletion of four amino acid residues. The amino acid alterations can also include a combination of substitutions, insertions, and deletions. These amino acid alterations can also be located in the same and / or different regions relative to the lasso substrate binding site of the cyclase. For example, an amino acid alteration may be introduced into a cyclase region predicted to interact with the loop of the pre-folded lasso substrate or lasso peptide product, and then additional alterations can be introduced into a different cyclase region that is predicted to interact with the ring, and / or tail of the pre-folded lasso substrate or lasso peptide product.

[0123] In some embodiments, the engineered lasso cyclase is a variant of a parent lasso cyclase which contains at least one amino acid alteration. In some embodiments, the engineered lasso cyclase further includes one amino acid alteration relative to a parent lasso cyclase, and that amino acid alteration involves changing a lysine to an alanine residue. In some embodiments, the engineered lasso cyclase further includes two amino acid alterations relative to a parent lasso cyclase. In some embodiments, the engineered lasso cyclase further includes two amino acid alterations relative to a parent lasso cyclase, and that amino acid alteration involves changing two lysine residues to alanine. In some embodiments, the engineered lasso cyclase is a variant of the parent lasso cyclase McjC. In some embodiments, the engineered lasso cyclase is a variant of the parent lasso cyclase McjC (SEQ ID NO: 18) which catalyzes the formation of microcin J25 and contains at least one amino acid alteration. In some embodiments, the engineered lasso cyclase is a variant of the parent lasso cyclase McjC and contains alteration of at least one lysine residue. In some embodiments, the engineered lasso cyclase is a variant of the parent lasso cyclase McjC and contains alteration of at least two lysine residues. In some embodiments, the engineered lasso cyclase is a variant of the parent lasso cyclase McjC and contains alteration of lysine residue number 252 (K252). In some embodiments, the engineered lasso cyclase is a variant of the parent lasso cyclase McjC and contains alteration of lysine residue number 388 (K388). In some embodiments, the engineered lasso cyclase is a variant of the parent lasso cyclase McjC and contains alteration of two lysine residues numbered K252 and K388. In some embodiments, the engineered lasso cyclase is a variant of theparent lasso cyclase McjC and contains alteration of lysine residue number 252 to alanine (K252A). In some embodiments, the engineered lasso cyclase is a variant of the parent lasso cyclase McjC and contains alteration of lysine residue number 388 to alanine (K388A). In some embodiments, the engineered lasso cyclase is a variant of the parent lasso cyclase McjC and contains alteration of two lysine residues numbered K252 and K388 to alanine (K252A and K388A).

[0124] Numerous exemplary alterations for an engineered lasso cyclase with improved properties are shown in Table 2. In certain embodiments, engineered lasso cyclases are used to promote or catalyze formation of lasso peptide products. In other embodiments, engineered lasso cyclases are used to promote or catalyze formation of lasso peptide products that are produced in low titer or are not produced when using the parent or wild-type lasso cyclase. In other embodiments, engineered lasso cyclases are used to promote or catalyze formation of lasso peptide products that are produced in low titer when using the parent or wild-type lasso cyclase, and the titer is <20 mg / L. In other embodiments, engineered lasso cyclases are used to promote or catalyze formation of lasso peptide products that are produced in low titer when using the parent or wild-type lasso cyclase, and the titer is <10 mg / L. In other embodiments, engineered lasso cyclases are used to promote or catalyze formation of lasso peptide products that are produced in low titer when using the parent or wild-type lasso cyclase, and the titer is <5 mg / L. In other embodiments, engineered lasso cyclases are used to promote or catalyze formation of lasso peptide products that are produced in low titer when using the parent or wild-type lasso cyclase, and the titer is <1 mg / L. In other embodiments, engineered lasso cyclases are used to promote or catalyze formation of lasso peptide products that are produced in low titer when using the parent or wild-type lasso cyclase, and the titer is <0.1 mg / L. In other embodiments, engineered lasso cyclases are used to promote or catalyze formation of lasso peptide products that are produced in low titer when using the parent or wild-type lasso cyclase, and the titer is <0.01 mg / L. In other embodiments, engineered lasso cyclases are used to promote or catalyze formation of lasso peptide products that are produced in low titer when using the parent or wild-type lasso cyclase, and the titer is not quantifiable (NQ).

[0125] In some embodiments, engineered lasso cyclases are used to promote or catalyze formation of non-natural lasso peptides. In some embodiments, engineered lasso cyclases are used to promote or catalyze formation of non-natural lasso peptides and the non-natural lasso peptides contain amino acid alterations or mutations relative to the wild-type lasso peptide. In some embodiments, engineered lasso cyclases are used to promote or catalyze formation ofnon-natural lasso peptides and the non-natural lasso peptides contain binding epitopes grafted into the loop or ring. In some embodiments, engineered lasso cyclases are used to promote or catalyze formation of non-natural lasso peptides and the non-natural lasso peptides contain both binding epitopes grafted into the loop or ring and amino acid alterations or mutations. In some embodiments, engineered lasso cyclases are used to promote or catalyze formation of non- natural lasso peptides that bind to and modulate biological receptors. In some embodiments, engineered lasso cyclases are used to promote or catalyze formation of non-natural lasso peptides that bind to and modulate integrin receptors. In some embodiments, engineered lasso cyclases are used to promote or catalyze formation of non-natural lasso peptides that derive from SEQ ID NO: 1 and that bind to and modulate integrin receptors. In some embodiments, engineered lasso cyclases are used to promote or catalyze formation of non-natural lasso peptides that bind to and modulate avP8 integrin receptor. In some embodiments, engineered lasso cyclases are used to promote or catalyze formation of non-natural lasso peptides that derive from SEQ ID NO: 1 and that bind to and modulate avP8 integrin receptors (Table 2). In some embodiments, engineered lasso cyclases are used to promote or catalyze formation of non- natural lasso peptides that derive from SEQ ID NO: 1 and contain an integrin binding epitope. In some embodiments, engineered lasso cyclases are used to promote or catalyze formation of non-natural lasso peptides that derive from SEQ ID NO: 1 and contain the integrin binding epitope Arg-Gly-Asp-Leu (RGDL) (SEQ ID NO: 25), such as SEQ ID NO: 2. In some embodiments, engineered lasso cyclases are used to promote or catalyze formation of non-natural lasso peptides that derive from SEQ ID NO: 1 and contain the integrin binding epitope Arg- Gly-Asp-Leu (RGDL) (SEQ ID NO: 25) and bear a loop reside deletion. In some embodiments, engineered lasso cyclases are used to promote or catalyze formation of non-natural lasso peptides that contain the integrin binding epitope Arg-Gly-Asp-Leu (RGDL) (SEQ ID NO: 25) and a deletion of the P16 residue, as in SEQ ID NO: 3. In some embodiments, engineered lasso cyclases are used to promote or catalyze formation of non-natural lasso peptides that contain the integrin binding epitope Arg-Gly-Asp-Leu (RGDL) (SEQ ID NO: 25) and a deletion of the P16 residue of SEQ ID NO 2, as in SEQ ID NO: 3. In some embodiments, engineered lasso cyclases are used to promote or catalyze formation of non-natural lasso peptides that contain the integrin binding epitope Arg-Gly-Asp-Leu (RGDL) (SEQ ID NO: 25), a deletion of the P16 residue, and further alterations to amino acid residues of SEQ ID NO 3, as in SEQ ID NOS: 4- 8. In some embodiments, an engineered lasso cyclase containing a K252 A mutation is used to promote or catalyze formation of non-natural lasso peptides. In some embodiments, anengineered lasso cyclase containing a K388 A mutation is used to promote or catalyze formation of non-natural lasso peptides. In some embodiments, an engineered lasso cyclase containing K252A and K388A mutations is used to promote or catalyze formation of non-natural lasso peptides. In some embodiments, an engineered lasso cyclase containing a K252 A mutation is used to promote or catalyze formation of non-natural lasso peptides corresponding to SEQ ID NOS: 2-8. In some embodiments, an engineered lasso cyclase containing a K388A mutation is used to promote or catalyze formation of non-natural lasso peptides corresponding to SEQ ID NOS: 2-8. In some embodiments, an engineered lasso cyclase containing K252A and K388A mutations is used to promote or catalyze formation of non-natural lasso peptides corresponding to SEQ ID NOS: 2-8.8. TABLES AND SEQUENCES

[0126] Table 1. Shows production titers for SEQ ID NOS: 1 and 5 using wild-type McjC (entry 1) and 14 different McjC mutants. Average titers represent biological triplicate 96 deep-well cultures of variants derived from single colonies. Quantification was performed via UV-HPLC in comparison to isolated lasso peptide standards, with masses verified by LCMS.

[0127] Table 2 Shows lasso peptide analog sequences, production titer (mg / L), and av08 integrin inhibition data. SEQ ID NO: 1 is native MccJ25. Sequence changes in SEQ ID NOS: 2-8 are bolded and deletions indicated with a dash, relative to SEQ ID NO: 1. Titers were determined by triplicate cultivations in 96-well plates and verified in 1 L shake flasks. WT McjC = wild-type MccJ25 cyclase. McjC-1 = K252A McjC cyclase mutant. MccjC-2 = K388A McjC cyclase mutant. McjC-3 = K252A / K388A McjC cyclase double mutant. ICso values for SEQ ID NOS: 2-8 were determined by ELISA and / or AlphaLISA assays using LAP protein as standard substrate against which SEQ ID NOS: 2-8 inhibited binding to avP8. ND = not detected. NQ = non-quantifiable mass spectrometry signal. X means no data was collected.

[0128] The sequences in Table 3 and Table 4 illustrate amino acid and / or nucleic acid sequences that can be used to generate the engineered lasso cyclases, recombinant nucleic acids, and / or compositions described herein, and to perform the methods described herein, including those described in the Examples. As needed, an RNA sequence can be readily deduced from the DNA sequence.

[0129] Table 3. Amino acid sequences, mutations vs parent lasso scaffold SEQ ID NO: 1, and SEQ ID NOS for lasso peptides produced, along with oligonucleotide sequences and SEQ ID NOS for each complete precursor peptide (leader and core). The oligonucleotide sequence for G1 of SEQ ID NO 1 is bolded. All precursor peptides are derived from SEQ ID NO: 1 and each has the following leader sequence attached to the N-terminus of the lasso peptide amino acid sequence: MIKHIHFDKLSSSKKNNVPHSAKGVIQIKKSASQLTK (SEQ ID NO 23) for SEQ ID NO 1.

[0130] Table 4. Amino acid sequences for lasso peptidase (B), lasso cyclase (C), and transporter (D) proteins and oligonucleotide sequences for corresponding B, C, and D genes clonedinto the plasmids for generating each biosynthetic enzyme and the engineered lasso cyclases thereof. Oligonucleotide sequence given for both E. coli codon-optimized (CO = “codon-optimized”, accession numbers for codon-optimized oligonucleotides listed as N / A since no accession numbers are available) and wild-type (“WT”, with NCBI accession number), where applicable. Translated amino acid sequences (with NCBI accession number) are identical between codon-optimized and wild-type starting oligonucleotide sequences.9. EXAMPLES

[0131] Examples related to the present invention are described below. In most cases, alternative techniques can be used. The examples are intended to be illustrative and are not limiting or restrictive to the scope of the invention. For example, where lasso peptides or engineered lasso cyclases are prepared following a protocol of a Scheme, it is understood that conditions may vary, for example, any of the solvents, reaction times, reagents, temperatures, supplements, work up conditions, or other reaction parameters may be varied.General Methods

[0132] Reagents used for molecular biology experiments are purchased from New England BioLabs (Ipswich, MA), Thermo Fisher Scientific (Waltham, MA), or Gold Biotechnology Inc. (St. Louis, MO). Other chemicals are purchased from Sigma-Aldrich (St. Louis, MO). Escherichia coli DH5a and BL21 (DE3) strains are used for plasmid maintenance, extract production, or lasso peptide production. All molecular biology and cell-free biosynthesis reactions are conducted using standard plates, vial, and flasks typically employed when working with biological molecules such as DNA, RNA and proteins. LC-MS / MS analyses (including Hi- Res analysis) are performed on an Agilent 6530 Accurate-Mass Q-TOF MS equipped with a dual electrospray ionization source and an Agilent 1260 LC system with diode array detector or an Agilent 1290 Infinity II HPLC System interfaced with an Agilent 6460C Triple Quadrupole LC / MS system. MS and UV data are analyzed with Agilent MassHunter Qualitative Analysis version B.05.00.For all E. coli transformations and pre-production starter cultures, Luria-Bertani (LB) solid [10 g / L casein peptone, 5 g / L yeast extract, lOg / L NaCl, 15 g / L agar, pH 7.0] and liquid [10 g / L casein peptone, 5 g / L yeast extract, lOg / L NaCl, pH 7.0] media were used, respectively. For production cultures, both in 96 deep-well plates and in shaking flasks, supplemented M9 liquid (3 g / L KH2PO4, 12.8 g / L Na2HPO4.7H2O, 0.5 g / L NaCl, 1 g / L NH4C1, 4 mL / L glycerol, 2 g / L casamino acids, 1 mL / L Teknova® vitamin mix, 0.49 g / L MgSCh, 0.015 g / L CaCh) medium was used.

[0133] Creation of MccJ25 biosynthetic operon expression vector. An expression vector (pLAM58, “B-vector”) containing the biosynthetic enzymes and transporter required for MccJ25 biosynthesis and export was built from synthetic DNA fragments. Briefly, the genesencoding mcjB (peptidase), mcjC (lasso cyclase), and mcjD (transporter) were synthesized (see Table 4 for sequences) together with their native promoter (Twist Biosciences). The genes and their promoter were then assembled into a pBR322 backbone containing a kanamycin (kanR resistance marker via overlap assembly (NEBuilder HiFi DNA Assembly, New England Biolabs). Correct assembly following transformation into E. coli DH5a (New England Biolabs) was validated via Sanger sequencing.

[0134] Cloning of Mcj A-derived Lasso Peptides. All McjA-derived lasso peptides (Table 2) were cloned in a similar manner via type Ils restriction-guided assembly of synthetic lasso precursor sequences into a receiver expression vector. The receiver vector (pLAM106) was constructed via overlap assembly (NEBuilder HiFi DNA Assembly, New England Biolabs) from DNAfragments encoding aP15a origin, chloramphenicol resistance cassette CmR, sacB countersei ection marker (Bacillus sublilis). and pl 19 promoter from the Anderson promoter collection (Identifier: BBa_J23119, iGEM Registry of Standard Biological Parts). Correct assembly following transformation into E. coli DH5a (New England Biolabs) was validated via Sanger sequencing (Genewiz, by Azenta).

[0135] Lasso peptide mcjA precursor sequences with flanking Bsal cut-sites and relevant overhang sequences were synthesized as eBlocks (Integrated DNA Technologies) and directly amplified using Q5 polymerase (2X master mix, New England Biolabs) using primers oLAM59 and 0LAM6O (Table 5). Following DNA clean up (ZYMO DNA clean and concen- trator-5, ZYMO Research), purified PCR products were used in type-IIs restriction / ligation assembly in a 15 mL reaction mix with pLAM106, T4 Ligase, IX T4 Ligase Buffer, Bsal-HF v2, IX NEB rCutsmart Buffer (all enzymes and buffers from New England Biolabs). Initial 20-minute digestion at 37°C was followed by 15 cycles of alternating 37°C incubation (10 minutes) and 16°C incubation (10 minutes). Reactions were then held at 16° C indefinitely prior to transformation into chemically competent E. coli cells. Transformations were carried out in either DH5aE. coli (New England Biolabs) for plasmid stocking orin BL21 E. coli (New England Biolabs) containing the pLAM58 biosynthetic plasmid. Completed transformations were plated on LB media containing the appropriate antibiotics (chloramphenicol or kanamycin + chloramphenicol) and 10% sucrose to select against undigested pLAM106 plasmids retaining the sacB cassette. Assemblies of mcjA variants into pLAM106 are referred to herein as “A-vectors.”

[0136] Generation of McjC site-directed mutants. A custom Golden Gate vector system was constructed for the generation of site-directed mutants of the cyclase mcjC and re-incorporation into the B-plasmid biosynthetic plasmid. Briefly, mcjC was amplified using primers Donor5 and Donor6 (Table 5), while the backbone of pLAM106 was amplified with primers Donor7 and Donor8 (Table 5). Both fragments were assembled via overlap assembly (NEBuilder HiFi DNA Assembly, New England Biolabs), replacing the sacB cassette with the mcjC coding sequence while retaining flanking Bsal cut sites and overhangs, generating donor vector pDonor2 to encode putative cyclase mutants. Similarly, a receiver vector pReceiver2 was generated from amplifying pLAM58 using primers Receives and Received and amplifying the sacB cassette from pLAM106 using primers Receives and Receive? (Table 5). Both fragments were assembled via overlap assembly (NEBuilder HiFi DNA Assembly, New England Biolabs), replacing the mcjC vector with the sacB cassette and introducing Bsal cut-sites and overhangs. Both resultant plasmids were fully sequenced to ensure fidelity.

[0137] Site directed mutations (SDMs) were made directly to the donor vector encoding mcjC using a partial-overlap primer-driven mutagenesis strategy described previously (Liu, H., Nai- smith, J.H. An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol. BMC Biotechnol. 2008, 8, 91. Doi: 10.1186 / 1472-6750-8-91). All mutagenesis primers are available in Table 5. Overlapping primers were added to a reaction containing the donor plasmid (~1 ng / uL) and Q5 polymerase master mix. Thermocycling conditions were optimized following the recommendations described previously (Doi: 10.1186 / 1472-6750-8-91), with sufficient extension time to amplify the entire circular backbone over 25-30 cycles. Following post-PCR clean-up and concentration (ZYMO DNA Clean & Concentrator, Zymo Research), reaction products were transformed into chemically competent Dh5a to promote nick repair and generate enough plasmid to verify SDMs via Sanger Sequencing (GeneWiz, Azenta).

[0138] Donor plasmids bearing the desired mutations were then included in Golden Gate reactions alongside the pLAM58-derived receiver vector to introduce mutant cyclases into the B-vector expression system in a scarless, error-free fashion. Reaction conditions identical to those described above for A-vector assembly were employed. Reaction products were then transformed into Dh5a E. coli and plated on sucrose-containing LB for counter selection. Correctly assembled B-plasmids were then purified and co-transformed with A-plasmids containing lasso peptide sequences of interest into the BL21 expression system for downstream lasso peptide production, quantification and purification.

[0139] Small scale production of SEQ ID NOS: 1-8 and engineered cyclase screening against select lassos. A preculture was generated by adding 600 mL of LB broth + 50 mg / mLKanamycin + 17 mg / mL Chloramphenicol to each well of a 96 deep-well cell culture plate. Single colonies for each engineered lasso peptide or screen cyclase variant were added to triplicate wells. Pre-cultures were grown overnight in shakers at 37°C, 200 rpm. After 14-16 hrs, small-scale production cultures were generated from the pre-cultures using supplemented M9 media containing the appropriate antibiotics. Supplemented, antibiotic-containing M9 media (630 mL) was added to each well of a new 96 deep-well plate and were inoculated with 20pL of the LB preculture. M9 cultures were allowed to grow for 3-4 hours at 30°C shaking at 200 rpm before increasing the speed to 900 rpm. Cultures were then allowed to grow for 3 days (~72 hours) at 30°C, 900 rpm prior to harvesting. After 3 days of shaking, the lasso peptides produced were extracted from the whole bacterial culture broth as described below and analyzed by LCMS for production titers. All engineered lasso peptides that were produced at titers > 0.5 mg / L were scaled up in shake flask cultivations, as described below.

[0140] Production of SEQ ID NOS: 1-8 (Table 2). High producing clones from small-scale production of Lassos 1-8 were selected and targeted for large scale culturing (> IL). M9 supplemented media was prepared from 5X M9 salts (Teknova, Hollister, CA, USA) by first autoclaving the IX M9 media and then adding the remaining components Precultures were generated by inoculating individual sequence verified E. coli colonies containing the “A” and “B” plasmids for each engineered lasso peptide from either a patch plate or a premade glycerol stock into 10 mL of LB broth in culture tubes with 50 mg / mL of kanamycin and 17 mg / mL of chloramphenicol and shaking overnight at 37°C, 200 rpm. Precultures should reach about OD600 of 2 to 3 after 16 hours incubation. Subsequently, 2 mL of the corresponding preculture was inoculated into 50 mL of LB broth in 250 mL baffled flasks with 50 mg / mL of kanamycin and 17 mg / mL of chloramphenicol to reach OD600 > 0.1. The pre-cultures were then allowed to grow for 4-5 hours at 37°C, 200 rpm to reach target OD between 2 to 3. These final precultures (50 mL) then were inoculated into 950 mL of M9 media supplemented with 50 mg / mL of kanamycin and 17 mg / mL of chloramphenicol in a 2 L baffled shake flask. Production cultures were grown for 3 days at 30°C, 200 rpm, after which lasso titer was assessed and engineered lasso peptides were isolated and purified as described below.

[0141] LCMS data acquisition: Samples were analyzed using an Agilent QQQ-LCMS system, which consisted of a 1290 Infinity II UHPLC, photodiode array, Jet Stream Source, and a 6460C QQQ. Samples were injected (1 pL) onto a Phenom enex Kinetex XB-C18 column (1.7 pm, 50 x 2.1 mm) operating at 0.4 mL / min with 1 minute at 5% acetonitrile (+ 0.1% (v / v) formic acid), a gradient of 5% to 95% over 5 minutes, and a 1 minute hold at 95%.

[0142] Standard curve: A Standard curve was generated using Lasso 2 and used to quantify all variants. Samples containing 20, 8, 3.2, 1.3, and 0.5 pg / mL of the purified lasso peptide (prepared by serial dilution of a 500 pg / mL DMSO stock into 75% methanol) were injected onto the LCMS. The peak area of the 220 nm UV absorbance peak was determined and plotted to produce the standard curve.

[0143] Analytical sample analysis: Samples were prepared from whole broth cultures by transferring 600 pL of each whole-broth sample to a deep well plate, lyophilizing, and resuspending in 300 pL 75% methanol. The plate was vortexed, sonicated, then centrifuged. The resulting supernatant was analyzed on the LCMS. 220 nm UV peaks that corresponded to MS peaks consistent with predicted peptide masses were integrated, and the resulting peak areas were quantified using the standard curve. UV and MS peak identity was confirmed by comparison to a negative control sample, and only peaks unique to each lasso peptide strain were used for analysis.Lasso Peptide Isolation

[0144] Lasso peptides shown in Table 2 were extracted from the whole cell broth by first centrifuging the broth in 750 mL Nalgene bottles, then separating the supernatant from the cell pellet. The resulting supernatant was then subjected to solid phase extraction (SPE). SPE columns were prepared as follows: For every liter of culture volume, 12.5 g of HP20ss resin (Itochu) was packed into an empty fritted column. The column was washed with 5 column volumes of MeOH, then equilibrated with 5 column volumes of deionized water. The clarified cell broths (via centrifugation at 5,000 x g) were loaded directly to a prepared HP20ss column and fractionated as follows: The column was washed with 125 mL deionized water, then eluted with 125 mL 30% MeOH / water, 125 mL 50% MeOH / water, 125 mL 75% MeOH / water, and 125 mL 100% MeOH. Each fraction was run on the LC-MS to determine which contained the lasso peptide and approximate the quantity. Fractions were run on the LCMS quantification method to determine which contained lasso peptide, and those fractions were pooled and concentrated to dryness on a rotary evaporator and lyophilizer. These fractions were then further purified by preparative HPLC (method below).

[0136] Preparative HPLC. Preparative HPLC was carried out using an Agilent 1100 or 1260 purification system (ChemStation software, Agilent) equipped with an autosampler, multiple wavelength detector, Prep-LC fraction collector and Phenomenex Luna 5pm C18(2) 150 x 30 mm preparative column. The 1260 system additionally included an Agilent MSD. Fractionscontaining lasso peptides were identified using the LCMS method described above prior to combining and lyophilizing. Product quality control (QC) was performed on the pooled and concentrated lasso fractions (see Product QC method below). The preparative HPLC method included the following:Column: Phenomenex Luna® preparative column 5 pM, C18(2) 100 A 150 x 30 mmFlow rate: 20 mL / minTemperature: RTMobile Phases: HPLC grade water, MeOH, acetonitrile, isopropyl alcohol, trifluoroacetic acid (TFA), in different percentages and used as gradientsInjection amount: variable 5-200 pLExample method: Solvent A is water with 0.05% TFA, solvent B is acetonitrile with 0.05% TFA. 24% - 26% B over 20.0 min, then 20% to 95% B over 1 minute followed by 95% B for 3 minutes. 5-minute post run equilibration time.

[0137] Semi-preparative HPLC. If necessary, semi -preparative HPLC purifications were performed on an Agilent 1100 Series Instrument with a multiple wavelength detector. The semipreparative HPLC method included the following:Column: Phenomenex Luna 5pm Cl 8(2) 250 x 10 mmFlow rate: 4 mL / minTemperature: RTMobile Phases: water, MeOH, acetonitrile, trifluoroacetic acid, in different percentages and used as gradientsInjection amount: variable 0.01 to 1.0 mL

[0138] Sample QC The purity of each eluted parent scaffold peptide and engineered lasso peptide shown in Table 1 was examined by LC-MS on an Agilent 6460C Triple Quadrupole LC / MS system (LC / TQ) equipped with a Jet stream source (AJS), an Agilent 1290 Infinity II LC system, and a diode array detector (DAD). Where possible, MSMS fragmentation was used to further characterize lasso peptides based on the rule described in Fouque, et al., Analyst, 2018, 143, 1157-1170, and to confirm amino acid sequences. Proton NMR and high-resolution LCMS data were acquired as described above to further confirm peptide structures. The analytical LCMS method for purity assessment included the following:Column: Phenomenex Kinetex 1.7 pm XB-C18 100 A, 50 x 2.1 mm column.Flow rate: 0.4 mL / minTemperature: 40 °CMobile Phase A: 0.1% formic acid in water (LCMS grade)Mobile Phase B: acetonitrile (LCMS grade)Injection amount: 1 pLHPLC Gradient: 5% B for 1.0 min, then 5 to 50% B over 7 minutes followed by 50 to 95% B over 2 min and 95% B for 2 min. 2-minute post run equilibration time.

[0139] After a blank subtraction, the peaks from the TIC, UV210, and UV280 signals were integrated, and purity was reported as the Area Sum % for each signal using Agilent Mas- sHunter Qualitative Analysis 10.0.

[0140] Lasso cyclase substrate binding site and catalytic pocket prediction. The McjC active site pocket volume for the AlphaFol d2 predicted structure was calculated using the CASTp web server (http: / / sts.bioe.uic.edu / castp / index.html). avP8 IC50 assays

[0141] The integrin inhibiting activity and selectivity of all parent scaffold peptides and engineered lasso peptides shown in Table 2 were determined using ELISA or AlphaLISA assays. Human integrins avP8 was purchased from R&D Systems (Minneapolis, MN) and biotinylated avP8 was purchased from Aero Biosystems (Newark, DE). LAP protein was used as the standard substrate for avP8 competitive binding and inhibition assays and was purchased from R&D Systems (WI, USA).ELISA assays

[0142] For the ELISA assays, clear flat-bottom immune-nonsterile 96-well plates were coated with 50 mL of 0.4 pg / ml LAP (R&D Systems, cat. no. 246-LP) in carbonate buffer (15 mM Na2COs, 35 mM NaHCCh, pH 9.6) overnight at 4 °C. The plates were then washed three times with 200 mL PBST (PBS, 0.05% Tween 20) containing 1 mM MgCh in a Tecan Hydroflex plate washer. The plates were blocked with 150 mL TSB buffer (20 mM Tris HC1 [pH 7.5], 150 mM NaCl, 1 mM CaCh, 1 mM MgCh, 1 mM MnCh, 1% BSA) for 1 hour at room temperature. The plates were washed three times with 200 mL PBST containing 1 mM MgCh. To each well, 50 mL of avP8 (R&D Systems, cat. no. 4135-AV-050), that was preincubated with serially diluted inhibitor (from 0.001 nM to 10 pM) for at least 30 minutes in TSB buffer, was added and incubated for 1 hour at room temperature. Each concentration of inhibitor was performed in duplicate. The plates were then washed three times in 200 pl PBST (10 mMNa2HPO4, pH 7.5, 150 mM NaCl, and 0.01% Tween 20) containing 1 mM MgCh. To each well, 50 mL of 0.5 pg / mL mouse anti-human integrin av (CD51) monoclonal antibody targeting the av subunit (R&D Systems, cat. no. MAB1219) was added and incubated for 1 hour at room temperature. The plates were then washed three times in 200 mL PBST containing 1 mM MgCh. To each well, 50 mL of goat anti-mouse IgG peroxidase conjugate (Sigma, cat. no. DC02L) at a concentration of 0.07 pg / ml was added and incubated for 1 hour at room temperature. The plates were then washed three times in 200 mL PBST containing 1 mM MgCh. To each well, 50 pL TMB substrate (ThermoFisher, cat. no. 34021) was added and incubated for 10-30 minutes at room temperature. The reaction was terminated with 50 pL 6N H2SO4. The plate was then measured at an OD450 in a Perkin Elmer Victor Nivo multimode plate reader (Perkin Elmer, San Jose, CA). The resulting inhibition curves are analyzed using GraphPad Prism software. The inflection point of these curves describes the IC50 value. The IC50 values for all integrin assays were calculated using non-linear regression analysis with GraphPad PRISM expressed as the concentration of inhibitor bound to the integrin.AlphaLISA assays

[0143] For AlphaLISA, 0.4 pg / ml biotinylated avP8 (Aero Biosystems, cat. no. IT6-H82E4) was incubated with 0.72 pg / ml LAP with serially diluted inhibitor (from 0.001 nM to 10 pM) in binding buffer (25 mM HEPES [pH 7.4], 137 mM NaCl, 1 mM MgCh, 1 mM MnCh, 2 mM CaCh, 2.7 mM KC1, and 0.05% Tween-20) in a total volume of 16 pl for 1.5-2 hrs in an Al- phaPlate-384 plate (Perkin Elmer, cat. no., 6005350). To each well, an 8 pl volume of antigoat IgG acceptor beads (Perkin Elmer, cat. No. AL107C) at a concentration of 100 pg / ml and anti-human LAP (R&D Systems, cat. no. AF-246-NA) at a concentration 3.84 pg / ml in binding buffer was added and incubated for 1 hr. To each well, an 8 pl volume of streptavidin beads (Perkin Elmer, cat. no. 67669992) at a concentration of 50 pg / ml was added and incubated for 45 minutes. The plate was then measured in a Perkin Elmer Victor Nivo multimode plate reader at excitation / emission of 680 nm / 615 nm. For each inhibitor, assays were done in duplicate and the resulting inhibitor curves were analyzed using GraphPad Prism software. The inflection point of these curves describes the IC50 value. The IC50 values for all integrin assays were calculated using non-linear regression analysis with GraphPad PRISM expressed as the concentration of inhibitor bound to the integrin.EXAMPLE 1. Selection of microcin J25 scaffold for discovery of integrin av[38 inhibitors

[0144] Microcin J25 (MccJ25, SEQ ID NO 1) contains 21-amino acids and represents one of the best characterized examples of a lasso peptide. Naturally occurring MccJ25 is produce by E. coli and has potent antimicrobial properties by virtue of binding to RNA polymerase inside the cells of certain other bacteria (Semenova, E., et al., J. BacterioL 2005, 187(11), 3859- 3863). The biosynthetic pathway and enzymes forming MccJ25 have been elucidated and pro- totypically involve a peptidase (McjB) and cyclase (McjC), along with an ABC-transporter (McjD) (Duquesne, S., et al., Chem. BioL, 2007, 14, 793-803) for exporting the antimicrobial lasso peptide. MccJ25 is a lasso peptide with an 11 -residue loop that can be substituted at most positions with at least one other amino acid (Yan, K-P., et al., ChemBioChem, 2012, 13, 1046- 1052; Pavlova, O., et al., J. Biol. Chem., 2008, 283, 25589-2559), making it an attractive scaffold for engineering novel lasso peptide sequences aimed at discovering new therapeutics (See FIG. 4 for MccJ25 structure and amino acid sequence and numbering). Accordingly, the lasso cyclase of the MccJ25 biosynthetic pathway, namely McjC, is considered tolerant to amino acid substitutions at defined positions across the scaffold. For example, the MccJ25 scaffold has been shown to be tolerant to incorporation of integrin-binding epitopes such as Arg-Gly-Asp (RGD; Hegemann, J.D., et. al., J. Med. Chem., 2014, 57, 5829-5834), and thus can serve as a versatile starting point for the discovery of potent inhibitors of integrins. One such integrin that could be inhibited by MccJ25 variants is av08, which is abundantly overexpressed on tumors (Takasaka, N. et al. JCI Insight 3, 2018. Doi: 10.1172 / jci. insight.122591) and tumor-associated regulatory T-cells (Worthington, J. J. et al. Immunity, 2015, 42, 903- 915). Integrin avP8 also is one of the primary activators of immunosuppressant TGF0 in the tumor microenvironment (Laine, A., et al., Nature Comm. 2021, 12:6228. Doi: 10.1038 / s41467-021-26352-2), where TGF0 is sequestered by LAP protein until it is released by binding to integrins such as avP8 (Dodagatta-Marri et al., Cell Reports, 2021, 36, 109309. Doi: 10.1016 / j.celrep.202L 109309).EXAMPLE 2. Using microcin J25 cyclase (McjC) for av[38 inhibitor production

[0145] Natural lasso cyclase McjC was engineered to enhance production of desired novel variants of microcin J25. The desired novel analogs of microcin J25 served as avP8 integrin inhibitors. The known avP8 integrin-binding epitope, Arg-Gly-Asp-Leu (RGDL (SEQ ID NO: 25) - an epitope derived from the TGFP-LAP protein), was introduced into MccJ25 loop. RGDL (SEQ ID NO: 25) was grafted into positions 12-15 of SEQ ID NO: 1 by replacing GIGT(SEQ ID NO: 28) to generate SEQ ID NO: 2, which was found to be a modest competitive inhibitor of integrin av08 (ICso = 271 nM measured vs LAP protein by ELISA assay; Table 2). The lasso peptide loop size and binding epitope position can greatly impact how well an epitope binds to the target protein of interest.

[0146] Further improvement of avP8 inhibition was achieved by deletion and mutagenesis of multiple residues across SEQ ID NO: 2 (FIG. 5). Deletion of P16 in SEQ ID NO: 2 afforded SEQ ID NO: 3 with improved avP8 ICso = 116 nM (Table 2). Evolution experiments involving mutagenesis of residues in the loop and ring of SEQ ID NO: 3 were conducted. The isoleucine at position 16 of SEQ ID NO: 3 was mutated to glutamine (I16Q) to yield SEQ ID NO: 4, which showed improved inhibition of avP8, with ICso = 16 nM (Table 2).

[0147] Unfortunately, certain desired avP8 integrin inhibitor sequences were produced poorly or in quantities too small to be quantified. Marked decreases in lasso analog production titers were observed when multiple mutations and specific changes were introduced to enhance the potency of avP8 inhibitors, such as SEQ ID NO: 4 which was produced at 9x lower titer relative to SEQ ID NO: 3. Introduction of an A3R mutation in SEQ ID NO: 4 led to SEQ ID NO: 5, which showed significantly improved avP8 inhibition (ICso = 5.9 nM), but with a further 3.5x concomitant reduction in production titer. Decreased production of SEQ ID NOS: 4 and 5 was postulated to be due to lower wild-type cyclase tolerance to specific substitutions (e.g., I16Q) and / or lower acceptance of non-natural substrates as the number of mutations increased and the more the sequence diverged from the natural lasso peptide. Since desired analogs, such as SEQ ID NOS: 4 and 5, were not produced or were produced at low levels using the wild-type cyclase (McjC), cyclase engineering was implemented to broaden the amino acid tolerance of McjC and to enhance production of promising analogs that inhibit integrin avP8 with high potency.EXAMPLE 3. Engineering McjC for improved integrin inhibitor production

[0148] Cyclase McjC is composed of 513 amino acids and its structure has not been determined experimentally. The tertiary structure of cyclase McjC was predicted by introducing the cyclase sequence into AlphaFold 2 (Yang, Z., et al., Sig. Transduct. Target. Ther., 2023, 8, 115. Doi: 10.1038 / s41392-023-01381-z) (FIG. 6). The algorithm CASTp 3.0 (Tian, W., et al., Nucleic Acids Res., 2018, 46 (Web Server issue), W363-W367. Doi: 10.1093 / nar / gky473) was used to predict the amino acid residues that constitute thesubstrate binding site and catalytic pocket of the McjC structure predicted by AlphaFold2. Prefolded MccJ25 core peptide and final lasso product MccJ25 were docked into the substrate binding site of the cyclase structure predicted by AlphaFold2 and CASTp 3.0 using MOE. Analysis of the enzymatic pocket with docked pre-folded core peptide substrate and MccJ25 allowed a group of 15 different residues to be predicted to be within interacting distance (< 6 Angstroms) of the incipient lasso peptide and identified as amino acids that may impact the catalytic folding process. Ten cyclase amino acid side chains, including K252 and K388, were found to extend into the McjC catalytic pocket and were predicted to potentially impact interaction between enzyme and the incipient loop of the lasso peptide as it is being formed (FIG. 7A). The identified 10 residues were mutated to alanine, along with 5 additional residues that were believed to be important for catalytic activity, including the known loss-of-function mutant D302A which is involved in ATP binding (Yan, K-P, et al., ChemBioChem, 2012, 13, 1046-1052). The 15 McjC variants were screened for improved functional activity by monitoring production levels of SEQ ID NO: 1 (MccJ25) and SEQ ID NO: 5 when the genes for the biosynthetic pathway were co-expressed with each McjC mutant in E. coli (Table 1). The functional screen revealed two substitutions in McjC, K252A and K388A, that significantly improved the production of SEQ ID NO: 5. Improvement in production of SEQ ID NO: 5 by substituting large positively charged lysine residues with small neutral alanine residues was consistent with the model and the prediction that identified residues in the pocket could interact with the incipient lasso peptide loop and ring, each of which contain a positively charged arginine residue (FIG. 7B).EXAMPLE 4. Use of engineered McjC variants for improved integrin inhibitor production

[0149] Three engineered McjC variants bearing the mutations K252A, K388A, and the double mutant (K252A / K388A) were tested for production of a full series of eight substrates and compared with wild-type McjC (Table 2). Wild-type MccJ25 (SEQ ID NO: 1) was produced at >3-fold higher titers using the McjC double variant (0.57 g / L), which is 70-fold higher than other literature reports (Pan, S. J., et al., Protein. Expr. Purify 2010, 71, 200-206). Similarly, the McjC variants produced SEQ ID NOS: 4 and 5 at 10- and 20-fold higher titers, respectively. SEQ ID NOS 6-8, which were recalcitrant to production using wild-type McjC, were now produced with greatly improved titers that allowed isolation and testing for integrin inhibition. SEQ ID NOS: 6-8 bearing the expanded QRGDLQ epitope (SEQ ID NO: 27) displayed ~4-fold more potent av08 inhibition vs SEQ ID NO: 5 (Table 2). The engineered McjC cyclases enabled ample production of SEQ ID NO: 8, which contains an unprecedented 10 mutations and a deletion relative to parent SEQ ID NO: 1, including the QRGDLQ epitope (SEQ ID NO: 27) at loop positions 11-16 and four ring mutations (A3R, H5S, V6I, and P7I), thus affording the most potent av08 inhibitor with ICso = 1.5 nM. This improvement in ICso was only accessible through these cyclase variants, highlighting the importance of cyclase engineering to develop lasso peptides as potential novel therapeutics.

[0150] Table 5. Primers used in these Examples. All sequences are 5’ to 3’. Cloning methods: SDM, site directed mutagenesis (Quikchange); BEL, blunt end ligation; OEP, overlap extension PCR. _F, forward primer; _R, reverse primer.EXAMPLE 5. McjC variants Inhibition of avp8

[0151] ELISA assays were performed as described herein for SEQ ID NOS: 1, 2, 3, 5, 6, 7, and 8 in the presence of avP8 and LAP (natural ligand). An AlphaLISA assay was performed as described herein for SEQ ID NO: 4 in the presence of biotinylated avP8 and LAP. For each assay, duplicates at each inhibitor concentration were performed. Curves and ICso values were calculated using GraphPad PRISM. Representative ICso curves for lasso peptide inhibition of avP8 are shown in FIG. 8.ReferencesMontalban-Lopez, M. et al. New developments in RiPP discovery, enzymology and engineering. Nat. Prod. Rep. 2021, 38, 130-239.Duquesne, S., Destoumieux-Garzon, D., Zirah, S., Goulard, C., Peduzzi, J., and Rebuffat, S. Two Enzymes Catalyze the Maturation of a Lasso Peptide in Escherichia coli. Chem. BioL, 2007, 14, 793-803.Yan, K-P., et al., Dissecting the Maturation Steps of the Lasso Peptide Microcin J25 in vitro. ChemBioChem, 2012, 13, 1046 - 1052. Doi: 10.1002 / cbic.201200016.Wang et al., Recent Advances and Perspectives on Expanding the Chemical Diversity of Lasso Peptides, Front. Bioeng. Biotechnol. 2021 9:741364. doi: 10.3389 / fbioe.2021.741364Hegemann, J. D. et al., Lasso Peptides: An Intriguing Class of Bacterial Natural Products. Acc. Chem. Res. 2015, 48, 1909-1919.Hegemann, J. D., et al. Caulosegnins I— III: AHighly Diverse Group of Lasso Peptides Derived from a Single Biosynthetic Gene Cluster. J. Am. Chem. Soc., 2013, 135, 210-222.Ducasse, R. et al. Sequence Determinants Governing the Topology and Biological Activity of a Lasso Peptide, Microcin J25. ChemBioChem, 2012, 13, 371-380.Zong, C., et al., Construction of Lasso Peptide Fusion Proteins. ACS Chem. Biol. 2016, 11, 61-68.Hills, E., et al., Comprehensive Mutational Analysis of the Lasso Peptide Klebsidin. ACS Chem. BioL, 2022, 17(4), 998-1010. Doi: 10.1021 / acschembio.2c00148.Pavlova, O., et al., Systematic Structure- Activity Analysis of Microcin J25. J. Biol. Chem., 2008, 283, 25589-2559. Doi: 10.1074 / jbc.M803995200.Thokkadam, A. et al., High-Throughput Screen Reveals the Structure-Activity Relationship of the Antimicrobial Lasso Peptide Ubonodin. ACS Cent. Sci., 2023, 9, 540-550. Doi: 10.1021 / acscentsci.2c01487.Inokoshi, J., et al., Structure-activity analysis of Gram-positive bacterium-producing lasso peptides with antimycobacterial activity. Sci. Report, 2016, 6:30375. Doi: 10.1038 / srep30375.Knappe, T. A., et al., Insights into the Biosynthesis and Stability of the Lasso Peptide Capistruin. Chemistry & Biology, 2009, 16, 1290-1298. Doi:10.1016 / j.chembiol.2009.11.009.Liu, T. et al., Rational generation of lasso peptides based on biosynthetic gene mutations and site-selective chemical modifications. Chem Sci., 2021, 12(37), 12353-12364. Doi: 10.1039 / dlsc02695j.Si, Y, et al., Cell-Free Biosynthesis to Evaluate Lasso Peptide Formation and Enzyme- Substrate Tolerance. J. Am. Chem. Soc., 2021, 143, 5917-5927. Doi: 10.1021 / jacs. lc01452.Zyubko, T., et al., Efficient in vivo synthesis of lasso peptide pseudomycoidin proceeds in the absence of both the leader and the leader peptidase, Chem. Sci., 2019, 10, 9699-9707. Doi: 10.1039 / c9sc02370d.Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature, 2021, 596, 583-589. Doi: 10.1038 / s41586-021-03819-2.Yang, Z., et al. AlphaFold2 and its applications in the fields of biology and medicine. Sig. Transduct. Target. Ther., 2023, 8, 115. Doi: 10.1038 / s41392-023-01381-z.Johansson- Akhe, I., and Wallner, B. Improving peptide-protein docking with AlphaFold-Multimer using forced sampling. Front. Bioinform., 2022, 2:959160. Doi: 10.3389 / fbinf.2022.959160.Du, Z., et al., The trRosetta server for fast and accurate protein structure prediction. Nature Protoc., 2021, 16(12), 5634-5651. Doi: 10.1038 / s41596-021-00628-9.Tian, W, et al., CASTp 3.0: computed atlas of surface topography of proteins. Nucleic Acids Res., 2018, 46 (Web Server issue), W363-W367. Doi: 10.1093 / nar / gky473.Zhu X., Xiong Y, Kihara D. Large-scale binding ligand prediction by improved patch-based method Patch- Surfer2. 0. Bioinformatics, 2015, 31, 707-713. Doi: 10.1093 / bioinformat- ics / btu724.Yang J., Roy A., Zhang Y Protein-ligand binding site recognition using complementary binding-specific substructure comparison and sequence profile alignment. Bioinformatics. 2013, 29, 2588-2595. Doi: 10.1093 / bioinformatics / btt447.Krivak R., Hoksza D. P2Rank: machine learning based tool for rapid and accurate prediction of ligand binding sites from protein structure. J Cheminf, 2018, 10, 39. Doi: 10.1186 / sl3321- 018-0285-8.Wu Q., Peng Z., Zhang Y, Yang J. COACH-D: improved protein-ligand binding sites prediction with refined ligand-binding poses through molecular docking. Nucleic Acids Res., 2018, 46, W438-W442. Doi: 10.1186 / sl 3321 -018-0285-8.Jimenez J., Doerr S., Martinez-Rosell G., Rose A.S., De Fabritiis G. DeepSite: protein-binding site predictor using 3D-convolutional neural networks. Bioinformatics, 2017, 33, 3036- 3042. Doi: 10.1093 / bioinformatics / btx350.Yang, K.K., et al., Machine-leaming-guided directed evolution for protein engineering, Nature Methods, 2019, 16(8), 687-694. Doi: 10.1038 / s41592-019-0496-6.Hegemann, J.D., et. al., Rational Improvement of the Affinity and Selectivity of Integrin Binding of Grafted Lasso Peptides. J. Med. Chem., 2014, 57, 5829-5834.Doi: 10.1021 / jm5004478.Takasaka, N. et al. Integrin avP8-expressing tumor cells evade host immunity by regulating TGF-P activation in immune cells. JCI Insight 3, 2018. Doi: 10.1172 / jci. insight.122591.Worthington, J. J. et al. Integrin avP8-Mediated TGF-P Activation by Effector Regulatory T Cells Is Essential for Suppression of T-Cell-Mediated Inflammation. Immunity, 2015, 42, 903-915. Doi: 10.1016 / j.immuni.2015.04.012.Laine, A., et al., Regulatory T cells promote cancer immune-escape through integrin avP8- mediated TGF-P activation. Nature Comm. 2021, 12:6228. Doi: 10.1038 / s41467-021-26352- 2.Dodagatta-Marri et al., Integrin avP8 on T cells suppresses anti -turn or immunity in multiple models and is a promising target for tumor immunotherapy. Cell Reports, 2021, 36, 109309. Doi: 10.1016 / j.celrep.2021.109309.Severinov, K., et al., Low-molecular-weight post-translationally modified microcins, Mol Microbiol., 2007, 65(6), 1380-1394. Doi: 10.1111 / j. l365-2958.2007.05874.x.Semenova, E., et al., Structure-Activity Analysis of Microcin J25: Distinct Parts of the Threaded Lasso Molecule Are Responsible for Interaction with Bacterial RNA Polymerase.J. Bacteriol. 2005, 187(11), 3859-3863. Doi: 10.1128 / JB.187.11.3859-3863.2005.Pan, S. J., et al., Engineered gene clusters for the production of the antimicrobial peptide microcin J25. Protein. Expr. 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Claims

1. An engineered lasso cyclase that tolerates amino acid sequences of a lasso peptide that are not tolerated by a wild-type or parent lasso cyclase during enzymatically catalyzed cyclization of a lasso core peptide substrate leading to formation of a properly folded lasso peptide.

2. The engineered lasso cyclase of claim 1, wherein the engineered lasso cyclase forms a properly folded lasso peptide with at least 20% higher titer, rate, and / or yield relative to the wild-type or parent lasso cyclase.

3. The engineered lasso cyclase of claims 1 and 2, wherein the engineered lasso cyclase comprises a variant amino acid sequence of the parent lasso cyclase, wherein the parent lasso cyclase is McjC (SEQ ID NO: 18).

4. The engineered lasso cyclase of any one of claims 1 to 3, wherein the engineered lasso cyclase comprises a mutation at amino acid position K388.

5. The engineered lasso cyclase of claim 4, wherein the engineered lasso cyclase comprises a K388A mutation.

6. The engineered lasso cyclase of any one of claims 1 to 3, wherein the engineered lasso cyclase comprises a mutation at amino acid position K252.

7. The engineered lasso cyclase of claim 6, wherein the engineered lasso cyclase comprises a K252A mutation.

8. The engineered lasso cyclase of any one of claims 1 to 3, wherein the engineered lasso cyclase comprises a mutation at amino acid positions K252 and K388.

9. The engineered lasso cyclase of claim 8, wherein the engineered lasso cyclase comprises K252A and K388A mutations.

10. The engineered lasso cyclase of any one of claims 1 to 9, wherein the engineered lasso cyclase comprises one or more features in comparison to the wild-type or parent lasso cyclase, wherein the features comprise one or more of: i) higher cyclase stability to heat; ii) increased titer, rate, and / or yield of a lasso peptide product; and iii) increased acceptance of non-natural amino acids.

11. The engineered lasso cyclase of any one of claims 1 to 10, wherein the engineered lasso cyclase produces a lasso peptide comprising an amino acid sequence corresponding to any one of SEQ ID NOs: 1-8.

12. The engineered lasso cyclase of any one of claims 1 to 11, wherein the engineered lasso cyclase produces a lasso peptide comprising the amino acid sequence corresponding to any one of SEQ ID NOs: 1-8 at a greater titer than the wild-type or parent lasso cyclase.

13. The engineered lasso cyclase of claim 12, wherein the engineered lasso cyclase produces a lasso peptide comprising the amino acid sequence corresponding to any one of SEQ ID NOs: 4-8 at a 2-fold to 20-fold greater titer than the wild-type or parent lasso cyclase.

14. The engineered lasso cyclase of any one of claims 1 to 13, wherein the engineered lasso cyclase produces a lasso peptide that is an inhibitor of an integrin or an integrin receptor.

15. The engineered lasso cyclase of claim 14, wherein the lasso cyclase produces a lasso peptide that is an inhibitor of integrin avP6 or avP8.

16. A recombinant nucleic acid encoding the engineered lasso cyclase of any one of claims 1 to 15.

17. The recombinant nucleic acid of claim 16, wherein the recombinant nucleic acid comprises a nucleotide sequence encoding the engineered lasso cyclase operatively linked to a promoter.

18. A vector or plasmid comprising the recombinant nucleic acid of claim 16 or 17.

19. A non-naturally occurring microbial organism comprising the recombinant nucleic acid of claim 16 or 17, or the vector or plasmid of claim 18.

20. A method of producing an engineered lasso cyclase with improved properties or functional performance in comparison to the wild-type or parent lasso cyclase, the method comprising one or more of:(xi) building a computational model based on a known lasso cyclase structure or predicting a lasso cyclase structure;(xii) predicting a lasso cyclase substrate binding site and catalytic pocket;(xiii) docking an ensemble of pre-folded lasso core substrates in different conformational states that simulate transformation of substrate to lasso peptide product in the modeled cyclase substrate binding site and catalytic pocket;(xiv) identifying and selecting one or more cyclase residues that: a. are predicted by computational modeling and conformational dynamics simulations to extend into the substrate binding and catalytic pocket; b. are predicted to engage in atomic interactions (attractive, neutral, or repulsive) with the docked pre-folded lasso core substrates and / or lasso peptide product, whereby an interaction is defined as atoms of the cyclase and lasso substrate that approach within < 6 Angstroms of each other; c. reside < 6 Angstroms from a cyclase ATP binding site; d. are predicted to approach atoms in residues of a predicted pre-folded or folded loop of the lasso peptide product within a distance of < 6 Angstroms; or e. are predicted to approach atoms in residues of a predicted pre-folded or folded ring of the lasso peptide product within a distance of < 6 Angstroms;(xv) performing mutagenesis of the lasso cyclase at one or more cyclase amino acid residues selected in one or more of (a)-(e); and(xvi) screening of mutated cyclase variants and selecting cyclases with improved properties or functional performance relative to a wild-type or parent lasso cyclase.

21. The method of claim 20, wherein the improved properties or functional performance in comparison to the wild-type or parent lasso cyclase comprises one or more of: i) higher cyclase stability to heat; ii) increased tolerance to amino acid substitutions by a wider range of amino acids in a lasso core peptide substrate or lasso peptide product; iii) increased titer, rate, and / or yield of a lasso peptide product; and iv) increased acceptance of non-natural amino acids.

22. The method of claim 20 or 21, wherein the mutagenesis comprises one or more of alanine scanning, site-directed and random multi-site mutagenesis.

23. The method of any one of claims 20 to 22, wherein the method comprises building the computational model based on atomic coordinates of a lasso cyclase obtained from nuclear magnetic resonance (NMR) analysis, X-ray crystallography, neutron diffraction, or 3-dimensional electron microscopy (3D-EM).

24. The method of any one of claims 20 to 23, wherein the method comprises predicting a structure of target lasso cyclase using a protein structure prediction algorithm.

25. The method of claim 24, wherein the protein structure prediction algorithm comprises an algorithm selected from AlphaFold, AlphaFol d2, and trRosetta.

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