ACS Chemical Biology

Lanthipeptides represent the largest group of ribosomally synthesized and post-translationally modified peptides (RiPPs). Lanthipeptides offer promising avenues for discovering new antibacterial and antifungal agents. Here, we identify and structurally analyze the product of the tla BGC, which encodes a class II lanthipeptide in the thermophilic bacterium Thermoactinomyces sp. DSM 45891. Heterologous co-expression of the lanthipeptide synthetase TlaM resulted in modification of the two precursor peptides TlaA1 and TlaA2, which share 58% identity. TlaA1 was dehydrated seven times and TlaA2 six times. In both peptides, four thioether rings were formed with two overlapping DL-(methyl)lanthionine rings at the C-terminus. Both peptides also contain two central and N-terminal non-overlapping DL-methyllanthionines. These findings demonstrate that these peptides deviate from the general rule of stereoselective LL-(methyl)lanthionine formation from a DhxDhxXxxXxxCys motif (Dhx = dehydroalanine or dehydrobutyrine). AspN-cleaved TlaM-modified TlaA1 displayed anti-microbial activity against a subset of bacteria including Gram-negative ESKAPE pathogens. We named the lantibiotic thermolanthin.

Kdnases have been reported in a variety of organisms, including marine species such as trout and oysters, the opportunistic Gram-negative bacterium Sphingobacterium multivorum, and several fungal species of the genus Aspergillus, including Aspergillus terreus and Aspergillus fumigatus.. In particular, the Kdnase from the opportunistic airborne pathogen Aspergillus fumigatus (AfKdnase) plays an important role in fungal cell wall integrity and virulence, although the underlying mechanisms remain unclear. To better understand this class of enzymes, selective and sensitive tools are required for discovery, detection and visualization of active Kdnases in complex biological samples. In this work, we report the development of difluoro-Kdn mechanism-based probes functionalized with azide and biotin tags for labeling and detection of Kdnases. We show that the probes exhibit selectivity for Kdnase over the neuraminidases tested and efficiently label recombinantly expressed AfKdnase at micromolar concentrations. In addition, using the azide-bearing probe and click chemistry, we successfully visualized native Kdnases in A. fumigatus mycelia, demonstrating their utility for studying these enzymes in crude biological samples and highlighting their potential for discovering Kdnases in other organisms including fungal and bacterial species.

Multidrug efflux pumps transport antibiotics across the cellular membrane resulting in resistance conferred to the host organism. Efflux pump inhibitors (EPIs) potentiate the efficacy of antibiotics by blocking drug efflux and hold promise as adjuvant therapeutics in the fight against multidrug resistant pathogenic bacteria. A hurdle in the field has been the lack of selectivity of small molecule EPIs which often display off-target toxicity due to non-specific binding. To tackle this specificity challenge, we aimed to maximize an inhibitors binding surface area to efflux pumps by designing miniprotein EPIs using computational protein design and an E. coli co-expression assay to screen inhibition in cells. We used S. aureus NorA as a model efflux transporter since it confers drug resistance to fluoroquinolones, puromycin, and other cytotoxic compounds. Starting from a focused miniprotein library of only 86 members, we identified inhibitors in the screen that blocked NorA transport under active efflux conditions in vitro. Our most promising inhibitor I-23 was validated by solving a cryo-EM structure of the miniprotein in complex with NorA, which stabilized the transporter in the outward-open conformation. I-23 has a ferredoxin-like fold with one of its {beta}-hairpins inserted into the substrate binding pocket of NorA and other parts of the globular fold occupying the shallow pocket and making extensive intermolecular contacts with NorA. An arginine residue on the tip of the hairpin loop was positioned near an anionic patch required for NorA antibiotic efflux. The identified structural motifs in this work could be employed to explore the molecular properties of peptidoglycan penetration; full realization of the therapeutic potential of the designed miniprotein inhibitors will require determining the principles for facilitating passage of [~]7 to 8 kDa miniproteins across the peptidoglycan bacterial cell wall.

The LC3/GABARAP protein family is a promising target for selective inhibition of autophagy and for targeted protein degradation. LC3/GABARAP proteins are challenging targets for small-molecule drug development due to their long, shallow binding grooves. In this work, we evaluate multiple approaches to stabilizing the extended structure of the native binding motif, producing N-methylated peptides and stapled peptides with low nanomolar affinity. A crystal structure and molecular dynamics simulations support a model where the N-methylation pre-organizes the motif into an extended, strand-like structure. N-methylation allowed minimization of the binding motif to a tetrapeptide that retained sub-micromolar affinity while minimizing charge and overall molecular weight. The truncated, N-methylated tetrapeptide showed moderate passive permeability. These results highlight more drug-like space for the development of LC3/GABARAP ligands with high affinity and selectivity.

Iron is an essential component of cellular biology. Thus, irons low bioavailability is a key evolutionary pressure guiding microbial dynamics in the marine environment. Among marine bacteria, Microbulbifer is an underexplored and functionally versatile bacterial genus, which is commonly associated with sponges, algae, corals and sediments. Previously, genome analyses have revealed that Microbulbifer spp. can degrade polymers and synthesize natural products. Despite their recognized potential to produce secondary metabolites, siderophores are yet to be identified in Microbulbifer, and their iron acquisition strategies remain largely unknown. Here, we developed a comprehensive mass spectrometry-based query language (MassQL) code to determine siderophore production by Microbulbifer spp. in mono- and mixed culture with a marine pathogen, which can be replicated for discovery of these compounds in any organism. Using this workflow, we discovered a new metallophore, which we named bulbichelin, as well as a suite of previously unreported petrobactins containing unprecedented longer chain length acylation on the central spermidine moiety. We applied genome mining methods to describe the biosynthesis of these compounds. Using metal infusion mass spectrometry, we show that bulbichelins bind a variety of metals. Notably, neither of these compounds were produced in a co-culture of Microbulbifer with coral-derived pathogen Vibrio coralliilyticus Cn52-H1. This observation suggests that Microbulbifer uses alternate strategies in a mixed community, such as siderophore piracy for metal acquisition. Understanding how siderophores shape interspecies interactions between Microbulbifer spp. and other marine organisms will aid in unraveling the chemical and catalytic versatility of this genus and adaptation in nutrient deplete marine environment.

The killer-cell immunoglobulin-like receptors (KIR) are a family of activating and inhibitory HLA class I (HLA-I) binding receptors expressed on natural killer (NK) cells and subsets of T cells. The KIR detect HLA-I molecules in a peptide-dependent manner, with some KIR displaying exquisite peptide-specificity. Studying peptide recognition by KIR often uses TAP-deficient cell lines expressing single HLA-I alleles, which are heterogenous and time consuming to generate. Here, we established an alternative approach using peptide-exchange technologies hitherto developed for studying T cell recognition of HLA-I. We tested two methods; dipeptide-mediated peptide exchange and open-HLA-I, HLA-I molecules consisting of heavy chain-{beta}2m disulphide bonded dimers. We combined peptide-exchange technologies with SpyTag-SpyCatcher chemistry to allow rapid detection of KIR binding via HLA-I displayed on plates or cells. We demonstrated the fidelity of this system with peptides of known KIR specificity bound to HLA-C*05:01. We then screened a peptide library to identify novel strong KIR2DS4 binding peptides presented by HLA-C*04:01. Peptide-exchanged HLA-C was functionally competent, promoting activation of KIR2DS4+ NK cells and inhibiting activation of KIR2DL1+ NK cells. Together, we show that peptide-exchangeable HLA-I molecules are ligands for KIR, presenting a flexible, efficient system for examining the peptide-sequence dependent recognition of HLA-I by KIR.

Protein tyrosine kinases are important regulators of cell signaling, and aberrant kinase activity contributes to many human diseases, including cancers. All protein tyrosine kinases share a highly-conserved ATP binding pocket but diverge in their substrate binding sites in order to mediate distinct signaling events. Many potent and efficacious ATP-competitive tyrosine kinase inhibitors have been developed, however it remains challenging to achieve on-target selectivity across different kinases and target specific disease mutants, given the high degree of conservation in the ATP-binding pocket. By contrast, the variable substrate-binding site offers an opportunity for selective inhibition, provided molecules can be targeted to this site. Here, we present a modular strategy to design selective, peptide-based covalent inhibitors of tyrosine kinases with a distinct binding mode from existing ATP-competitive inhibitors. Using Src kinase as a model system, we demonstrate that Src-selective reactivity can be achieved by first designing an optimized substrate peptide and then strategically positioning an electrophile on the peptide to target a non-conserved cysteine on the kinase. We show that substrate-derived covalent peptides can inhibit kinase activity, bind simultaneously with an ATP-competitive inhibitor, and even inhibit the activity of kinases bearing a common drug resistance mutation. We further explore the application of this approach to develop an inhibitor of the cancer-relevant fibroblast growth factor receptor 1 kinase that shows selectivity for an oncogenic mutant over the wild-type enzyme. Our modular strategy to generate selective covalent peptides targeting protein tyrosine kinases provides a promising framework for future chemical probe and drug development efforts.

Polyketide biosynthesis is typically initiated by loading a starter unit onto an acyl carrier protein (ACP). In type I modular polyketide synthases (PKSs), responsible for the assembly of diverse bioactive metabolites in bacteria, this ACP is usually incorporated into a chain initiation module, alongside a starter unit loading domain. In several cases, the starter unit undergoes structural modification prior to the initiation of chain assembly. Gladiolin, an antibiotic with promising activity against bacterial and fungal pathogens, is assembled by a trans-acyltransferase (AT) PKS in Burkholderia gladioli. It appears to incorporate a succinyl starter unit, but the gladiolin PKS lacks a conventional loading module, making it unclear how this happens. The gladiolin biosynthetic gene cluster encodes an AT of unassigned function (GbnB), an ACP (GbnA), and an asparagine synthetase homolog (GbnC) with similarity to enzymes that amidate the malonyl-ACP starter unit in glutarimide antibiotic biosynthesis. Here, we elucidate a cryptic starter unit amidation mechanism in gladiolin biosynthesis involving these three proteins. GbnB loads a succinyl unit onto the phosphopantetheinyl arm of GbnA, which is subsequently amidated by GbnC using glutamine as the nitrogen donor. After the fully assembled polyketide chain is released, GbnM hydrolyzes the amide, yielding mature gladiolin. Phylogenetic analyses, coupled with gene cluster reannotation revealed analogous enzymatic machinery likely responsible for cryptic succinamyl and malonamyl starter unit incorporation into etnangien and sorangicin A, respectively. Retro-biosynthetic analyses suggest succinamyl and malonamyl starter units may be involved in the assembly of other metabolites, such as the sorangiolides and azumamides.

Rhamnogalacturonan-II (RG-II) is considered the most complex glycan in nature. It forms part of an intricate network of complex glycans in the plant cell wall where it plays a critical role in plant growth, development and defence. It has been identified as an important nutrient source for the human gut microbiota (HGM), a key modulator of human health and disease status. Increasing evidence also suggests that RG-II can modulate plant-microbe interactions. Given its importance and potential, detailed studies of RG-IIs structure-function relationships and metabolism are required to underpin future crop-improvement strategies and to harness its benefits for plant and human health. Progress in this field is however hampered by RG-IIs structural complexity and limited access to enabling tools, in particular chemically defined RG-II-derived oligosaccharide (CDRO) substructures. Achieving targeted, efficient, and scalable production of CDROs remains a significant challenge and is indeed one of the major reasons why RG-II and glycomic research in general, significantly lag behind genomic and proteomic research. Here, we have genetically engineered as well as screened a diverse set of genetic strains, including transposon (Tn) mutants of the prominent model human gut microbe Bacteroides thetaiotaomicron (B. theta) and its gut and plant-associated relatives for new CDRO-generating and/or RG-II-utilising strains. Several CDROs, some of which had never been produced before by any other means (including chemical synthesis), where generated and characterised by a combination of high-resolution mass spectrometry (MS), enzymatic profiling and 2D-NMR. In addition to expanding the CDRO toolbox, we identified key genetic strains that will serve as a base or platform for the production of an unprecedented amount of CDROs covering the complexity and diversity of chemical modifications in RG-II. CDROs were later exploited to gain new insights into the microbial metabolism of RG-II in the human gut, revealing key aspects of its chemical structure that drive or limit its metabolism in B. theta. Notably, we generated new evidence in support of an alternative operational paradigm for polysaccharide utilisation systems that are widespread in the Bacteroidota phylum. We confirmed the presence of pathways for the metabolism of RG-II and/or RG-II core sugars D-apiose (D-Apif), and 3-deoxy-D-manno-2-octulosonic acid (D-Kdo) in aerobic plant-associated microbes including fungi and Flavobacterium spp., highlighting their potential to be exploited as cost-effective alternatives to B. theta for the generation of CDROs.

Kinases have proven to be one of the most fertile target classes for new drug approvals. However, classical reversible inhibitors may not be capable of the levels of specificity or target modulation required across a broad spectrum of disease areas. Approaches that chemically modify kinase inhibitors in solvent exposed regions are unveiling a swathe of mechanisms to address kinase function in new ways. For example, by either covalently recruiting nucleophilic residues outside of the ATP-binding pocket to inhibit, or by recruiting secondary effector proteins to degrade. Here, we systematically assessed the impact of minimal electrophilic modifications to ATP-site binding scaffolds, leading us to identify molecules that can control the activity and abundance of the master autophagy regulator, Unc-51-like autophagy activating kinase 1 (ULK1).

Recombinant peptide production was pioneered in the 1970s for the generation of therapeutic peptides, with notable examples including insulin and somatostatin. These early methods required the use of cyanogen bromide (BrCN) for cleavage of the native peptide sequence from a fusion protein. Since that time, while numerous BrCN-dependent peptide methods continue to be reported, the accessibility and cost of site-specific proteases have improved dramatically. These developments have enabled alternative approaches to recombinant peptide generation that obviate the need for BrCN, an environmentally destructive toxin. We recently created an immobilized SUMO protease that can replace BrCN usage in recombinant peptide production workflows by releasing native peptides expressed as part of a SUMO-peptide fusion protein. We have used this approach to generate P113 peptide, the minimal active fragment of the antifungal peptide Histatin 5. In this report, we describe the creation and characterization of this immobilized SUMO protease and its application in the production of experimentally viable quantities of active P113 peptide.

The SARS CoV 2 accessory protein Orf9b is in a complex monomer-dimer equilibrium that influences its interactions with the host mitochondrial receptor Tom70. This interaction is critical for viral suppression of a Type-1 interferon response during infection. Modulating this equilibrium with a small molecule, either by stabilizing the Orf9b dimer or blocking its interaction with Tom70, represents a promising strategy for restoring interferon signaling and the antiviral response. To build tool molecules that could test this concept, we performed two screens: a crystallographic fragment screen against the Orf9b homodimer and a high-throughput fluorescence polarization screen for competitors of an Orf9b-derived peptide binding to Tom70. Fragment screening revealed two binding sites with potential to be developed into an inhibitor: one located at the peripheral dimer interface and the other just outside the lipid-binding channel that defines the central dimer interface. Functionalization of the fragments outside of the lipid-binding channel with hydrophobic moieties stabilized the Orf9b dimer thereby indirectly inhibiting association with Tom70. In parallel, the high throughput screen for competitive inhibitors of the Tom70:Orf9b interaction discovered a separate series of molecules. These molecules display dynamic structure activity relationship (SAR) and could be improved in the future to modulate the interaction between Tom70 and potentially a wide range of substrates. Collectively, these results demonstrate the feasibility of two distinct strategies to manipulate the Orf9b-Tom70 equilibrium, which is critical to the host response to SARS CoV 2 infection.

The self-labeling protein HaloTag is used to install a wide variety of functional small molecules in cells and living organisms with exquisite specificity with respect to cell type and subcellular localization. HaloTag is a core part of many biotechnology-based tools for sensing, tracking, and manipulating biological systems with a high degree of spatial and temporal control. Due to the limitations of fluorescent proteins and other self-labeling proteins, most of these tools have historically been restricted to a single channel. In this work, we used structure-guided rational design and directed evolution to produce an orthogonal HaloTag protein called OrthoTag which reacts selectively with a modified chloroalkane substrate. OrthoTag retains many of HaloTags superior properties, and reaction rate measurements show OrthoTag and its substrate have 60-fold mutual orthogonality to HaloTag. We demonstrate the application of OrthoTag for multiplexed labeling experiments in mammalian cells with minimal optimization. Going forward, OrthoTag can be directly incorporated into any HaloTag-based system to allow simultaneous measurement or manipulation of two biological targets or processes. The availability of multiple high-performance self-labeling proteins will enable the continued development of new multiplexed biotechnology methods.

Antibiotic resistance in Mycobacterium tuberculosis is a pressing global health challenge demanding new therapeutic strategies. The bacterial respiratory chain comprises promising antibacterial targets, with dual inhibition of the terminal oxidases cytochrome bcc:aa3 and cytochrome bd (cyt bd) showing bactericidal activity. While bcc:aa3 inhibitors such as Q203 have advanced clinically, cyt bd remains underexplored due to difficulties in assigning activity of the purified enzyme and structurally resolving the quinol substrate binding site. Here, we report a rapid in vitro screening platform for cyt bd inhibitors by engineering a minimal respiratory system that couples the activity of cyt bd to that of a type 2 NADH dehydrogenase. This coupled assay enables spectroscopic monitoring of NADH oxidation as a proxy for cyt bd activity, allowing rapid screening of over 10,000 compounds. Screening identified WSL017, a fragment with low micromolar potency against both M. tuberculosis and E. coli cyt bd. Kinetic and structural analyses revealed competitive inhibition at the quinol-binding site, providing the first structural insights into cyt bd inhibition by a non-quinone scaffold. WSL017 displayed growth inhibition of M. tuberculosis H37ra, corroborating oxidase inhibition as a promising therapeutic strategy. This work establishes a pipeline for cyt bd inhibitor discovery and highlights new opportunities for structure-guided drug development against cytochrome bd oxidases.

Microorganisms in the human gut influence the efficacy and metabolism of host-targeted small molecule therapeutics, including the frontline Parkinsons disease drug levodopa (L-dopa). Previous work has identified a mechanism-based inhibitor of gut bacterial decarboxylases that degrade L-dopa, -fluoromethyltyrosine (AFMT). However, early experiments with AFMT in rodent models suggested undesirable in vivo metabolism by host tyrosine hydroxylase, producing a metabolite likely to worsen Parkinsons phenotypes and prevent application as an L-dopa co-treatment. Here, we demonstrate oxidation of AFMT in vitro by recombinant human tyrosine hydroxylase. We then develop AFMT analogs that retain activity against bacterial decarboxylases but have reduced susceptibility to host hydroxylation. Suitable arenes for inhibitor design were identified using assays with commercially available noncanonical amino acids, which revealed aryl difluorination as a promising modification. Difluoroaryl AFMT derivatives are less prone to degradation by tyrosine hydroxylase in vitro yet still inhibit L-dopa metabolism by bacterial decarboxylases. This work exemplifies how substrate reactivity can streamline design of mechanism-based enzyme inhibitors, as well as how constraints posed by the host can be incorporated during development of microbiome-targeted therapeutics. The compounds reported here are promising starting points for future studies in animal models and further exploration of gut bacterial effects on L-dopa treatment efficacy.

Sirtuins (SIRTs), which remove protein lysine acyl modifications, play crucial roles in diverse cellular processes, including metabolism, gene transcription, DNA damage repair, cell survival, and stress response. Several sirtuins are considered non-oncogene addiction of cancer cells and promising targets for anticancer drug development. High-throughput screening (HTS) methods for sirtuins are critical for the development of potent and isoform-selective sirtuin inhibitors, which are needed to validate the therapeutic potential. Herein, we designed and synthesized a fluorescent polarization (FP) tracer, KP-SC-1. Using this high-affinity tracer, we developed a robust, high-throughput FP competition assay for screening SIRT1-3 inhibitors. The assay was validated by testing known SIRT1-3 inhibitors. The assay can detect NAD+-independent SIRT1-3 inhibitors, as well as NAD+-dependent inhibitors, such as Ex-527 and TM. Finally, our assay showed satisfactory stability and outstanding performance in a pilot library screening. Compared to previous assays, the FP assay uses much less SIRT1-3 enzymes, a feature important for high-throughput library screening. We believe that the FP assay developed here will accelerate the discovery and development of SIRT1-3 inhibitors.

Rhodoquinone (RQ) is a recently discovered component of the mammalian electron transport chain (ETC) with a high degree of tissue-specificity. Currently, a lack of pure analytical standards limits efforts to precisely quantify its levels using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and interrogate its biochemical functions within mammalian ETC complexes. Here, rhodoquinone-9 (RQ-9) and rhodoquinone-10 (RQ-10), and their isomeric by-products isorhodoquinone-9 (isoRQ-9) and isorhodoquinone-10 (isoRQ-10), were synthesized from ubiquinone-9 and ubiquinone-10 starting materials. Isomers were separated and purified by flash chromatography and structurally confirmed with nuclear magnetic resonance (NMR) spectroscopy. The chromatographic and fragmentation patterns of both the oxidized and reduced forms of these electron carriers were further characterized by LC-MS/MS, establishing signatures for their confident identification in lipidomics studies. LC-MS/MS analysis of murine kidney tissue with RQ-9 analytical standard spike-in corroborate the identity of the endogenous murine RQ-9 and enable absolute quantification of its levels. Thus, we synthesized and purified RQ-9 and RQ-10 analytical standards that will enable absolute quantification in mammalian tissues and in vitro reconstitution studies on RQ-9 and RQ-10 in the mammalian ETC.

In light of the "silent" AMR pandemic, new avenues to combat pathogenic bacteria are needed. In this work, we screened a large molecule library (n=35 684 unique compounds) with the aim of identifying molecules being able to bind and block translation of the prfA-thermosensor transcript in the bacterial pathogen Listeria monocytogenes. Using a thiazole-orange displacement approach, 468 ([~]1.3% of all molecules) showed the ability to reduce fluorescence. After dose response testing, 32 compounds remained promising and eight of them showed sufficient purity and availability to be further validated. Interestingly, four compounds, being structurally very similar, showed specificity for prfA at a varying degree. All four compounds carried 3 aromatic rings with one connecting amine between two of the rings and an amide linking an aliphatic amine side chain. The most selective compounds, M5, showed a Kd of [~]0.8 {micro}M for the prfA RNA at 35{degrees}C. However, none of the eight most efficient compounds were able to inhibit prfA translation in vitro, suggesting that the molecules are able to bind but not affect the stability of the overall structure. Through this work, we have been able to identify a set of molecules, able to bind the prfA thermosensor RNA selectively, but without affecting translation. These molecules could constitute an important scaffold for further drug development.

Homeodomain-interacting protein kinase 4 (HIPK4) remains an understudied member of the dark kinome. While genetic knockout studies suggest roles for HIPK4 in spermiogenesis and cutaneous squamous cell carcinoma, whether these cellular functions can be recapitulated by pharmacological inhibition remains to be determined. However, such investigations have been hampered by a lack of high-quality chemical tools. To address this, we employed a rational design strategy utilizing macrocyclization of a bosutinib-based scaffold. Systematic optimization led to the discovery of AZ137 (28e), a potent and selective HIPK4 inhibitor (IC50 = 11 nM; cellular EC50 = 76 nM). AZ137 exhibits exceptional selectivity across three comprehensive orthogonal panels, high solubility, and no detectable cytotoxicity. Its cellular activity was confirmed in cell-based assays of HIPK4-dependent F-actin remodeling. Together with a negative control compound, this probe set provides a foundational framework for the validating HIPK4 as a therapeutic target and a high-quality resource to elucidate its roles in normal physiology and disease. For Table of Contents Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/720179v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@12438borg.highwire.dtl.DTLVardef@11083beorg.highwire.dtl.DTLVardef@1395fb4org.highwire.dtl.DTLVardef@1ba3db8_HPS_FORMAT_FIGEXP M_FIG C_FIG

The contemporary shift toward multispecific antibodies, antibody-drug conjugates (ADCs), and bespoke glycoengineered therapeutics have exposed the limitations of standard genomic engineering tools. This paper presents a novel iterative engineering paradigm utilizing the Leap-In Transposase(R) platform. By leveraging a suite of three mutually orthogonal transposase-transposon systems, we demonstrate the sequential modification of the Chinese Hamster Ovary (CHO) genome to achieve three distinct functional outcomes: (i) First, the creation of a glutamine synthetase (GS)-deficient host (CHO-K1-GS) via targeted knockdown, (ii) Second, the integration of multiple copies of a model therapeutic IgG1 for expression, and (iii) Third, the subsequent knockdown of the fucosylation pathway to modulate the glycan profile of the expressed IgG1. Genetic stability (copy number & sequence) of each integration event was confirmed using Targeted Locus Amplification (TLA) and Next-Generation Sequencing (NGS). Functional stability (expression levels, metabolic phenotype, and glycan phenotypes) was confirmed using standard cell culture and analytical techniques. Crucially, the truly orthogonal nature of the transposase-transposon pairs prevents cross-mobilization and ensures the structural and functional integrity of previously integrated cargo. This study establishes a "What You See Is What You Get" (WYSIWYG) methodology that provides a robust, scalable, and predictable framework for developing next-generation complex biopharmaceutical manufacturing cell lines.