Biology subjects

Budisa, N.

Publications and source records attributed to Budisa, N..

7 recordsLinked to original sources

bioRxiv2026

Architecture Matters: Design Rules for Multigene IDO1/PD L1 Cassettes in Human Skin Cells

Allogeneic cell therapies require the coordinated expression of multiple immunomodulatory genes, yet multigene circuits that function in permissive cell lines often fail in differentiated human tissues for unclear reasons. Here, we systematically dissect how transcriptional architecture governs functional immunoregulation in engineered human keratinocyte and fibroblast lines. Using site-specific large-cargo integration (eePASSIGE) as an enabling tool, we determined that genomic insertion efficiency was not the limiting factor for phenotype; rather, promoter arrangement and gene order dictated expression hierarchy. A single-promoter EF1-IDO1-T2A-GFP design that expressed robustly in HEK293T cells was nearly silent in skin-derived cells, preventing reporter-based enrichment. In dual- and tri-modular cassettes, we observed severe transcriptional interference: a downstream CMV promoter driving GFP or PD-L1/iCasp9 (via EMCV-IRES) markedly suppressed the upstream EF1-IDO1 unit, despite intact integration (resulting in [~]175-625-fold attenuation), demonstrating strong promoter interference within the circuit. Functionally, co-culture assays revealed a hierarchical immunomodulatory logic: high IDO1 expression proved to be a requisite threshold for T-cell suppression, whereas PD-L1 provided measurable benefit only against highly activated, PD-1+ T cells in vitro. Collectively, these data establish a site-specific framework for generating immune-tuned skin cells and define essential design rules for avoiding promoter interference in next-generation translational skin substitutes.

bioengineering↗

bioRxiv2024

Antibiotic-free whole-cell biocatalytic fermentation: Escherichia coli with surface-displayed PETases for sustainable plastic degradation

Plastic pollution has increasingly burdened the environment, driving the need for natural degradation platforms that utilize microbial enzymes to break plastics down into monomers. In this study, we introduce a novel approach using Escherichia coli as a fermentative, antibiotic-free whole-cell biocatalyst with surface-displayed, genomically integrated PETases for efficient plastic degradation. PETases, a class of esterases, catalyze the hydrolysis of polyethylene terephthalate (PET) into mono-2-hydroxyethyl terephthalate (MHET). Surface display of these enzymes was achieved via gene fusions with an N-terminal cysteine (Cys) triacylated anchor, mediated by the Braun lipoprotein (Lpp) signal peptide. To circumvent issues associated with plasmids, - such as genetic instability and reliance on antibiotics - we used a Type I-F CRISPR-associated transposase to insert the genes directly into specific E. coli genome sites. Proper enzyme display and activity on the E. coli surface were confirmed through enzyme activity tests, Western blotting, and flow cytometry, with cells retaining PET degradation ability over multiple generations. High-performance liquid chromatography (HPLC) analysis assessed degradation efficiency, identifying byproducts such as bis (2-hydroxyethyl) terephthalate and terephthalic acid. This study establishes a proof-of-concept for efficient plastic degradation using engineered bacteria as robust, sustainable, and genomically stable whole-cell biocatalysts, providing a promising platform for addressing plastic waste management. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=194 SRC="FIGDIR/small/624590v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@1320dd8org.highwire.dtl.DTLVardef@12b025forg.highwire.dtl.DTLVardef@a8b0b3org.highwire.dtl.DTLVardef@e8d2c7_HPS_FORMAT_FIGEXP M_FIG C_FIG One-sentence AbstractEscherichia coli was engineered as a fermentative, antibiotic-free whole-cell biocatalyst, featuring surface-displayed and genomically integrated PETases for efficient plastic degradation. This innovative approach has the potential to transform plastic recycling by enabling sustainable, large-scale degradation of plastic waste through environmentally friendly microbial systems.

synthetic biology↗

bioRxiv2023

Genomically integrated orthogonal translation in Escherichia coli, a new synthetic auxotrophic chassis with altered genetic code, genetic firewall, and enhanced protein expression

In the last three decades, genetic code engineering has expanded protein biosynthesis options from the natural set of 20 canonical amino acids to over 250 non-canonical amino acids (ncAAs). This progress involves rewiring of protein translation by establishing Orthogonal Translation Systems (OTS) through orthogonal pairs. Traditionally encoded on plasmid vectors, these systems are often unstable and burdensome in large-scale fermentations. To moving forward from academia to reliable technology, it is crucial to integrate OTS genetic modules stably into a molecular chassis with a defined genome background. Here, we demonstrate genomically integrated OTS in Escherichia coli, creating a synthetic auxotrophic chassis with an altered genetic code. Using CRISPR-associated transposase tool (CASTs), we targeted multiple genome sites, inserting OTS components (enzymes, tRNA genes) non-disruptively. Our OTS system demonstrated site-specific incorporation of m-oNB-Dopa through in-frame amber stop codon readthrough, enabling the expression of smart underwater bioglues. Simple metabolic labelling, introducing fluoroproline analogs enhancing conformational stability during orthogonal translation, further bolstered system robustness. These chassis, equipped also with synthetic auxotrophy for m-oNB-Dopa, serve as a built-in genetic barrier (genetic firewall), ensuring safe bioproduction in genetically isolated settings. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/567690v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@a7a3e0org.highwire.dtl.DTLVardef@1f7d3c5org.highwire.dtl.DTLVardef@1703dfcorg.highwire.dtl.DTLVardef@1f584bf_HPS_FORMAT_FIGEXP M_FIG C_FIG One-sentence AbstractCRISPR-assisted transposition has enabled the development of a robust and biosafe Escherichia coli-based chassis with genomically integrated orthogonal translation components, enhancing synthetic protein production and laying the foundation for the transition of this research field from academia to reliable technology.

synthetic biology↗

bioRxiv2023

Escherichia coli adapts metabolically to 6- and 7-fluoroindole, enabling proteome-wide fluorotryptophan substitution

Nature has scarcely evolved a biochemistry around fluorine. However, modern science proved fluorinated organic molecules to be suitable building blocks for biopolymers, from peptides and proteins up to entire organisms. Here, we conducted adaptive laboratory evolution (ALE) experiments to introduce fluorine into living microorganisms. By cultivating Escherichia coli with fluorinated indole analogues, we successfully evolved microbial cells capable of utilizing either 6-fluoroindole or 7-fluoroindole for growth. Our improved ALE protocols enabled us to overcome previous challenges and achieve consistent and complete adaptation of microbial populations to these unnatural molecules. In the ALE experiments, we supplied fluoroindoles to auxotrophic E. coli bacteria, exerting strong selective pressure that led to microbial adaptation and growth on monofluorinated indoles. Within the cells, these indoles were converted into corresponding amino acids (6- and 7-fluorotryptophan) and incorporated into the proteome at tryptophan sites. This study is a first step and establishes a strong foundation for further exploration of the mechanisms underlying fluorine-based life and how a formerly stressor (fluorinated indole) becomes a vital nutrient. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=64 SRC="FIGDIR/small/559291v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1a3b17org.highwire.dtl.DTLVardef@1d716aorg.highwire.dtl.DTLVardef@10126d5org.highwire.dtl.DTLVardef@9c8a43_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

"Cold" Orthogonal Translation: Psychrophilic Pyrrolysyl-tRNA Synthetase as Efficient Tool for Expanding the Genetic Code

Using orthogonal translation systems (OTSs) is one of the most efficient strategies for producing unnatural proteins through the incorporation of non-canonical amino acids (ncAAs) into the genetic code. Traditionally, efforts to expand substrate specificity start with a (hyper-)stable enzyme capable of withstanding the structural changes induced by necessary mutations. In contrast, we propose a radically different approach for the PylRS system: by starting with enzymes that evolved to cope with instability in order to adapt to cold conditions, potentially offering greater resilience to mutational changes. By finding and further engineering a psychrophilic ("cold") OTS from Methanococcoides burtonii, we developed an alternative to the widely used mesophilic and thermophilic systems. This novel OTS demonstrated exceptional in vivo efficiency for a broad range of substrates, even at very low ncAA concentrations and low cultivation temperatures. The general versatility of the PylRS system across a wide range of applicable host organisms suggests that the Cold-OTS has the potential to also improve protein yields in these hosts and help to drive the transformation of the expanded genetic code from an academic pursuit into a high-value, chemistry-driven biotechnology.

synthetic biology↗

bioRxiv2021

Evolving a mitigation of the stress response pathway to change the basic chemistry of life

Billions of years of evolution have produced only slight variations in the standard genetic code, and the number and identity of proteinogenic amino acids have remained mostly consistent throughout all three domains of life. These observations suggest a certain rigidity of the genetic code and prompt musings as to the origin and evolution of the code. Here we conducted an adaptive laboratory evolution (ALE) to push the limits of the code restriction, by evolving Escherichia coli to fully replace tryptophan, thought to be the latest addition to the genetic code, with the analog L-{beta}-(thieno[3,2-b]pyrrolyl)alanine ([3,2]Tpa). We identified an overshooting of the stress response system to be the main inhibiting factor for limiting ancestral growth upon exposure to {beta}-(thieno[3,2-b]pyrrole ([3,2]Tp), a metabolic precursor of [3,2]Tpa, and Trp limitation. During the ALE, E. coli was able to "calm down" its stress response machinery, thereby restoring growth. In particular, the inactivation of RpoS itself, the master regulon of the general stress response, was a key event during the adaptation. Knocking out the rpoS gene in the ancestral background independent of other changes conferred growth on [3,2]Tp. Our results add additional evidence that frozen regulatory constraints rather than a rigid protein translation apparatus are Lifes gatekeepers of the canonical amino acid repertoire. This information will not only enable us to design enhanced synthetic amino acid incorporation systems but may also shed light on a general biological mechanism trapping organismal configurations in a status quo. SIGNIFICANCE STATEMENTThe (apparent) rigidity of the genetic code, as well as its universality, have long since ushered explorations into expanding the code with synthetic, new-to-nature building blocks and testing its boundaries. While nowadays even proteome-wide incorporation of synthetic amino acids has been reported on several occasions1-3, little is known about the underlying mechanisms. We here report ALE with auxotrophic E. coli that yielded successful proteome-wide replacement of Trp by its synthetic analog [3,2]Tpa accompanied with the selection for loss of RpoS4 function. Such laboratory domestication of bacteria by the acquisition of rpoS mitigation mutations is beneficial not only to overcome the stress of nutrient (Trp) starvation but also to evolve the paths to use environmental xenobiotics (e.g. [3,2]Tp) as essential nutrients for growth. We pose that regulatory constraints rather than a rigid and conserved protein translation apparatus are Lifes gatekeepers of the canonical amino acid repertoire (at least where close structural analogs are concerned). Our findings contribute a step towards understanding possible environmental causes of genetic changes and their relationship to evolution. Our evolved strain affords a platform for homogenous protein labeling with [3,2]Tpa as well as for the production of biomolecules5, which are challenging to synthesize chemically. Top-down synthetic biology will also benefit greatly from breaking through the boundaries of the frozen bacterial genetic code, as this will enable us to begin creating synthetic cells capable to utilize an expanded range of substrates essential for life.

synthetic biology↗

bioRxiv2019

Laboratory evolution of Escherichia coli enables life based on fluorinated amino acids

Organofluorine compounds are toxic to various living beings in different habitats. On the other hand, fluorine incorporation into single proteins via related amino acid analogues has become common practice in protein engineering. Thus, an essential question remains: can fluorinated amino acids generally be used as xeno-nutrients to build up biomass, or do large amounts of fluorine in the cells render them nonviable? To gain information about the effect of long-term exposure of a cellular proteome to fluorinated organic compounds, we constructed an experiment based on bacterial adaptation in artificial fluorinated habitats. We propagated Escherichia coli (E. coli) in the presence of either 4- or 5-fluoroindole as essential precursors for the in situ synthesis of tryptophan (Trp) analogues. We found that full adaptation requires astonishingly few genetic mutations but is accompanied by large rearrangements in regulatory networks, membrane integrity and quality control of protein folding. These findings highlight the cellular mechanisms of the evolutionary adaption process to unnatural amino acids and provide the molecular foundation for novel and innovative bioengineering of microbial strains with potential for biotechnological applications.\n\nOne Sentence SummaryLaboratory evolution enabled for the first time Escherichia coli to use fluorinated indoles as essential precursors for protein synthesis by introducing few genetic mutations but large rearrangements in regulatory networks, membrane integrity and quality control of protein folding.

synthetic biology↗