Biology subjects

Kulhanek, D. L.

Publications and source records attributed to Kulhanek, D. L..

4 recordsLinked to original sources

bioRxiv2026

A Quantitative Two-Channel Genetic Reporter for Selenocysteine Biosynthesis and Incorporation

Selenocysteine (Sec), the 21st amino acid, is a rare non-canonical amino acid that represents an attractive target for protein engineering due to its desirable chemical properties such as high affinity for metals, strong nucleophilicity, and reversible covalent bond formation. To bypass the natural constraints on Sec placement within proteins, several strategies have been developed to rewire the native translational machinery to enable site-specific incorporation. However, these usually abolish the quality control mechanism that excludes the serine-charged selenocysteinyl-tRNA (Ser-tRNASec), the immediate biosynthetic precursor, from translation resulting in heterogenous protein species. This challenge is confounded by a lack of genetic tools to accurately report the selenylation state of the tRNA pool as most are blind to competing process of Ser incorporation, which can only be observed using analytical methods. To resolve this issue, we have developed a new fluorescent reporter, Selenocysteine Adjusted Ratiometric Chromophore (SeARCh), which exhibits two distinct spectral outputs dependent on the incorporation of either Ser (red) or Sec (green). Using SeARCh, we define several factors which influence the observed Sec:Ser ratio and construct a new hybrid biosynthetic pathway with improved performance, achieving 90% Sec incorporation. Furthermore, SeARCh displays unusually complex mass spectra due to the isotope distribution of selenium and heterogenous nature of the protein in solution and we report specific methods to account for this behaviour and precisely quantify the rare Ser-containing species found at high Sec incorporation efficiencies. Our findings suggest that the equilibrium between selenoprotein and tRNASec expression levels is a key driver of incorporation efficiency and implies a process that is broadly biosynthetically constrained. Collectively these tools represent a significant advance in the metrology of selenocysteine biosynthesis and incorporation and can be used to inform and standardize future engineering efforts.

synthetic biology↗

bioRxiv2025

Mapping the phenotypic landscape of a transcriptional repressor using Deep Mutational Scanning and Growth-based Quantitative Sequencing

CymR is a TetR-family transcriptional repressor that recognizes a well-defined operator sequence in the promoter PcymRC. The native ligand cumate and several structurally related aromatic acids bind at an allosteric site and induce a conformational change in CymR, resulting in release from the DNA operator and de-repression of the promoter. The amino acid residues that contribute to these core functions have not been mapped, nor has the protein been subjected to extensive mutagenesis to modify its function. Here, for the first time, we integrate Deep Mutational Scanning (DMS) with Growth-based Quantitative Sequencing (GROQ-Seq) to evaluate a comprehensive phenotypic landscape of CymR variants, including single amino acid insertions and deletions. We measure this library across a concentration gradient of small molecule inducers to construct an induction curve for all library members. From this analysis, we identify amino acids throughout the protein that are essential for repressor function and discover several mutations that improve the sensitivity of CymR to the ligand perillic acid. In addition, rarely investigated insertion mutants are revealed to be a key driver of novel phenotypes, including several regions of CymR where insertions result in an inverted phenotype and the isolation of variants exhibiting an unusual band-stop phenotype. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/693801v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@121b16org.highwire.dtl.DTLVardef@b0478aorg.highwire.dtl.DTLVardef@128f2ecorg.highwire.dtl.DTLVardef@164a426_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2024

A modular toolkit for environmental Rhodococcus, Gordonia, and Nocardia enables complex metabolic manipulation.

Soil-dwelling Actinomycetes are a diverse and ubiquitous component of the global microbiome, but largely lack genetic tools comparable to those available in model species such as E. coli or Pseudomonas putida, posing a fundamental barrier to their characterization and utilization as hosts for biotechnology. To address this, we have developed a modular plasmid assembly framework along with a series of genetic control elements for the previously genetically intractable Gram-positive environmental isolate Rhodococcus ruber C208 and demonstrate conserved functionality in diverse environmental isolates of Rhodococcus, Nocardia and Gordonia. This toolkit encompasses Mycobacteriale origins of replication, broad-host range antibiotic resistance markers, transcriptional and translational control elements, fluorescent reporters, a tetracycline-inducible system, and a counter-selectable marker. We use this toolkit to interrogate the carotenoid biosynthesis pathway in Rhodococcus erythropolis N9T-4, a weakly carotenogenic environmental isolate and engineer higher pathway flux towards the keto-carotenoid canthaxanthin. This work establishes several new genetic tools for environmental Mycobacteriales and provides a synthetic biology framework to support the design of complex genetic circuits in these species. IMPORTANCESoil-dwelling Actinomycetes, particularly the Mycobacteriales, include both diverse new hosts for sustainable biomanufacturing and emerging opportunistic pathogens. Rhodococcus, Gordonia and Nocardia are three abundant genera with particularly flexible metabolisms and untapped potential for natural product discovery. Among these, Rhodococcus ruber C208 was shown to degrade polyethylene, Gordonia paraffinivorans can assimilate carbon from solid hydrocarbons, and Nocardia neocaledoniensis (and many other Nocardia) possesses dual isoprenoid biosynthesis pathways. Many species accumulate high levels of carotenoid pigments, indicative of highly active isoprenoid biosynthesis pathways which may be harnessed for fermentation of terpenes and other commodity isoprenoids. Modular genetic toolkits have proven valuable for both fundamental and applied research in model organisms, but such tools are lacking for most Actinomycetes. Our suite of genetic tools and DNA assembly framework were developed for broad functionality and to facilitate rapid prototyping of genetic constructs in these organisms.

microbiology↗

bioRxiv2023

Interrogating the function of bicistronic translational control elements to improve consistency of gene expression

Context independent gene expression is required for genetic circuits to maintain consistent and predicable behavior. Previous efforts to develop context independent translation have leveraged the helicase activity of translating ribosomes via bicistronic design translational control elements (BCDs) located within an efficiently translated leader peptide. We have developed a series of bicistronic translational control elements with strengths that span several orders of magnitude, maintain consistent expression levels across diverse sequence contexts, and are agnostic to common ligation sequences used in modular cloning systems. We have used this series of BCDs to investigate several features of this design, including the spacing of the start and stop codons, the nucleotide identity upstream of the start codon, and factors affecting translation of the leader peptide. To demonstrate the flexibility of this architecture and their value as a generic modular expression control cassette for synthetic biology, we have developed a set of robust BCDs for use in several Rhodococcus species.

synthetic biology↗