Biology subjects

Hellinger, J.

Publications and source records attributed to Hellinger, J..

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

Engineering a Bright Near-Infrared Fluorescent Protein by Screening a Comprehensive Phenotypic Landscape

Near-infrared fluorescent proteins (niRFPs) are valuable markers for tracking cellular phenomena as they offer greater imaging depth, lower background, and minimal invasiveness relative to other fluorescent probes. The small ultra red fluorescent protein (smURFP) is the brightest niRFP currently reported and has been the subject of several mutagenesis studies to improve its biophysical characteristics. Here, we demonstrate a systematic approach to exploring the mutational landscape of smURFP using a comprehensive deep mutational scanning (DMS) library to identify novel smURFP mutations which confer greater in vivo fluorescence. By observing changes in relative abundance between naive and fluorescence sorted populations, we provide analysis of the enrichment of all possible single-codon substitutions, insertions, and deletions for smURFP. Enriched populations yielded a series of seven single-codon substitutions which confer a three-fold increase in in vivo fluorescence in E. coli when combined. Finally, we assess the potential underlying mechanisms for increased fluorescence by characterizing the biophysical and photophysical properties of the mutant niRFP sequences. We confirm that two of the derived smURFP sequences have a higher molecular brightness than wild type and yield the brightest niRFP reported.

synthetic biology↗

bioRxiv2025

Directed evolution of L-DOPA sensing and production enables efficient incorporation of an expanded genetic code into proteins

3,4-dihydroxyphenylalanine (L-DOPA) is a catechol-containing amino acid derived from L-tyrosine that can be introduced into proteins primarily as a post-translational modification. It can also be introduced in a site-specific manner into proteins via orthogonal tRNA synthetase:tRNA pairs. However, despite the interest in expanded genetic codes containing L-DOPA, efforts to improve metabolism relating to its production have been limited. Here, we report and engineer a LysR-family transcription factor, PP_2251, that can serve as a biosensor for L-DOPA, thereby allowing the optimization of genetic circuits for vastly improved L-DOPA production via compartmentalized partnered replication (CPR), an emulsion PCR-based directed evolution scheme. Using this biosensor, a promiscuous E. coli flavin-dependent monooxygenase (HpaB) was optimized via CPR for enhanced L-DOPA biosynthesis. Engineered HpaB variants could support efficient translational incorporation of L-DOPA into proteins with 80% incorporation efficiency and yields in excess of 250 mg.L-1. Improvements in L-DOPA production are not only of great interest for protein engineering and genetic code expansion but should impact the production of DOPA-derived natural products including catecholamine neurotransmitters and benzylisoquinoline alkaloids (BIAs), molecules of intense pharmaceutical interest.

synthetic biology↗

bioRxiv2020

Social signaling via bioluminescent blinks drives schooling behavior in the flashlight fish Anomalops katoptron

The bioluminescent flashlight fish Anomalops katoptron live in schools of several hundred specimens. To understand how flashlight fish, integrate bioluminescent signaling into their schooling behavior, we analyzed movement profiles and blink frequencies. Isolated specimen of A. katoptron show a high motivation to align with fixed or moving artificial light organs. Depending on presented frequencies A. katoptron responds with a reduction in swimming speed and its own blink frequency. Higher presented blink frequencies reduce the nearest neighbor distance. In the natural environment A. katoptron is changing its blink frequencies and nearest neighbor distance in a context specific manner. Blink frequencies are increased from day to night and during avoidance behavior, while nearest neighbor distance is decreased with increasing blink frequencies. A. katoptron changes its blink frequencies by modifying light organ occlusion. Our results suggest that visually transmitted information via specific blink patterns determine intraspecific communication and group cohesion in schooling A. katoptron.Competing Interest StatementThe authors have declared no competing interest.View Full Text

animal behavior and cognition↗