Biology subjects

Martin, V. J. J.

Publications and source records attributed to Martin, V. J. J..

5 recordsLinked to original sources

bioRxiv2026

Biosensor-guided genome-wide CRISPR screens enable cis,cis-muconic acid overproduction in yeast

Classical pathway metabolic engineering relies on enhancing strain productivity through the introduction of rationally identified genetic perturbations, which while useful, are time-consuming and miss unintuitive targets. Genome-wide CRISPR screens offer an alternative by efficiently sampling genomes, enabling the engineering of novel phenotypes, and are further enhanced when combined with high-throughput strain interrogation methods. Here, using Saccharomyces cerevisiae, we engineered small molecule biosensors into population enrichment tools using zeocin resistance and fluorescence as signal outputs. We then introduced the multi-functional genome-wide CRISPR (MAGIC) library that targets every gene for activation, interference, or deletion, thus creating biosensor-aided MAGIC, or senMAGIC. As a case study, we utilized senMAGIC to identify single genetic determinants that improve productivity of the nylon precursors protocatechuic and cis,cis-muconic acids (PCA and ccM). These were then randomly assembled as triple gRNA-tRNA arrays for multiplexed gene perturbations to identify combinatorial effects. Top gRNA hits were then confirmed using direct genomic integrations in senMAGIC-free production strains, resulting in identifying known as well as novel targets that increase PCA and ccM titres. Our results demonstrate senMAGIC as a robust automation-friendly whole-genome evolution tool and highlight limitations of the method, providing a roadmap for future multifunctional CRISPR screens to optimize host metabolism for efficient bioprocesses.

synthetic biology↗

bioRxiv2026

Heterologous engineering of receptors using OrthoRep (HERO) for directed evolution of GPCRs in yeast

Engineering G protein-coupled receptors (GPCRs) for biosensing applications remains challenging due to structural and functional constraints when such proteins are expressed in a heterologous host. OrthoRep offers the continuous accumulation and selection of signal-enhancing mutations in yeast, yet it has not been applied to GPCR evolution. We apply OrthoRep-driven mutagenesis to evolve opioid GPCRs and improve yeast-based biosensors: first, we express the human -opioid receptor (OPRM1) on the OrthoRep p1 plasmid and couple ligand activation to growth. Serial cell passaging of the biosensor generated functionally diverse mutants. However, mutations that decouple receptor activation from selection arose over longer passaging campaigns, halting evolution. We resolve this issue using a site-specific recombinase that re-introduces evolved variants onto the p1 landing pad of an unmutated biosensor, allowing continued selection. This led to an ~18-fold improvement in sensitivity of a mutant OPRM1 over wt-OPRM1. Additional polymerases were added to the platform, broadening the mutational profiles available. Assembling these components forms the platform we call HERO (Heterologous Engineering of Receptors using OrthoRep). Lastly, we deploy HERO using automation to improve the human {delta}-opioid receptor's weak response to an agonist by ~40-fold. Sensitivity also improved for a structurally dissimilar ligand assayed on the same evolved variants.

synthetic biology↗

bioRxiv2025

Sequencing of a dairy isolate unlocks Kluyveromyces marxianus as a host for lactose valorization

The use of genetically modified non-conventional yeast provides significant potential for the bioeconomy by diversifying the tools available for the development of sustainable and novel products. In this study, we sequenced and annotated the genome of Kluyveromyces marxianus Y-1190 to establish it as a platform for lactose valorization. The strain was chosen for rapid growth on lactose-rich dairy permeate, high transformation efficiency, and ease of culturing in bioreactors. Genomic sequencing revealed that K. marxianus Y-1190 possesses single nucleotide polymorphisms associated with efficient lactose metabolism. The strain is diploid with notable genomic heterogeneity, which appears to be critical for its robust growth and acid tolerance. To further exploit this platform strain, we developed protocols for gene and chromosome manipulation using CRISPR editing, constructed and validated a series of promoters compatible with MoClo vectors, and designed synthetically inducible promoters for K. marxianus. These tools enable precise control over gene expression, allowing for the tailored optimization of metabolic pathways and production processes. The synthetic promoters provide flexibility for dynamic expression tuning, while the CRISPR-based editing protocols facilitate targeted genetic modifications with high efficiency. Together, these advancements significantly enhance the genetic toolbox for K. marxianus, positioning it as a versatile platform for industrial biotechnology. These tools open new opportunities for the sustainable production of bio-based chemicals, fuels, and high-value products, leveraging lactose-rich feedstocks to contribute to a circular economy.

synthetic biology↗

bioRxiv2023

Endogenous tagging using split mNeonGreen in human iPSCs for live imaging studies

Endogenous tags have become invaluable tools to visualize and study native proteins in live cells. However, generating human cell lines carrying endogenous tags is difficult due to the low efficiency of homology-directed repair. Recently, an engineered split mNeonGreen protein was used to generate a large-scale endogenous tag library in HEK293 cells. Using split mNeonGreen for large-scale endogenous tagging in human iPSCs would open the door to studying protein function in healthy cells and across differentiated cell types. We engineered an iPS cell line to express the large fragment of the split mNeonGreen protein (mNG21-10) and showed that it enables fast and efficient endogenous tagging of proteins with the short fragment (mNG211). We also demonstrate that neural network-based image restoration enables live imaging studies of highly dynamic cellular processes such as cytokinesis in iPSCs. This work represents the first step towards a genome-wide endogenous tag library in human stem cells.

cell biology↗

bioRxiv2022

Imaging tools generated by CRISPR/Cas9 tagging reveal cytokinetic diversity in mammalian cells

Cytokinesis is required to physically separate the daughter cells at the end of mitosis. This process occurs via the ingression of an actomyosin ring that assembles in anaphase and pulls in the overlying plasma membrane as it constricts. Mechanistic studies have uncovered different pathways that regulate the assembly and position of the ring in mammalian cells, but the majority of these studies were done using HeLa cells with overexpressed transgenes, and the relative requirement for these mechanisms among the majority of cell types is not known. Here, we used CRISPR/Cas9 gene editing to endogenously tag cytokinesis proteins, anillin, Ect2 and RhoA, as well as other cellular components, with fluorescent proteins. These tools enabled the visualization of cytokinesis by live imaging to quantitatively study these proteins at endogenous levels. As a proof-of-concept, tagging anillin in multiple mammalian cell lines revealed cytokinetic diversity, which will be useful for studies of how mechanisms controlling cytokinesis vary among cell types. We also successfully tagged multiple cellular components in the same cell line, demonstrating the versatility of these tagging tools.

cell biology↗