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Functional and structural characterization of an IclR family transcription factor for the development of dicarboxylic acid biosensors

Author(s): Pham C; Nasr MA; Skarina T; Di Leo R; Kwan DH; Martin VJJ; Stogios PJ; Mahadevan R; Savchenko A;

Prokaryotic transcription factors (TFs) regulate gene expression in response to small molecules, thus representing promising candidates as versatile small molecule-detecting biosensors valuable for synthetic biology applications. The engineering of such biosensors requires thorough in vitro and i ...

Article GUID: 38696354


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

Author(s): Husser MC; Pham NP; Law C; Araujo FRB; Martin VJJ; Piekny A;

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. Usin ...

Article GUID: 38652106


Genome sequencing of 15 acid-tolerant yeasts

Author(s): Bagley JA; Pyne ME; Exley K; Kevvai K; Wang Q; Whiteway M; Martin VJJ;

We report draft genome sequences for 15 non-conventional Saccharomycotina yeast strains obtained from public culture repositories. Included in our collection are eight strains of Pichia with broad tolerance to dicarboxylic acids. The genome sequences of these strains will enable comparative genomics of acid-tolerant phenotypes and strain engineering of no ...

Article GUID: 37747226


Screening non-conventional yeasts for acid tolerance and engineering Pichia occidentalis for production of muconic acid

Author(s): Pyne ME; Bagley JA; Narcross L; Kevvai K; Exley K; Davies M; Wang Q; Whiteway M; Martin VJJ;

Saccharomyces cerevisiae is a workhorse of industrial biotechnology owing to the organism's prominence in alcohol fermentation and the suite of sophisticated genetic tools available to manipulate its metabolism. However, S. cerevisiae is not suited to overproduce many bulk bioproducts, as tox ...

Article GUID: 37652930


CRAPS: Chromosomal-Repair-Assisted Pathway Shuffling in Yeast

Author(s): Dykstra CB; Pyne ME; Martin VJJ;

A fundamental challenge of metabolic engineering involves assembling and screening vast combinations of orthologous enzymes across a multistep biochemical pathway. Current pathway assembly workflows involve combining genetic parts ex vivo and assembling one pathway configuration per tube or well. Here, we present CRAPS, Chromosomal-Repair-Assisted Pathway ...

Article GUID: 37584634


Divergent directed evolution of a TetR-type repressor towards aromatic molecules

Author(s): Nasr MA; Martin VJJ; Kwan DH;

Reprogramming cellular behaviour is one of the hallmarks of synthetic biology. To this end, prokaryotic allosteric transcription factors (aTF) have been repurposed as versatile tools for processing small molecule signals into cellular responses. Expanding the toolbox of aTFs that recognize new inducer molecules is of considerable interest in many applicat ...

Article GUID: 37377432


Pathway elucidation and microbial synthesis of proaporphine and bis-benzylisoquinoline alkaloids from sacred lotus (Nelumbo nucifera)

Author(s): Pyne ME; Gold ND; Martin VJJ;

Sacred lotus (Nelumbo nucifera) has been utilized as a food, medicine, and spiritual symbol for nearly 3000 years. The medicinal properties of lotus are largely attributed to its unique profile of benzylisoquinoline alkaloids (BIAs), which includes potential anti-cancer, anti-malarial and anti-arrhythmic compounds. BIA biosynthesis in sacred lotus differs ...

Article GUID: 37004909


Cytokinetic diversity in mammalian cells is revealed by the characterization of endogenous anillin, Ect2 and RhoA

Author(s): Husser MC; Ozugergin I; Resta T; Martin VJJ; Piekny AJ;

Cytokinesis is required to physically separate the daughter cells at the end of mitosis. This crucial process requires the assembly and ingression of an actomyosin ring, which must occur with high fidelity to avoid aneuploidy and cell fate changes. Most of our knowledge of mammalian cytokinesis was generated using over-expressed transgenes in HeLa cells. ...

Article GUID: 36416720


The MyLo CRISPR-Cas9 Toolkit: A Markerless Yeast Localization and Overexpression CRISPR-Cas9 Toolkit

Author(s): Bean BDM; Whiteway M; Martin VJJ;

The genetic tractability of the yeast Saccharomyces cerevisiae has made it a key model organism for basic research and a target for metabolic engineering. To streamline the introduction of tagged genes and compartmental markers with powerful CRISPR-Cas9-based genome editing tools we constructed a Markerless Yeast Localization and Overexpression (MyLO) CRI ...

Article GUID: 35708612


Functional expression of opioid receptors and other human GPCRs in yeast engineered to produce human sterols

Author(s): Bean BDM; Mulvihill CJ; Garge RK; Boutz DR; Rousseau O; Floyd BM; Cheney W; Gardner EC; Ellington AD; Marcotte EM; Gollihar JD; Whiteway M; Martin VJJ;

The yeast Saccharomyces cerevisiae is powerful for studying human G protein-coupled receptors as they can be coupled to its mating pathway. However, some receptors, including the mu opioid receptor, are non-functional, which may be due to the presence of the fungal sterol ergosterol instead of ch ...

Article GUID: 35610225


A Versatile Transcription Factor Biosensor System Responsive to Multiple Aromatic and Indole Inducers

Author(s): Nasr MA; Timmins LR; Martin VJJ; Kwan DH;

Allosteric transcription factor (aTF) biosensors are valuable tools for engineering microbes toward a multitude of applications in metabolic engineering, biotechnology, and synthetic biology. One of the challenges toward constructing functional and diverse biosensors in engineered microbes is the limited toolbox of identified and characterized aTFs. To ov ...

Article GUID: 35316041


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