Heterologous reporter expression in the planarian Schmidtea mediterranea through somatic mRNA transfection

Planarians have long been studied for their regenerative abilities, but they possess limited genetic tools due to challenges in gene delivery, expression, and detection, despite decades of work. We developed a toolbox for heterologous protein expression in planarian cells and in live animals. Specifically, we identified and optimized nanotechnological and chemical transfection methods to efficiently deliver mRNA encoding nanoluciferase into somatic cells, including planarian adult stem cells (neoblasts). The use of a luminescent reporter allowed us to quantitatively measure protein expression through spectroscopy and microscopy, thus overcoming the strong autofluorescent background of planarian tissues. Using this platform, we investigated the use of endogenous untranslated region (UTR) sequences and codon usage bias to post-transcriptionally alter gene expression. Our work provides a strong foundation for advancing exogenous gene expression and for the rapid prototyping of genetic constructs to accelerate the development of transgenic techniques in planarians.

Download Full-text

A Novel Regulated Hybrid Promoter That Permits Autoinduction of Heterologous Protein Expression inKluyveromyces lactis

Applied and Environmental Microbiology ◽

10.1128/aem.00542-19 ◽

2019 ◽

Vol 85 (14) ◽

Cited By ~ 2

Author(s):

Hassan Sakhtah ◽

Juliane Behler ◽

Alana Ali-Reynolds ◽

Thomas B. Causey ◽

Saulius Vainauskas ◽

...

Keyword(s):

Gene Expression ◽

Carbon Source ◽

Protein Expression ◽

Recombinant Proteins ◽

Heterologous Protein ◽

Kluyveromyces Lactis ◽

Heterologous Protein Expression ◽

Heterologous Proteins ◽

Content Type ◽

Hybrid Promoter

ABSTRACTThe yeastKluyveromyces lactishas been a successful host for the production of heterologous proteins for over 30 years. Currently, the galactose-/lactose-inducible and glucose-repressibleLAC4promoter (PLAC4) is the most widely used promoter to drive recombinant protein expression inK. lactis. However, PLAC4is not fully repressed in the presence of glucose and significant protein expression still occurs. Thus, PLAC4is not suitable in processes where tight regulation of heterologous gene expression is required. In this study, we devised a novelK. lactispromoter system that is both strong and tightly controllable. We first tested several different endogenousK. lactispromoters for their ability to express recombinant proteins. A novel hybrid promoter (termed P350) was created by combining segments of twoK. lactispromoters, namely, the strong constitutive PGAP1promoter and the carbon source-sensitive PICL1promoter. We demonstrate that P350is tightly repressed in the presence of glucose or glycerol and becomes derepressed upon depletion of these compounds by the growing cells. We further illustrate the utility of P350-controlled protein expression in shake flask and high-cell-density bioreactor cultivation strategies. The P350hybrid promoter is a strong derepressible promoter for use in autoinduction of one-step fermentation processes for the production of heterologous proteins inK. lactis.IMPORTANCEThe yeastKluyveromyces lactisis an important host for the expression of recombinant proteins at both laboratory and industrial scales. However, the system lacks a tightly regulated promoter that permits controlled expression of heterologous proteins. In this study, we report the engineering of a highly regulated strong hybrid promoter (termed P350) for use inK. lactis. P350is tightly repressed by glucose or glycerol in the medium but strongly promotes gene expression once the carbon source has been consumed by the cells. This feature permits heterologous protein expression to be “autoinduced” at any scale without the addition of a gratuitous inducer molecule or changing feed solutions.

Download Full-text

Fungal Light-Oxygen-Voltage Domains for Optogenetic Control of Gene Expression and Flocculation in Yeast

mBio ◽

10.1128/mbio.00626-18 ◽

2018 ◽

Vol 9 (4) ◽

Cited By ~ 10

Author(s):

Francisco Salinas ◽

Vicente Rojas ◽

Verónica Delgado ◽

Javiera López ◽

Eduardo Agosin ◽

...

Keyword(s):

Gene Expression ◽

Protein Expression ◽

Heterologous Protein ◽

Dynamic Range ◽

Industrial Applications ◽

Yeast Cells ◽

Heterologous Protein Expression ◽

Control Of Gene Expression ◽

Content Type ◽

Chemical Inducers

ABSTRACT Optogenetic switches permit accurate control of gene expression upon light stimulation. These synthetic switches have become a powerful tool for gene regulation, allowing modulation of customized phenotypes, overcoming the obstacles of chemical inducers, and replacing their use by an inexpensive resource: light. In this work, we implemented FUN-LOV, an optogenetic switch based on the photon-regulated interaction of WC-1 and VVD, two LOV (light-oxygen-voltage) blue-light photoreceptors from the fungus Neurospora crassa. When tested in yeast, FUN-LOV yields light-controlled gene expression with exquisite temporal resolution and a broad dynamic range of over 1,300-fold, as measured by a luciferase reporter. We also tested the FUN-LOV switch for heterologous protein expression in Saccharomyces cerevisiae, where Western blot analysis confirmed strong induction upon light stimulation, surpassing by 2.5 times the levels achieved with a classic GAL4/galactose chemical-inducible system. Additionally, we utilized FUN-LOV to control the ability of yeast cells to flocculate. Light-controlled expression of the flocculin-encoding gene FLO1, by the FUN-LOV switch, yielded flocculation in light (FIL), whereas the light-controlled expression of the corepressor TUP1 provided flocculation in darkness (FID). Altogether, the results reveal the potential of the FUN-LOV optogenetic switch to control two biotechnologically relevant phenotypes such as heterologous protein expression and flocculation, paving the road for the engineering of new yeast strains for industrial applications. Importantly, FUN-LOV’s ability to accurately manipulate gene expression, with a high temporal dynamic range, can be exploited in the analysis of diverse biological processes in various organisms. IMPORTANCE Optogenetic switches are molecular devices which allow the control of different cellular processes by light, such as gene expression, providing a versatile alternative to chemical inducers. Here, we report a novel optogenetic switch (FUN-LOV) based on the LOV domain interaction of two blue-light photoreceptors (WC-1 and VVD) from the fungus N. crassa. In yeast cells, FUN-LOV allowed tight regulation of gene expression, with low background in darkness and a highly dynamic and potent control by light. We used FUN-LOV to optogenetically manipulate, in yeast, two biotechnologically relevant phenotypes, heterologous protein expression and flocculation, resulting in strains with potential industrial applications. Importantly, FUN-LOV can be implemented in diverse biological platforms to orthogonally control a multitude of cellular processes.

Download Full-text

FUN-LOV: Fungal LOV domains for optogenetic control of gene expression and flocculation in yeast

10.1101/285296 ◽

2018 ◽

Author(s):

Francisco Salinas ◽

Vicente Rojas ◽

Verónica Delgado ◽

Javiera López ◽

Eduardo Agosin ◽

...

Keyword(s):

Gene Expression ◽

Protein Expression ◽

Heterologous Protein ◽

Dynamic Range ◽

Industrial Applications ◽

Yeast Cells ◽

Heterologous Protein Expression ◽

Control Of Gene Expression ◽

Chemical Inducers ◽

Cellular Processes

AbstractOptogenetic switches permit accurate control of gene expression upon light stimulation. These synthetic switches have become a powerful tool for gene regulation, allowing modulation of customized phenotypes, overcoming the obstacles of chemical inducers and replacing their use by an inexpensive resource: light. In this work, we implemented FUN-LOV; an optogenetic switch based on the photon-regulated interaction of WC-1 and VVD, two LOV (Light Oxygen Voltage) blue-light photoreceptors from the fungus Neurospora crassa. When tested in yeast, FUN-LOV yields light-controlled gene expression with exquisite temporal resolution, and a broad dynamic range of over 1300-fold, as measured by a luciferase reporter. We also tested the FUN-LOV switch for heterologous protein expression in Saccharomyces cerevisiae, where Western blot analysis confirmed strong induction upon light stimulation, surpassing by 2.5 times the levels achieved with a classic GAL4/galactose chemical inducible system. Additionally, we utilized FUN-LOV to control the ability of yeast cells to flocculate. Light-controlled expression of the flocculin encoding gene FLO1, by the FUN-LOV switch, yielded Flocculation in Light (FIL), whereas the light-controlled expression of the co-repressor TUP1 provided Flocculation in Darkness (FID). Overall, the results revealed the potential of the FUN-LOV optogenetic switch to control two biotechnologically relevant phenotypes such as heterologous protein expression and flocculation, paving the road for the engineering of new yeast strains for industrial applications. Importantly, FUN-LOV’ s ability to accurately manipulate gene expression, with a high-temporal dynamic range, can be exploited in the analysis of diverse biological processes in various organisms.ImportanceOptogenetic switches are molecular devices which allow the control of different cellular processes by light, such as gene expression, providing a versatile alternative to chemical inducers. Herein, we report a novel optogenetic switch (FUN-LOV) based on the LOV-domain interaction of two blue-light photoreceptors (WC-1 and VVD) from the fungus N. crassa. In yeast cells, FUN-LOV allowed tight regulation of gene expression, with low background in darkness and a highly dynamic and potent control by light. We used FUN-LOV to optogenetically manipulate, in yeast, two biotechnologically relevant phenotypes: heterologous protein expression and flocculation, resulting in strains with potential industrial applications. Importantly, FUN-LOV can be implemented in diverse biological platforms to orthogonally control a multitude of cellular processes.

Download Full-text