Hilgers, F. | Search bioRxiv

bioRxiv2026

A multichromatic UV-RGB optogenetic toolbox for control of gene expression in Pseudomonas putida

Optogenetics uses light to provide precise, reversible, and non-invasive control over bacterial functions including gene expression with high spatiotemporal resolution. Although many optogenetic systems have been developed for Escherichia coli, only a limited number is available for other prokaryotes, such as pseudomonads. Here, we establish a toolbox of genomically integrated optogenetic gene cassettes for light-responsive regulation of target gene expression in Pseudomonas putida with UV-A, blue, green, and red light. Using transposon Tn7-mediated chromosomal integration, we implemented four optogenetic systems: the photocaged IPTG/Ptac-LacI system, the LOV-based Dusk switch, the cyanobacteriochrome system CcaS/R, and different bacteriophytochrome-based REDusk variants. Benchmarking with the mCherry reporter demonstrated high dynamic ranges of up to [~]270-fold, low basal expression, and largely homogeneous population responses in P. putida. Spatial illumination further enabled patterned single- and dual-color gene expression. As a proof of concept, we applied the toolbox for light-controlled regulation of pyoverdine (PVD) biosynthesis in P. putida. The expression of the alternative sigma factor PfrI, which upregulates the production of the siderophore during iron-limitation, was placed under optogenetic control in a {Delta}pfrI background. The red-light responsive switches resulted in the strongest induction of PVD synthesis and enabled spatial control of siderophore-mediated microbial interactions. To demonstrate transferability, light-dependent pyoverdine production was further established in the human pathogen Pseudomonas aeruginosa PAO1. Together, this optogenetic plug-and-play toolbox enables non-invasive, spatiotemporal reprogramming of gene expression and cellular processes in pseudomonads and expands the available optogenetic repertoire beyond established model organisms.

synthetic biology↗

bioRxiv2020

Ustilago maydis serves as a novel production host for the synthesis of plant and fungal sesquiterpenoids

AbtractSesquiterpenoids are important secondary metabolites with various pharma- and nutraceutical properties. In particular, higher basidiomycetes possess a versatile biosynthetic repertoire for these bioactive compounds. To date, only a few microbial production systems for fungal sesquiterpenoids have been established. Here, we introduce Ustilago maydis as a novel production host. This model fungus is a close relative of higher basidiomycetes. It offers the advantage of metabolic compatibility and potential tolerance for substances toxic to other microorganisms. We successfully implemented a heterologous pathway to produce the carotenoid lycopene that served as a straightforward read-out for precursor pathway engineering. Overexpressing genes encoding enzymes of the mevalonate pathway resulted in increased lycopene levels. Verifying the subcellular localisation of the relevant enzymes revealed that initial metabolic reactions might take place in peroxisomes: despite the absence of a canonical peroxisomal targeting sequence, acetyl-CoA C-acetyltransferase Aat1 localised to peroxisomes. By expressing the plant (+)-valencene synthase CnVS and the basidiomycete sesquiterpenoid synthase Cop6, we succeeded in producing (+)-valencene and -cuprenene, respectively. Importantly, the fungal compound yielded about tenfold higher titres in comparison to the plant substance. This proof of principle demonstrates that U. maydis can serve as promising novel chassis for the production of terpenoids.

microbiology↗