Light-based control of metabolic flux through assembly of synthetic organelles

Zhao, Evan M.; Suek, Nathan; Wilson, Maxwell Z.; Dine, Elliot; Pannucci, Nicole L.; Gitai, Zemer; Avalos, José L.; Toettcher, Jared E.

doi:10.1038/s41589-019-0284-8

Cited by 190 publications

(194 citation statements)

References 46 publications

(93 reference statements)

Supporting

Mentioning

194

Contrasting

Order By: Relevance

“…They also reveal two distinct classes of optoNBs, those for which binding occurs in the light and others for which it occurs in the dark. The ability to induce binding by switching to dark conditions is rare, as most previously-developed optogenetic tools exhibit light-induced binding; yet the few existing light-suppressible optogenetic tools available 23,24 have already proven useful for probing T cell signaling 25 , controlling metabolic flux 26,27 , and studying the consequences of protein phase separation 15 . Photoswitchable domain insertion thus holds promise for engineering light-based control of protein-protein interactions.…”

Section: Initial Optonbs Exhibit Weak Light-switchable Binding As Welmentioning

confidence: 99%

Optogenetic control of protein binding using light-switchable nanobodies

Gil

Zhao

Wilson

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

A growing number of optogenetic tools have been developed to control binding between two engineered protein domains. In contrast, relatively few tools confer light-switchable binding to a generic target protein of interest. Such a capability would offer substantial advantages, enabling photoswitchable binding to endogenous target proteins in vivo or light-based protein purification in vitro. Here, we report the development of opto-nanobodies (OptoNBs), a versatile class of chimeric photoswitchable proteins whose binding to proteins of interest can be enhanced or inhibited upon blue light illumination. We find that OptoNBs are suitable for a range of applications: modulating intracellular protein localization and signaling pathway activity and controlling target protein binding to surfaces and in protein separation columns. This work represents a first step towards programmable photoswitchable regulation of untagged, endogenous target proteins. Highlights 1. Opto-Nanobodies (OptoNBs) enable light-regulated binding to a wide range of protein targets. 2. We identify an optimized LOV domain and two loop insertion sites for light-regulated binding. 3. OptoNBs function in vivo: when expressed in cells and fused to signaling domains, OptoNBs enable light-activated and light-inhibited Ras/Erk signaling. 4. OptoNBs function in vitro: Target proteins can be reversibly bound to OptoNB-coated beads and separated through size-exclusion chromatography.3

show abstract

Section: Initial Optonbs Exhibit Weak Light-switchable Binding As Welmentioning

confidence: 99%

Optogenetic control of protein binding using light-switchable nanobodies

Gil

Zhao

Wilson

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…As examples, light has been used to control expression of enzymes involved in biofuel synthesis 13,14 and to regulate bacterial growth via metabolic control. 15,16 In addition, it has been used to enable light-activated drug release from hydrogels 17 and patterning of Escherichia coli onto multiple materials, 18 indicative of the wide ranging potential of optogenetic approaches. At present, the current bacterial optogenetic toolset primarily includes two-component systems 8,19,20 and split proteins.…”

Section: Introductionmentioning

confidence: 99%

Light-Inducible Recombinases for Bacterial Optogenetics

Sheets

Wong

Dunlop

2019

Preprint

View full text Add to dashboard Cite

Optogenetic tools can provide direct and programmable control of gene expression. Lightinducible recombinases, in particular, offer a powerful method for achieving precise spatiotemporal control of DNA modification. However, to-date this technology has been largely limited to eukaryotic systems. Here, we develop optogenetic recombinases for Escherichia coli which activate in response to blue light. Our approach uses a split recombinase coupled with photodimers, where blue light brings the split protein together to form a functional recombinase. We tested both Cre and Flp recombinases, Vivid and Magnet photodimers, and alternative protein split sites in our analysis. The optimal configuration, Opto-Cre-Vvd, exhibits strong blue light-responsive excision and low ambient light sensitivity. For this system we characterize the effect of light intensity and the temporal dynamics of light-induced recombination. These tools expand the microbial optogenetic toolbox, offering the potential for precise control of DNA excision with light-inducible recombinases in bacteria.

show abstract

“…We used a previously developed δ -integration ( δ -INT) vector, pYZ23 63 , to integrate multiple copies of gene cassettes into genomic YARCdelta5 δ-sites, the 337 bp long-terminal-repeat of S. cerevisiae Ty1 retrotransposons (YARCTy1-1, SGD ID: S000006792). The selection marker in pYZ23 is the shBleMX6 gene, which encodes a protein conferring resistance to zeocin and allows selection of different numbers of integration events by varying zeocin concentrations 91, 92 . Resistance to higher concentrations of zeocin correlates with a higher number of gene cassettes integrated into δ-sites.…”

Section: Methodsmentioning

confidence: 99%

Genetically encoded biosensors for branched-chain amino acid metabolism to monitor mitochondrial and cytosolic production of isobutanol and isopentanol in yeast

Zhang¹,

Hammer²,

Carrasco-López³

et al. 2020

Preprint

Self Cite

View full text Add to dashboard Cite

22Branched-chain amino acid (BCAA) metabolism can be harnessed to produce many valuable 23 chemicals. Among these, isobutanol, which is derived from valine degradation, has received 24 substantial attention due to its promise as an advanced biofuel. While Saccharomyces cerevisiae is 25 the preferred organism for isobutanol production, the lack of isobutanol biosensors in this organism 26 has limited the ability to screen strains at high throughput. Here, we use a transcriptional regulator of 27 BCAA biosynthesis, Leu3p, to develop the first genetically encoded biosensor for isobutanol 28 production in yeast. Small modifications allowed us to redeploy Leu3p in a second biosensor for 29 isopentanol, another BCAA-derived product of interest. Each biosensor is highly specific to 30 isobutanol or isopentanol, respectively, and was used to engineer metabolic enzymes to increase titers. 31The isobutanol biosensor was additionally employed to isolate high-producing strains, and guide the 32 construction and enhancement of mitochondrial and cytosolic isobutanol biosynthetic pathways, 33 including in combination with optogenetic actuators to enhance metabolic flux. These biosensors 34 promise to accelerate the development of enzymes and strains for branched-chain higher alcohol 35 production, and offer a blueprint to develop biosensors for other products derived from BCAA 36 metabolism. 37 38 Key Words: Branched-chain amino acid metabolism biosensor, LEU3, isobutanol biosensor, 39 isopentanol biosensor, metabolic engineering, high-throughput screen, enzyme engineering, 40 mitochondrial and cytosolic isobutanol pathways 41 42 particular, S. cerevisiae benefits from higher tolerance to alcohols than many bacteria 21 . In addition, 60BCHAs are naturally produced in S. cerevisiae as products of BCAA degradation. Accordingly, 61 metabolic pathways for BCHA production have been engineered by rewiring BCAA biosynthesis and 62 the Ehrlich degradation pathway (Supplementary Figure 1). The upstream BCAA biosynthetic 63 pathway is comprised of three mitochondrially localized enzymes: acetolactate synthase (ALS, 64 encoded by ILV2), ketol-acid reductoisomerase (KARI, encoded by ILV5), and dehydroxyacid 65 dehydratase (DHAD, encoded by ILV3) 22 . Ilv2p, Ilv3p, and Ilv5p convert pyruvate to the valine 66 4 precursor a-ketoisovalerate (a-KIV), which can be exported to the cytosol and converted to 67 isobutanol via the downstream BCAA Ehrlich degradation pathway 23 , comprised of a-ketoacid 68 decarboxylases (a-KDCs) and alcohol dehydrogenases (ADHs). Alternatively, a-KIV can be 69 converted to α-ketoisocaproate (a-KIC) by sequential reactions carried out by a-isopropylmalate 70 synthase (encoded by LEU4 and LEU9), isopropylmalate isomerase (encoded by LEU1), and 3-71 isopropylmalate dehydrogenase (encoded by LEU2). The LEU4 gene produces two forms of α-72 isopropylmalate synthase -a short form located in the cytosol, and a long form localized in 73 mitochondria along with Leu9p 22 . The same Ehrlich degradation pathway converts a-KIC to ...

show abstract

Light-based control of metabolic flux through assembly of synthetic organelles

Cited by 190 publications

References 46 publications

Optogenetic control of protein binding using light-switchable nanobodies

Optogenetic control of protein binding using light-switchable nanobodies

Light-Inducible Recombinases for Bacterial Optogenetics

Genetically encoded biosensors for branched-chain amino acid metabolism to monitor mitochondrial and cytosolic production of isobutanol and isopentanol in yeast

Contact Info

Product

Resources

About