Modular and Molecular Optimization of a LOV (Light-Oxygen-Voltage)-Based Optogenetic Switch in Yeast.

Optogenetic switches allow light-controlled gene expression with reversible and spatiotemporal resolution. In Saccharomyces cerevisiae, optogenetic tools hold great potential for a variety of metabolic engineering and biotechnology applications. In this work, we report on the modular optimization of the fungal light-oxygen-voltage (FUN-LOV) system, an optogenetic switch based on photoreceptors from the fungus Neurospora crassa. We also describe new switch variants obtained by replacing the Gal4 DNA-binding domain (DBD) of FUN-LOV with nine different DBDs from yeast transcription factors of the zinc cluster family. Among the tested modules, the variant carrying the Hap1p DBD, which we call "HAP-LOV", displayed higher levels of luciferase expression upon induction compared to FUN-LOV. Further, the combination of the Hap1p DBD with either p65 or VP16 activation domains also resulted in higher levels of reporter expression compared to the original switch. Finally, we assessed the effects of the plasmid copy number and promoter strength controlling the expression of the FUN-LOV and HAP-LOV components, and observed that when low-copy plasmids and strong promoters were used, a stronger response was achieved in both systems. Altogether, we describe a new set of blue-light optogenetic switches carrying different protein modules, which expands the available suite of optogenetic tools in yeast and can additionally be applied to other systems.

Photoperiodic Responses and Characterization of the Cmvvd Gene Encoding a Blue Light Photoreceptor from the Medicinal Caterpillar Fungus Cordyceps militaris (Ascomycetes).

Light is a necessary environmental factor for production of conidia and pigment, formation of stroma, and development of Cordyceps militaris, a well-known edible and medicinal mushroom. In this study, an obvious rhythm loop was observed in certain strains of C. militaris under conditions of alternating 12-hour intervals of dark and light. A possibly related gene, Cmvvd, the homologue of the blue-light photoreceptor of Neurospora crassa, was cloned from the genome of C. militaris. The protein CmVVD is predicted to be 203 amino acids in length and is characterized by the presence of a light, oxygen, or voltage domain. Analysis of the CmVVD sensor domain (light, oxygen, or voltage) suggested that it is a blue-light receptor. Cysteine 108 is essential for the in vivo function of VIVID (VVD) in N. crassa photoadaptation. However, proline is in this position instead in all of the tested CmVVD proteins, suggesting that CmVVD may have a different function or may function in ways different from VVD in N. crassa. Genetic variation analysis of CmVVD in 6 representative strains indicated that 3 informative sites exist. Cmvvd messenger RNA was able to be induced by light, and the expression level increased over 10 times after irradiation and was maintained at high levels in the nascent fruiting body. The light-induced expression of Cmvvd was abolished in Cmwc-1 mutants, suggesting that the expression of Cmvvd is dependent on the photoreceptor CmWC-1 or on a functional CmWC-1/WC-2 complex. This article will help to open the still-unexplored field of circadian rhythms for this fungus.

Protein conformational changes of Agrobacterium phytochrome Agp1 during chromophore assembly and photoconversion.

Phytochromes are widely distributed photochromic biliprotein photoreceptors. Typical bacterial phytochromes such as Agrobacterium Agp1 have a C-terminal histidine kinase module; the N-terminal chromophore module induces conformational changes in the protein that lead to modulation of kinase activity. We show by protein cross-linking that the C-terminal histidine kinase module of Agp1 mediates stable dimerization. The fragment Agp1-M15, which comprises the chromophore module but lacks the histidine kinase module, can also form dimers. In this fragment, dimer formation was stronger for the far-red-absorbing form Pfr than for the red-absorbing form Pr. The same or similar behavior was found for Agp1-M15Delta9N and Agp1-M15Delta18N, which lack 9 and 18 amino acids of the N-terminus, respectively. The fragment Agp1-M20, which is derived from Agp1-M15 by truncation of the C-terminal "PHY domain" (191 amino acids), can also form dimers, but dimerization is independent of irradiation conditions. The cross-linking data also showed that the PHY domain is in tight contact with Lys 16 of the protein and that the nine N-terminal amino acids mediate oligomer formation. Limited proteolysis shows that the hinge region between the chromophore module and the histidine kinase and a part of the PHY domain become exposed upon Pr to Pfr photoconversion.

Rhodobacter capsulatus photoactive yellow protein: genetic context, spectral and kinetics characterization, and mutagenesis.

A gene for photoactive yellow protein (PYP) was previously cloned from Rhodobacter capsulatus (Rc), and we have now found it to be associated with genes for gas vesicle formation in the recently completed genome sequence. However, the PYP had not been characterized as a protein. We have now produced the recombinant RcPYP in Escherichia coli as a glutathione-S-transferase (GST) fusion protein, along with the biosynthetic enzymes, resulting in the formation of holo-RcPYP following cleavage of the GST tag. The absorption spectrum (with characteristic peaks at 435 and 375 nm) and the photocycle kinetics, initiated by a laser flash at 445 nm, are generally similar to those of Rhodobacter sphaeroides (RsPYP) but are significantly different from those of the prototypic PYP from Halorhodospira halophila (HhPYP), which has a single peak at 446 nm and has slower recovery. RcPYP also is photoactive when excited with near-ultraviolet laser light, but the end point is then above the preflash baseline. This suggests that some of the PYP chromophore is present in the cis-protonated conformation in the resting state. The excess 435 nm form in RcPYP, built up from repetitive 365 nm laser flashes, returns to the preflash baseline with an estimated half-life of 2 h, which is markedly slower than that for the same reaction in RsPYP. Met100 has been reported to facilitate cis-trans isomerization in HhPYP, yet both Rc and RsPYPs have Lys and Gly substitutions at positions 99 and 100 (using HhPYP numbering throughout) and have 100-fold faster recovery kinetics than does HhPYP. However, the G100M and K99Q mutations of RcPYP have virtually no effect on kinetics. Apparently, the RcPYP M100 is in a different conformation, as was recently found for the PYP domain of Rhodocista centenaria Ppr. The cumulative results show that the two Rhodobacter PYPs are clearly distinct from the other species of PYP that have been characterized. These properties also suggest a different functional role, that we postulate to be in regulation of gas vesicle genes, which are known to be light-regulated in other species.

Heterologous production of Halorhodospira halophila holo-photoactive yellow protein through tandem expression of the postulated biosynthetic genes.

The photoactive yellow protein (PYP) is a bacterial photoreceptor which is the structural prototype for the PAS domain superfamily of regulators and receptors. PYP is known to have a unique p-hydroxycinnamic acid chromophore, covalently attached to a cysteine. To date, it has not been shown how holo-PYP is formed in vivo. Two genes, nearby pyp, were postulated to encode the biosynthetic enzymes, but only one was previously isolated and shown to have the requisite activity. By using a dual plasmid system, one expressing the PYP from Halorhodospira halophila and the other expressing a two-gene operon, consisting of tyrosine ammonia lyase and p-hydroxycinnamic acid ligase, we are able to present evidence that a functionally active holo-PYP can be synthesized in Escherichia coli. Plasmids containing only one of the two enzymes failed to produce holoprotein. Thus, the two genes have been shown to be both necessary and sufficient for production of holoprotein, although the activating group remains unknown. This expression system not only holds great potential for mutagenesis studies but also opens new possibilities in the search for (a) signaling partner(s) of the PYP.

Characterization of a bacterial tyrosine ammonia lyase, a biosynthetic enzyme for the photoactive yellow protein.

During genome sequence analysis of Rhodobacter capsulatus, nearby open reading frames were found that encode a photoactive yellow protein (PYP) and a hypothetical biosynthetic enzyme for its chromophore, a tyrosine ammonia lyase (TAL). We isolated the TAL gene, overproduced the recombinant protein in Escherichia coli, and after purification analyzed the enzyme for its activity. The catalytic efficiency for tyrosine was shown to be approximately 150 times larger than for phenylalanine, suggesting that the enzyme could in fact be involved in biosynthesis of the PYP chromophore. To our knowledge it is the first time this type of enzyme has been found in bacteria.