Genetically encodable bioluminescent system from fungi

Significance We present identification of the luciferase and enzymes of the biosynthesis of a eukaryotic luciferin from fungi. Fungi possess a simple bioluminescent system, with luciferin being only two enzymatic steps from well-known metabolic pathways. The expression of genes from the fungal bioluminescent pathway is not toxic to eukaryotic cells, and the luciferase can be easily co-opted to bioimaging applications. With the fungal system being a genetically encodable bioluminescent system from eukaryotes, it is now possible to create artificially bioluminescent eukaryotes by expression of three genes. The fungal bioluminescent system represents an example of molecular evolution of a complex ecological trait and with molecular details reported in the paper, will allow additional research into ecological significance of fungal bioluminescence.

replacement of zeocin resistant gene (Sh bl) with the kanamycin (aphA1), blasticidin (bsd) or hygromycin (hph) resistant genes, respectively. The aphA1 gene was amplified from the pET28 (Novagen) vector using the primer pair 5'-AAGGGAACATGTCTcatattcaacgggaaacgtcttg-3' (PciI site underlined) and 5'-AAAAACACGAAGTGTTAGAAAAACTCATCGAGCATCAAA-3' (DraIII site underlined) and cloned into pGAPZ resulting in pGAP-Kan plasmid. bsd gene was amplified from the pPic6αA vector (Invitrogen) with the primer pair 5'-TCCTCACCATGGCCAAGCCTTTGTCTCAAG-3' (NcoI site underlined) and 5'-ACAGACCACGAAGTGTTAGCCCTCCCACACATAACCA-3' (DraIII site underlined) and cloned into the corresponding sites of the recipient plasmid resulting in pGAP-Bsd. hph gene was amplified from the pcDNA3.1/Hygro(+) vector (Invitrogen) using the primer pair 5'-AACCCATCATGAaaaagcctgaactcaccg-3' (PagI site underlined) and 5'-TCTTTAGCGCTTTAttcctttgccctcggacg-3' (AfeI site underlined). pGAPZ was digested with DraIII, the overhanging nucleotides were removed with Klenow fragment, then linear DNA was subsequently digested with NcoI and used for cloning of PCR product HPH resulting in pGAP-Hyg. To generate GAP-pPic9 vector containing only HIS selective marker, GAP-pPic9K vector was digested with XhoI and BbvCI sites to remove the kanamycin resistance cassette. The ends of the DNA were blunted with Klenow fragment and re-ligated to generate the desired construct.
Clone nnLuz-H3H with the highest specific activity of both enzymes was used for the insertion of genes polyketide synthase (hisps) from N. nambi and 4'-phosphopantetheinyl transferase (npgA) from Aspergillus nidulans. The predicted 5.1 kb hisps gene without introns was ordered as a linear synthetic DNA and cloned into pGAPZ vector using Gibson assembly. Correctness of the recombinant hisps gene was verified by Sanger sequencing. Gene of 4'-phosphopantetheinyl transferase npgA was also ordered as linear synthetic DNA with flanking BstBI/SpeI restriction sites at the ends and cloned into the corresponding sites of pGAP-Bsd. The resulting plasmids NpgA/pGAP-Bsd and HispS/pGAPZ were linearized by AvrII restriction site. Both constructs were electroporated into nnLuz-H3H strain obtained above. Transformed cells were grown on YPDS plate (1 M sorbitol, 2% peptone, 1% yeast extract, 2 % glucose, 2% agar) supplemented with 100 ug ml -1 zeocin and 200 ug ml -1 blasticidin S. Colonies were sprayed with a 3.3 mg ml −1 solution of caffeic acid and analyzed by Fusion-Pulse.7 (Vilber Lourmat) equipped with DARQ-7 camera. The brightest clone nnLuz-H3H-HispS-NpgA was incubated with shaking for 72 h at 30°C in YPD medium (2% peptone, 1% yeast extract, 2 % glucose) supplemented with 0.75% caffeic acid. This allowed direct observation of bioluminescence with eyes or by a camera (Fig. 3A).
Whole genome sequencing of the engineered glowing yeast strain. We sequenced genome of the autoluminescent strain to confirm insertion of the genes. Genomic DNA was extracted and purified according to the protocol from the kit for expression of recombinant proteins in Pichia pastoris (Pichia Expression Kit manual, Invitrogen, cat. K1710-01). Library preparation was performed with TruSeq Nano DNA Low Throughput Library Prep Kit (Illumina, Cat. #20015964). We used multiplexed paired-end whole genome sequencing on Illumina NovaSeq 6000 device and NovaSeq 6000 S2 Reagent Kit (Illumina, Cat.#20012861) according to manufacturer's manual. Average length of the DNA fragments was about 350 bp before ligation of sequencing adapters. Raw sequencing reads were mapped to P. pastoris GS115 reference genome and additional sequences of all seven inserted constructs by BWA-MEM algorithm (7). Coverage calculations have been performed with bedtools. Copy number estimations for each of the inserted genes are summarized in Table S3. Alignments of Pichia pastoris genome sequencing reads are available at Figshare (doi: 10.6084/m9.figshare.6738953).
Expression in E. coli and purification of nnLuz. The coding sequence of nnLuz was amplified using the primers 5'-GGATCCCGCATTAACATTAGCCTCTC-3' (BamHI site underlined) and 5'-AAGCTTTCACTTGGCGTTTTCTACAATCTTTC-3' (HindIII site underlined), and cloned into the pET-23b vector (Novagen). E. coli cells BL21 DE3 CodonPlus (Stratagene) transformed with the plasmid nnLuz/pET23 were cultivated with vigorous shaking at 37°C in LB medium containing 200 ug/ml ampicillin and 34 ug/ml chloramphenicol. When the culture reached an optical density of 0.6 at 600 nm, luciferase synthesis was induced with 0.4 mM IPTG. After induction, the cultivation was continued for 16 h at 25°C. Expression of nnLuz in E. coli resulted in accumulation of most of the recombinant protein in insoluble inclusion bodies. To purify the protein, cells were harvested by centrifugation at 4 °C. The cells were suspended in 50 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 10 mM 2-ME, pH 8.0, and disrupted by sonication, using a Sonics VibraCell ultrasonic processor (Sonics and Materials Inc). After a 20-minute centrifugation at 10,000xg the pellet (inclusion bodies) was dissolved in 8M Urea, 50mM Tris-HCl, 1 mM TCEP, pH 8.0 buffer (buffer A) for 12 hours at room temperature. The solution was centrifuged at 30, 000 x g for 30 min at room temperature and supernatant was applied onto Ni Sepharose excel column (GE Healthcare Life Sciences), equilibrated with buffer A. After washing the column subsequently with buffer A, then with 0.5M NaCl in buffer A and with 10 mM imidazole in buffer A, luciferase was eluted with 250 mM imidazole in buffer A. Pooled fractions were buffer-exchanged on PD-10 column (GE Healthcare) into 10 mM Na-Acetate, 6M Urea pH 5.5 buffer. In vitro refolding of luciferase was performed by 50-fold dilution into 20 mM MES-NaOH, 5% glycerol, 1 mM DTT pH 6.2 buffer at 10 o C. After centrifugation at 30,000xg for 30 min at 4C supernatant was concentrated by ultrafiltration using 10 kDa membrane (Ultracel, Millipore) and stored at -70C. Deletion of the transmembrane region did not lead to notable solubilization of nnLuz in E. coli cytoplasm.The purified and in vitro refolded nnLuz was soluble and stable in at slightly acidic solutions (around pH 6.0) without detergents. However, for the luminescent activity of recombinant nnLuz the presence of detergents was strictly required.
Extraction of native nnLuz from Neonothopanus nambi mycelium. 10 g of Neonothopanus nambi mycelium was washed with water, dried by removing excess of water and frozen in liquid nitrogen. Frozen material was ground in a mortar and suspended in 100 ml of buffer (100 mM sodium phosphate buffer, 5 mM EDTA, 5 mM TCEP, 0.5 mM PMSF, pH 7.4). After a 30 minute incubation in ice-water, the suspension was sonicated for 10 minutes using Sonics VibraCell ultrasonic processor (Sonics and Materials inc), centrifuged at 5,000 g (4°C) for 15 min. The supernatant was then collected and centrifuged again at 140,000 g (4°C) for 1 hour. The supernatant was discarded and the pellet (microsomal fraction) containing the nnLuz was suspended in water by a Dounce grinder and stored at -70 °C.
Denaturing polyacrylamide gel electrophoresis and amino acid sequence analysis. SDS-PAGE was performed according to [ref. (8)] using a 10-25% gradient polyacrylamide gel. Gel staining was done according to the silver staining protocol from (9), or using a standard Coomassie G250 staining. Protein bands were excised from the gel and were identified by LC-MS. In-gel trypsinolysis was done according to [ref. (10)]. Mass spectrometry analysis was performed on the Ultimate 3000 Nano LC System (Thermo Fisher Scientific), connected to a Q Exactive HF mass spectrometer (Thermo Fisher Scientific). For data analysis, we used Mascot software (Matrix Science).
Western blotting. After SDS-PAGE the proteins were electrophoretically transferred onto a nitrocellulose membrane. After blocking with 5% fat-free dry milk in 20 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, pH 8.0 buffer the membrane was incubated with the mouse anti-HisTag antibody conjugated to horseradish peroxidase (Abcam). Immobilon Western kit (Millipore) was used for visualization. Signal was detected by Vilber Lourmat Fusion system. Luminescence measurements. Luminescence spectra were recorded using Varian Cary Eclipse fluorescence spectrophotometer. For the bioluminescence spectra, 1 cm agar square containing cultured Neonothopanus nambi mycelium was overlaid with 50 ul of 20 uM solution of luciferin in water and was placed in a cuvette facing emission PMT. For the chemiluminescence spectra of Pichia pastoris microsomal fraction, 50 ul of microsomal suspension was mixed with 1 ml of 100 mM sodium phosphate, 0.1% DDM, pH 7.5 buffer. Obtained data were corrected for the kinetics of luminescence intensity during spectra acquisition.
pH and temperature dependence of recombinant nnLuz chemiluminescence. For measuring the pH dependence of purified nnLuz chemiluminescence, 5 ul of luciferase solution (obtained by dissolving Pichia pastoris microsomal fraction as described in the above section) was mixed with 100ul of 200 mM buffer of indicated pH, 0.1% DDM and was placed into Promega Glomax 20/20 luminometer. Immediately after that, 25ul of 25 uM fungal luciferin in 2 mM formic acid, 0.1% DDM was injected and the light intensity was recorded at 1 sec intervals. The results are shown in Fig. S10.
For the temperature dependence of purified nnLuz stability, 25ul of luciferase solution (obtained by dissolving Pichia pastoris microsomal fraction as described in the above section) in 20 mM MES-NaOH, 5% glycerol, 1 mM DTT pH 6.2 buffer was incubated at indicated temperature for 10 min. After that, luciferase activity was measured with Promega Glomax 20/20 luminometer by injecting 25ul of 15 uM fungal luciferin in 2mM formic acid, 0.1% DDM into a vial containing 5 ul of luciferase sample in 100ul of 200 mM sodium phosphate buffer, 0.5 M Na 2 SO 4 , 0.1% DDM, pH 8.0. The results are shown in Fig. S10.

Сonversion of hispidin into luciferin in vitro.
For this experiment we used cells lysate of Pichia pastoris strain carrying a genomic copy of h3h gene. 5ul of the lysate was added to 40 ul of the PBS buffer (pH 7.4) containing 1 mM NADPH and 1 uM hispidin. After 5 min of incubation at 25°C 100 ul of assay buffer (100 mM sodium phosphate, 0.1% DDM, pH 7.5 buffer) and 0.5 ul Pichia pastoris carrying genomic copies of nnluz gene microsomal fraction were added to induce light emission. Pichia pastoris wild type cells were used in the same experiment as a negative control. Kinetics of bioluminescence was registered by a custom made luminometer (Oberon-K, Russia), results are shown in Fig. S21.

In vivo experiments
Heterologous expression of nnluz gene in the nervous system of developing Xenopus laevis embryo. The nnluz gene was subcloned into pCS2+ vector by NheI (blunted)/EcoRI (blunted) and SalI/XhoI cloning. Resulted plasmid was linearized by Acc65 restriction site and used in in vitro transcription to obtain nnLuz mRNA, using SP6 mМessage mМachine Kit (Ambion). mRNA was purified with the CleanRNA Standard Kit (Evrogen) and injected in both blastomeres (~500 pg per blastomere) of two-cell-stage Xenopus leavis embryo. RLD (rhodamine lysin dextran) was used as a tracer. The fungal luciferin solution (660 ug/ml in DMSO) was injected into the blastocoel cavity of gastrula-stage embryos (stage 10.5). Luminescence was detected after luciferin injection with the DM6000 microscope (Leica).
Whole-body imaging in mice. Mus musculus carcinoma CT26 cells were injected subcutaneously into the back of a female 10-weeks-old mouse (BALB/c strain, animal facility of Pirogov Russian National Research Medical University, Moscow, Russia). Cells expressing nnLuz were injected into the left side of the back, and cells expressing Photinus pyralis luciferase were injected into the right side. Before injection, the mouse was anesthetized by isoflurane/air (2,5/97,5 %) inhalation anesthesia. Ten minutes later, a mixture of fungal (0.5 mg) and firefly (0.5 mg) luciferin solutions was administered via intraperitoneal injection. Luminescence was visualized by IVIS Spectrum CT (PerkinElmer). The mouse was kept under sterile barrier conditions, usual light/dark cycle, and was given food and water ad libitum. The animal was treated humanely with regard to alleviation of suffering. All experiments were approved by the local ethical committee of RNRMU and were carried out in accordance to EU Directive 2016/63/EU.

Determination of LC50 of fungal luciferin to HEK293T cells.
HEK293T cells were seeded into 96-well plate, supported with 200 ul of full DMEM (described previously), 5 000 cells per well. Cells were incubated for 24 hours at 37ºC with 5% CO 2 , then fungal luciferin in DMSO was added to each well. Concentration of fungal luciferin was 0 ug/ml, 10 ug/ml, 25 ug/ml, 50 ug/ml, 100 ug/ml, 250 ug/ml, 500 ug/ml, 1000 mg/ml, each concentration of luciferin was added to 3 wells for preparing sample replicas. The concentration of DMSO was equal in each well (1%). Cells with luciferin and DMSO were incubated for 48 hours and then viability test using alamarBlue (ThermoFisher) was performed. The results are shown in Fig. S18.

Transfection efficiency calculation.
Approximately 300 000 HEK293T cells were seeded to 35 mm Fluorodish (World Precision Instruments). 24 hours after (at the 80% fo confluency) the cotransfection with FuGENE HD (Promega) of constructions nnLuz-C1 and pTurboFP635-C in equimolar quantities was performed due to the recommendation of the manufacturer. 6 hours after the transfection the growing medium was changed to fresh aliquot of full DMEM and after overnight incubation at 37°C and 5% CO2 the microscopy was carried out. The pictures of the cells (20x objective) were taken in 3 channels: brightfield, fluorescence in the red channel (ex. 560 nm, em. 607-682 nm), luminescence in the green channel (>505 nm). 11 sets of pictures of different areas with the cells were taken in total. The amount of all cells, cells visible in green channel and cells visible in red channel was counted manually for each set of pictures and the level of transfection was counted as average of all data (Fig. S19).
Relative growth rates of wild type strain of P. pastoris (GS115) versus autoluminescent strain of P. pastoris. Cells were grown in liquid YPD medium with 100 ug ml -1 of zeocin overnight. The next day, cell suspension was diluted with fresh YPD medium up to optical density of 0.07 at 600 nm and cultivated with shaking for 21.5 h at 30°C. The aliquots of samples were measured at time points of 0, 2.5, 5, 6.5, 8, 10, 13.5, 14.5, 16, 17.5, 20 and 21.5 hours. Each cell line was analyzed in three biological replicates and each sample was measured in triplicates (Fig. S20).

Bioinformatic analysis of evolution
Gene prediction. For those genomes lacking a gene prediction, Augustus v3.1 [ref. (11)] was used to obtain the predictions using the parameters pre-calculated for Coprinus cinereus. We then used blastp to locate the proteins that belonged to the cluster. The proteins were then manually curated to ensure they were correctly predicted. Missing proteins were also searched for using exonerate (12) and added to the proteome if found. A complete list of proteomes used can be found in Table S1.
Species tree reconstruction. First a blast search was performed using each gene encoded in Neonothopanus nambi against a database containing 57 complete proteomes of Agaricales species. The blast results were first filtered (overlap > 0.3 and e-value < 1e-05) and then scanned in order to obtain those proteins that contained a single hit in at least 40 of the species. 36 proteins fulfilled this requirement. Muscle v3.8 [ref. (13)] was then used to align each of the 36 protein families, trimal v1.4 [ref. (14)] was used to filter the alignment (gt of 0.5) and then the alignments were concatenated into a single multiple sequence alignment. RAxML (version 8.0.3) (15) was then used to reconstruct the species tree using the PROTGAMMALG model.  (17). A consensus of the six resulting alignments was obtained using M-coffee (18) and then this alignment was filtered using trimAl v1.4 (-gt 0.1 and -cs 0.16667) (14). The trimmed alignment was then used to reconstruct the gene tree. First a model selection process was performed reconstructing a neighbour joining tree using BIONJ (19) and calculating the likelihoods using seven different model (JTT, WAG, MtREV, VT, LG, Blosum62, Dayhoff). The model with the best likelihood according to the AIC criterion (20) was then used to reconstruct a maximum likelihood tree using phyml v3.0 [ref. (21)]. Finally, a bootstrap analysis was run using 100 repetitions.
Gene trees were visualized using ETE v3.0 [ref. (22)]. Nodes with bootstraps below 50 were collapsed as they were deemed unreliable. Gene trees were rooted based on the species tree reconstructed above. As the age of the duplication event that occurred in the luciferase ancestor was not clear we added four additional homologs found in Eurotiomycetes (Ascomycota) to serve as outgroups.
Mapping of duplication and loss events. ETE v3.0 [ref. (22)] was used to analyse the gene trees. Duplications and speciation events were detected using a species-overlap algorithm which, for each node, checks whether there are overlapping species at either side of the node. If such overlap exists, the node is considered a duplication node; if not, it is considered a speciation node. The duplication node that led to the formation of luz, h3h and hisps was mapped onto the species tree by assuming that it occurred in the common ancestor of the species found at both sides of the node. Loss events were then detected by assuming that if all the members of a clade had lost the copy of the protein in question, then this loss should have occurred at the base of the clade.

Reconstruction of figure showing gene order. The nnLuz and nnH3H proteins for
Neonothopanus nambi were located in a single contig that contained 8 proteins in total. Blast searches were performed from these 8 proteins to all the other genomes of interest. In the image, each square represents a gene that is found in the Neonothopanus nambi luciferase contig and that has a homolog in the other species. In grey are genes whose involvement in luciferin biosynthesis is unknown, while the coloured genes represent the luciferase (luz), hispidin-3-hydroxylase (h3h), caffeylpyruvate hydrolase (cph) and hispidin synthase (hisps) genes. The thinner lines represent genes that are found in other species but not in Neonothopanus nambi. The green lines are coloured to indicate homology between the different species while the black ones indicate no homolog was found of these genes in the region of interest. Figure          Figure S10. Activity of nnLuz in various pH and temperature conditions. Total light emission within 100 s period following the addition of the luciferin, in buffers with various pH (A) or after 10 min incubation of recombinant purified nnLuz at various temperatures (B). The data represent the mean ± SD for at least three measurements. All measurements were obtained on a Promega Glomax 20/20 luminometer. See Supplementary materials section "pH and temperature dependence of recombinant nnLuz chemiluminescence" for details.   Figure S14. In vivo assay for nnH3H activity in Pichia pastoris with negative controls. Clones of P. pastoris cells, carrying genomic copies of nnLuz and nnh3h genes in incident light and in the darkness before and after spraying with solutions of caffeic acid (0.018 M L -1 ), hispidin (0.215 OD ml -1 ) and fungal luciferin (100 µg ml -1 ) in PBS with 1% DMSO. The same dish was used for sequential spraying with caffeic acid and hispidin. Each dish was sprayed by 450-500 ul of solution.   Clones of wild type P. pastoris cells in incident light and in darkness before and after spraying with solutions of caffeic acid (0.018 M L -1 ), hispidin (0.215 OD ml -1 ) and fungal luciferin (100 µg ml -1 ) in PBS with 1% DMSO. The same dish was sprayed sequentially by caffeic acid, hispidin and fungal luciferin. Each dish was sprayed by 450-500 ul of solution.