Annotation of the results obtained in 2019
The purpose of the reporting phase was to obtain fully autonomous bioluminescent organisms that do not require the addition of exogenous luciferin for the glowing, using Pichia pastoris yeast. For this purpose we have selected the genes responsible for the biosynthesis of caffeic acid from Tyrosine (coumaroyl-3-hydroxylase and tyrosine-ammonia lyase), optimized their nucleotide sequence, cloned and coexpressed with the genes of the fungal bioluminescent system (luciferase, hispidin-3-hydroxylase, hispidin synthase, phosphopantetheinyl transferase). As a result we obtained a yeast strain demonstrating stable undamping luminescence visible to the naked eye when cultured under standard conditions for Pichia pastoris. We also showed that the previously discovered CPH gene actually encodes caffeoyl-pyruvate hydrolase, an enzyme that encircles the caffeic acid cycle converting oxyluciferin to caffeic acid. We also conducted preliminary experiments on the transfer of the bioluminescent system of fungi into mammalian cells. The HEK293NT cell line transiently transfected with plasmids encoding the luciferase, hispidin-3-hydroxylase, hispidin synthase and phosphopantetheinyl transferase genes showed well detectable luminescence in response to the addition of caffeic acid. Thus, the fungal bioluminescent system can be used for imaging laboratory mammals, including the creation of fully autonomous bioluminescent cancer cell lines and transplantable tumors.

 

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Annotation of the results obtained in 2017
We have discovered and sequenced the genes coding for luciferases of bioluminescent fungi Armillaria gallica, Armillaria mellea, Armillaria ostoyae, Mycena chlorophos, Mycena citricolor, Neonothopanus nambi, Omphalotus olearius, Panellus stipticus. The functions of the proteins encoded by these genes were confirmed by heterologous expression in human cells HEK293T. By analyzing the genome context of the discovered luciferases we found a cluster of genes coding for the complete metabolic cascade responsible of fungal bioluminescence. We obtained genetic constructs for the expression of hispidin synthase (HispS), hispidin-3-hydroxylase (H3H) and luciferase (Luz) in Pichia pastoris. The project news are available online at http://www.ibch.ru/structure/groups/TotalSynthesis

 

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Annotation of the results obtained in 2018
During the first year of work on the project, we identified and cloned genes that presumably encode enzymes of the fungal luciferin biosynthesis, namely the gene of hispidin synthase (HispS) and hispidin-hydroxylase (H3H). We assumed that HispS synthesizes hispidin from caffeic acid and malonyl-CoA, and H3H oxidizes hispidin to 3-hydroxyhydispidin, which, as was established earlier, is the fungal luciferin. We also suggested that for hispidin-synthase to work correctly the enzyme 4-phosphopantheteynyltransferase is needed so the corresponding gene (Npga from Aspergillus nidulans) was also cloned. The main task of the reporting stage was the experimental confirmation of the functions of these enzymes and the study of their properties, as well as the optimization of conditions for their functioning in a model organism. The amino acid sequence analysis of hispidin-hydroxylase revealed its similarity to 3-hydroxybenzoate 6-monooxygenases, which oxidize 3-hydroxybenzoates using NADH and oxygen. The conversion of hispidin to luciferin occurs in a similar way (by oxidation of the aromatic ring). Using data on the tertiary structure of hispidin-hydroxylase homologues (salicylate hydroxylase and others), we proposed a model of the tertiary structure of this protein. Using the obtained model, we assumed the possible structure of the site connecting the cofactor and the active center itself. The estimated amino acids of the active site that we have identified are conservative for both the nearest homologues of hispidin-synthase from higher fungi, and 3-hydroxybenzoate 6-monooxygenases. Analysis of the primary sequence of hispidin-synthase revealed two domains: one is homologous to acyl-CoA ligases, the second is homologous to styrylpyrone synthase. Such a multidomain structure is characteristic for polyketide synthases, which synthesize a variety of secondary metabolites. To confirm the supposed functions of these genes, we expressed them in eukaryotic heterologous systems. Pichia pastoris yeasts and mammalian cell culture HEK293T were chosen as model organisms for the expression of the studied genes. To detect the formation of luciferin, fungal luciferase was used. We found that upon co-expression of hispidin-hydroxylase and luciferase in model organisms, when hispisin is added to the cultures, well-detected luminescence occurs. However, upon individual genes expression into the model organism while further treatment with hispidin, the luminescence is missed. Thus, the H3H gene is indeed the gene of hispidin hydroxylase. Then we checked the work of hispidin-synthase. To do this, luciferase, hispidin-hydroxylase and hispidin-synthase were co-expressed in the model organisms. However, when caffeic acid was added to the model cultures, there is no luminescence detected, that indicate the inactivity of hispidin-synthase. To solve this problem, we co-expressed four genes — luciferase, hispidin-hydroxylase, hispidin-synthase, and 4-phosphopantheteinyl transferase in yeasts. Then upon treatment of the resulting strain with caffeic acid, bright luminescence appeared, visible to the naked eye. In the absence of at least one of the genes listed above, the addition of caffeic acid did not lead to the appearance of luminescence. Thus, the HispS gene cloned in this work is indeed the gene of hispidin synthase, and the protein encoded by it requires a posttranslational modification — phosphopantheteinylinization. The addition of the acetyl-CoA-carboxylase (ACC1) gene, which synthesizes malonyl-CoA, into the model system did not lead to an increase of the luminescent signal. We also tried to increase the efficiency of the bioluminescent system by merging luciferase and hispidin-synthase into one polypeptide chain. To do this, we have cloned the corresponding open reading frames, connecting them with the sequence encoding the linker region. Three different variants of connections of luciferase and hispidin- hydroxylase were created; however, the activity (luminescence) of such proteins expressed in mammalian cells was lower than while co-expression of individual plasmids encoding luciferase and hispidin-hydroxylase. This effect was observed in both cases with the addition of hispidin and with the addition of luciferin that indicates a decrease in the activity of luciferase upon formation of the fusion protein. We also obtained the sample of hispidin-hydroxylase and demonstrated its enzymatic activity in vitro. Thus, as a result of the implementation of the reporting phase of the project, we described and experimentally confirmed the pathway of biosynthesis of luciferin of higher fungi from the caffeic acid and demonstrated the possibility of the transfer of the corresponding cascade to eukaryotic organism. Adding to this cascade previously studied genes of biosynthesis of caffeic acid from tyrosine allows the creation of a genetically encoded autonomous bioluminescent system that does not require the addition of exogenous luciferin to glow. The results of the work are published at PNAS journal: https://www.pnas.org/content/pnas/115/50/12728.full.pdf.

 

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