INFORMATION ABOUT PROJECT,
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COMMON PART


Project Number17-14-01169

Project titleDevelopment of the first genetically encoded autonomous bioluminescent system of eukaryotes

Project LeadYampolsky Ilia

AffiliationShemyakin - Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences,

Implementation period2017 - 2019

Research area 04 - BIOLOGY AND LIFE SCIENCES, 04-202 - Proteomics; structure and functions of proteins

Keywordsbioluminescence, bioluminescent fungi, luciferin, luciferase, autonomous bioluminescence, glowing plants


 

PROJECT CONTENT


Annotation
Current project is focused on the development of a new genetically encoded autonomous bioluminescent system of eukaryotes. Bioluminescence is a phenomenon of cold light emission by living organisms. Usually visible light is emitted as a result of chemical oxidation of a luciferin molecule by air’s oxygen under catalysis of an enzyme, called luciferase. A vast range of modern analytical methods, such as tests for different analytes in vitro and in vivo, immunoassays, high throughput drug screening, analysis of reporter genes and bioluminescent imaging are based on bioluminescence. To date, only bacterial bioluminescent system can be completely encoded in a living organism using lux operon. Other bioluminescent systems can only be applied upon exogenous addition of the substrate of bioluminescence (luciferin) before each analytical trial. Bacterial bioluminescent system cannot be widely applicable in eukaryotic organisms not only because of the problems associated with expression of bacterial genes but also due to the toxicity of bacterial luciferin. Autonomous bioluminescent system suitable for use in eukaryotic hosts will be applied in vast majority of modern analytical bioluminescent methods. The only currently promising candidate of this type is a bioluminescent system of higher fungi (basidiomycota). Due to recent advances in the study of fungal bioluminescent system made by authors of the project, major substrates and proteins responsible for their synthesis were identified. In this regard, the development of a unique autonomous bioluminescent system and new heterologous eukaryotic systems on its based becomes a realistic perspective, with the development of autonomously luminescent plant as a first logical step. There is also a real possibility of expression of fungal bioluminescent system in other hosts, such as yeast and mammals. The first genetically encoded autonomously bioluminescent system, suitable for use in eukaryotic organisms, will be obtained as a result of this project. The absolute uniqueness of the system (possibility of expression in eukaryotic cells) guarantees development of new analytical techniques and bioluminescent methods on its base (including bioimaging). Also, the implementation of this project will make a significant contribution to modern biochemistry and proteomics in the field of luminescent species.

Expected results
Development of the first autonomous bioluminescent system of eukaryotes will be the main result of this project. The realization of this outcome will be achieved through the establishment of the primary structure of genes, encoding proteins of fungal bioluminescent system, study and optimization of vector sequences for expression of genes in the host organisms, and assembly of a vector with the cassette of genes of autonomous bioluminescent system. New culture of autonomously luminescent eukaryotic cells (presumably plant cells) will be obtained as a final result. The developed genetically encoded autonomous bioluminescent system of eukaryotes based on eight species (Armillaria gallica, Armillaria mellea, Armillaria ostoyae, Mycena chlorophos, Mycena citricolor, Neonothopanus nambi, Omphalotus olearius, Panellus stipticus) and all its components will be fully patented. The absolute uniqueness of this system (its ability to be expressed in eukaryotic cells) guarantee the development of a wide range of new competitive analytical bioluminescent methods on its base (including bioimaging). The results will be published in leading scientific journals such as journals of Nature Publishing Group, Journal of American Chemical Society, Angewandte Chemie, Photochemical and Photobiological Sciences, Biochemistry etc.


 

REPORTS


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

 

Publications

1. Минлянг Юань, Ксяоджи Ма, Тьяну Джианг, Юки Гао, Яньян Куи, Чаочао Жанг, Ксинге Янг, Юн Хуанг, Люпеи Ду, Илья Ямпольский, Миньонг Ли Prolonged bioluminescence imaging in living cells and mice using novel pro-substrates for Renilla luciferase Organic & Biomolecular Chemistry, Org. Biomol. Chem., 2017, Advance Article (year - 2017).

2. Н. Маркина, А. Гороховатский, А. Котлобай, К. Саркисян, Ю. Мокрушина, И.Ямпольский Hispidin-3-hydroxylase: a luciferin biosynthesis enzyme of glowing fungi The FEBS Journal, The FEBS Journal 284 (Suppl. 1) (2017) 106 (year - 2017).

3. Тьяну Джанг, Ксинге Янг, Юбин Жоу, Илья Ямпольский, Миньонг Ли New bioluminescent coelenterazine derivatives with various C-6 substitutions Organic & Biomolecular Chemistry, Org. Biomol. Chem., 2017, 15, 7008-7018 (year - 2017).


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.

 

Publications

1. Котлобай А.А., Саркисян К.С., Мокрушина Ю.А., Марсет-Хубен М., Серебровская Е.О., Маркина Н.М., Сомермейер Л.Г., Гороховацкий А.Ю., Введенский А., Пуртов К.В., Петушков В.Н., Ямпольский И.В. и др. Genetically encodable bioluminescent system from fungi Proceedings of the National Academy of Sciences of the United States of America, PNAS, 2018, Vol. 115, No 50, 12728-12732 (year - 2018).

2. Палкина К., Маркина Н., Мокрушина Ю., Чепурных Т., Саркисян К., Ямпольский И. Biosynthesis of hispidin by plant type III polyketide synthases in yeast and mammalian cell cultures FEBS Open Bio, FEBS Open Bio 8 (Suppl. S1) (2018) 107–496, p 172, P.07-024-Wed (year - 2018).

3. Шимомура О., Стевани К., Каськова З.М., Царькова А.С., Ямпольский И.В. Bioluminescence: Chemical Principles and Methods (Third Edition) Chapter 9 Luminous Fungi World Scientific Publishing Co. Pte. Ltd. Singapore, - (year - 2019).

4. - Гриб-светильник: ученые смогут создать живые предметы обихода Газета Известия, https://iz.ru/816549/mariia-nediuk/grib-svetilnik-uchenye-smogut-sozdat-zhivye-predmety-obikhoda (year - ).

5. - Российские биологи создали дрожжи, светящиеся в темноте РИА Новости, https://ria.ru/20181127/1533601876.html?referrer_block=index_archive_2 (year - ).

6. - Ученые выяснили, как светятся грибы, и создали светящиеся дрожжи Газета.ру, https://www.gazeta.ru/science/news/2018/11/27/n_12340837.shtml (year - ).

7. - Российские ученые создали светящиеся дрожжи телеканал 360tv.ru, https://360tv.ru/news/nauka/rossijskie-uchenye-sozdali-svetjaschiesja-drozhzhi/ (year - ).

8. - Ученые нашли способ делать клетки светящимися с помощью ферментов биолюминесцентных грибов ТАСС, https://tass.ru/nauka/5840835 (year - ).

9. - Ферменты из светящихся грибов заставили засветиться дрожжи Индикатор, https://indicator.ru/news/2018/11/27/griby-zastavili-drozhzhi-svetitsya/ (year - ).

10. - Российские ученые смогут с помощью светящихся грибов обнаружить рак МК.РУ, https://www.mk.ru/science/2018/11/27/rossiyskie-uchenye-smogut-s-pomoshhyu-svetyashhikhsya-gribov-obnaruzhit-rak.html (year - ).

11. - Ученые расшифровали биолюминесцентную систему грибов на генетическом и биохимическом уровнях Наука в Сибири, http://www.sbras.info/news/uchenye-rasshifrovali-biolyuminestsentnuyu-sistemu-gribov-na-geneticheskom-i-biokhimicheskom-ur (year - ).