Annotation of the results obtained in 2021
Considerable attention is paid to the search for new and improvement of existing bioluminescent systems that will allow visualization of intracellular processes in vivo. Several years ago, the described bioluminescent system of fungi using the example of N. nambi, the first eukaryotic system with a deciphered mechanism of luciferin biosynthesis, showed the possibility of its use for working with various model organisms, such as yeast, plants, and mammalian cells (Mitiouchkina et al. 2020). Despite this, the fungal bioluminescence system requires optimization, increase in brightness and further study to expand its functionality. Most of the research in the current reporting year was devoted to the study of the stage of biosynthesis of the precursor of fungal luciferin — hispidin. The biosynthesis of this polyketide is carried out by large multi-domain enzymes, a representative of which is N. nambi nnHispS. This enzyme is believed to belong to a family of hybrid enzymes composed in part of type I polyketide synthase domains combined with non-ribosomal peptidyl synthase domains (Hai, Huang, and Tang 2020). Due to the fact that nnHispS was recently described and has a complex domain organization that complicates its study, there is currently little information about this protein. In the course of our work this year, using bioinformatics methods, we managed to predict the most probable domain structure of nnHispS, as well as the boundaries of the ketosynthase domain. In addition, the alignment of the amino acid sequences of nnHispS and its homologs from related bioluminescent fungi made it possible to predict the positions of amino acid substitutions for the consensus mutagenesis. These nnHispS mutations were analyzed both individually and in various combinations. The Pichia pastoris yeast line was chosen as a heterologous system for this work. The analysis was performed based on the bioluminescent signal level with the addition of caffeic acid. It should be noted that all nnHispS mutants were functional, although some combinations of mutations led to a decrease in the bioluminescent signal level. As a result of the work, none of the mutants led to an increase in the brightness of the yeast luminescence when the substrate was added. Thus, the optimization of hispidin biosynthesis was performed using a different approach, namely, bicodon optimization of the nnHispS gene. Analysis of the codon sequence and optimization of their combinations made it possible to obtain a new variant of the nnHispS gene, which in yeast cells showed an increase in bioluminescence in comparison with the initial form of the nnHispS gene by an order of magnitude. This result probably reflects the importance of the folding stage and the formation of the spatial structure of domains for such a large enzyme as nnHispS, which is reflected by the increase in the efficiency of hispidin biosynthesis in the heterologous system of yeast cells. Biosynthesis of hispidin, presumably, can be determined by the level of malonyl-CoA, an additional substrate of nnHispS. Therefore, an increase in the level of malonyl-CoA is another direction in which work has been carried out in the framework of optimizing the biosynthesis of hispidin. During the work on this project, two approaches were tested: the addition of the antibiotic cerulenin, which blocks the biosynthesis of fatty acids by inhibition of fatty acid synthase (Leonard et al. 2008), and the introduction into the system of an additional gene of acetyl-CoA carboxylase (Tong 2005), which synthesizes malonyl-CoA. Unfortunately, both of these approaches did not lead to an increase in the brightness of the bioluminescent signal, which may have several explanations. First, the availability of malonyl-CoA may not be a limiting factor for hispidin biosynthesis. Secondly, malonyl-CoA can be an allosteric regulator of nnHispS or another enzyme of the bioluminescent system of fungi, which, with increasing concentration, has a suppressive effect on the system. This year, we quantitatively evaluated the luciferase luminescence from different fungi (Armillaria fuscipes, Armillaria gallica, Armillaria mellea, Armillaria ostoyae, Mycena chlorophos, Neonothopanus gardneri, Omphalotus olearius, Panellus stipticus), as well as the mutant form of luciferase for this, using already developed by our team test system for assessing the brightness of luciferases based on a culture of P. pastoris yeast cells. The comparison was performed by normalizing the signal from the studied luciferases to the signal from the reference firefly luciferase FFLuc. According to the obtained data, the luciferase of the N. nambi fungus had the highest bioluminescent signal exceeding the signal from the rest of the luciferases — 5-10 times, and the mutant variant of this luciferase — by 3 times. We have confirmed the functionality of this test system and the possibility of its use for quantitative assessment of the fluorescence of various luciferases and their mutant forms in order to search for brighter candidates.

 

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Annotation of the results obtained in 2020
To date, only two bioluminescent systems are known, for which the pathways of biosynthesis of the main components, luciferase and luciferin, have been described. One of them is represented by the bioluminescent system from bacteria, and the other one was found in fungi. The bioluminescent system of fungi was deciphered and successfully reconstructed in a model organism by our group as part of a project supported by the Russian Science Foundation in 2017-2019. Compared with an autonomous bioluminescent system based on bacterial luciferase, the bioluminescent system of fungi has a number of advantages, in particular, in terms of spectral characteristics (the maximum emission of the bacterial system is 490 nm (blue light), and the bioluminescent system of fungi is 525 nm (yellow light)) and the non-toxicity of the substrates of the system for cells. However, at present, the bioluminescence system of fungi is unambiguously inferior to the bacterial system in terms of brightness: for example, using the bacterial system it is already possible to register the glow of single cells, while such brightness is not yet available for the bioluminescence system of fungi. Therefore, the improvement of the autonomous bioluminescent system developed by us, based on luciferase and genes of fungal luciferin biosynthesis, is an urgent scientific problem with significant potential for practical application. During the first year of the project, using the heterologous expression of the 3 main genes of the caffeic acid cycle enzymes and the gene for the auxiliary enzyme phosphopantetheinyltransferase in the yeast Pichia pastoris, we determined that both stages of hispidin biosynthesis from caffeic acid can have a limiting effect. Each of the considered stages has a potential reserve of opportunities to increase the overall efficiency of the system. The data obtained during the work demonstrate the relevance of mutagenesis of hispidin-3-hydroxylase (H3H) and hispidin synthase (HispS) in order to optimize the biosynthesis of fungal luciferin. In this connection, it was decided to carry out mutagenesis (H3H) — an enzyme that catalyzes the reaction of luciferin formation — in order to optimize the activity of this enzyme and, accordingly, the biosynthesis of fungal luciferin. As a result, we obtained a library of random mutations of the h3h gene in the vector for expression in Pichia pastoris. The resulting mutant forms of H3H were tested in the heterologous system of Pichia pastoris, which expressed the fungal luciferase gene. We managed to find a substitution resulting in a more efficient conversion of hispidin to luciferin compared to the wild type. The rest of the variants showed less efficiency than the wild type H3H. In addition, we analyzed mutant clones carrying a dysfunctional version of a h3h gene. Sequencing of the h3h sequences of these clones made it possible to determine the sites of their grouping on the enzyme sequence, which, apparently, are directly related to its activity. We have studied the ability of cerulenin, an antifungal antibiotic that inhibits the biosynthesis of fatty acids, to indirectly increase hispidin synthesis with an increase in the intracellular concentration of malonyl-CoA. Using drop tests, we showed that cerulenin concentrations of 1-10 μM are able to inhibit the growth of Pichia pastoris cells, and low concentrations (0.5 μM) led to an approximately 2-fold increase in the luminescent signal in yeast expressing genes of the caffeic acid cycle and a gene of phosphopantetheinyltransferase, in response to the addition of caffeic acid. Presumably, this effect is associated with the greater availability of malonyl-CoA, which is necessary for the biosynthesis of hispidin from caffeic acid. Thus, the addition of cerulenin can be considered as one way of modulating the bioluminescent signal for the caffeic acid cycle system in Pichia pastoris. This year, we created a test system based on Pichia pastoris for quantitative comparison of the brightness of luciferases from various luminous basidiomycetes, as well as their artificially modified forms. It is the result of two successive transformations and contains the genes for N. nambi luciferase and firefly luciferase independently inserted into the genome. The conditions for measuring the bioluminescence of firefly luciferase and the working concentration of D-luciferin were determined. A linear correspondence between the number of yeast cells at serial dilution and the level of bioluminescent signal when measured for both firefly luciferase and fungal luciferase in this test system was shown. We also created a second version of the test system for the quantitative assessment of the brightness of fungal luciferases for mammalian cell lines. The test was carried out on HEK293T cells. Monocistronic variants of vectors were tested, in particular, vectors with three types of linkers were created. In addition, for each linker vector variants were created in which each of the luciferases (both fungal luciferase and firefly luciferase) was located both at the C-terminus and at the N-terminus of the protein. The comparison involved 6 variants of vectors of the test system. According to the comparison results, it was found that the luciferase located in the first position has a higher level of luminescent activity than that located at the 3'-end of the construct. We managed to get a working test system in which the luciferase genes were expressed separately. We made a choice in favor of a test system based on a bicistronic matrix, in which the expression of luciferase genes was carried out under different promoters and did not depend on the efficiency of linker cleavage.

 

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