Hallucinogenic yeast

D-lysergic acid can now be obtained using baker's yeast

Arseniy Belosokhov, "Elements"

Fig. 1. A spikelet of rye affected by ergot (Claviceps purpurea). Black horns are sclerotia of the fungus formed in place of the ovary. They contain toxins that, when eaten, can cause ergotism. The main source of infection is bread baked from poorly sorted flour. In the Middle Ages, epidemics of ergotism occurred regularly. Photos from the website commons.wikimedia.org

The relationship between people and lysergic acid has many different episodes: it left its mark in the sad history of medieval epidemics, and now its derivatives are used, for example, for stimulation during childbirth, in the treatment of Parkinsonism, in psychotherapy, as well as for recreational purposes. However, obtaining biologically active stereoisomers of lysergic acid is associated with many technical difficulties, which is why the synthesis of pharmacologically pure drugs is associated with high costs. In a recent work by scientists from London and Singapore, a complete biosynthetic chain was successfully reproduced, allowing the production of D-lysergic acid from the culture of the baker's yeast Saccharomyces cerevisiae. The results of this study may have a great impact on the further pharmacological industry of drugs using lysergic derivatives.

Lysergic acid and its derivatives play an important role both in history and in modern medicine. The natural source of these substances, referred to as alkaloids, is the ergot fungus (Claviceps purpurea). It belongs to hypocrine ascomycetes that parasitize cereals and form large black sclerotia full of alkaloids to protect against eating. If such sclerotia are ground into flour together with non-harvested grain, then the bread baked from it will be toxic: its regular consumption causes ergotism. In the Middle Ages, this disease was called "Anthony's fire" (gangrenous form) or "witch's writhe" (convulsive form). In the gangrenous form, ergot alkaloids stimulate the narrowing of blood vessels, which is why the tissues of the extremities may lose sensitivity and eventually die off. In the convulsive form, the nervous system is affected, which leads to severe spasms, convulsions and psychoses. Both forms of the disease can be fatal.

Fig. 2. The main composition of the Isenheim altar, which appears before the viewer only if both the outer and inner doors are open. In the central part there are wood-carved sculptures of Saints Augustine, Anthony and Jerome (from left to right). Below them are Jesus and the 12 apostles. On the side doors are episodes from the life of St. Anthony: a meeting with Paul the Hermit (left) and the temptation of Anthony (right). In the lower left corner of the right sash, the figure of a man sick with "Antony fire" is visible. The altar is painted by Matthias Grunewald, the wood carving is made by Nikolaus Hagenauer. The altar itself was created in 1512-1516, until 1793 it was located in the monastery of St. Anthony in Isenheim. Photos from the website khanacademy.org

In the modern world, ergot alkaloids are used against migraines, in the treatment of Parkinson's disease (A. Lieberman et al., 1976. Treatment of Parkinson's Disease with Bromocriptine), senile dementia (B. Winblad et al., 2008. Therapeutic use of nicergoline), arterial hypertension (R. M. Tarnowsky, 1954. Clinical Evaluation of Combined Hydrogenated Ergot Alkaloids (Hydrogine) in Arterial Hypertension), to stimulate uterine contractions during natural childbirth and to stop Uterine bleeding during cesarean section (N. Sharma et al,. 2016. Ergot Alkaloids: A Review on Therapeutic Applications). In popular culture, lysergic alkaloids are best known as natural precursors of synthetic diethylamide d-lysergic acid (LSD).

Now the production of D-lysergic acid (D-lysergic acid, DLA) as a precursor for pharmacological preparations, it is available in two ways: by extraction from Claviceps purpurea and by chemical synthesis. Both methods are associated with certain inconveniences.

More than half of the DLA for pharmacological needs is produced by a biotechnological method, in which commercial strains of Claviceps purpurea are grown in a submerged culture in bioreactors. This method gives a fairly high yield of the product. The problem, however, is that in the "velvet" conditions of a sterile bioreactor, mushroom strains tend to quickly "degenerate" and lose the ability to produce the required substances, since they are needed by the fungus to survive in harsh conditions of interspecific competition and lose their evolutionary meaning when growing a culture in vitro.

A more traditional method of obtaining DLA involves artificial infection of cereals and the collection of Claviceps purpurea sclerotia "in the field". At the same time, the culture retains its biochemical potential, but such production is more expensive and gives a much lower yield. There is also a significant risk that the properties of culture will change during natural evolutionary processes, which will shift the spectrum of substances produced by it.

Both biological methods of producing DLA have another important drawback: Claviceps purpurea does not produce only D-lysergic acid. In fact, mushrooms synthesize a number of alkaloids: ergotamine, ergotoxin (peptide type), ergometrine, ergometrinin (alkanamide type), ergine, erginine (amide type) and methylcarbinolamide α-lysergic acid. Their content varies greatly in different strains, and their close chemical relationship makes them difficult to separate and significantly increases the cost of extraction of pure D-lysergic acid.

In the chemical synthesis of DLA, complex chains of reactions are used, including from 8 to 19 stages of chemical transformations. At the same time, the most effective chain of 19 steps gives a reaction yield of about 12%, and the simplest (of 8 steps) — only 10.6%. In both cases, cleaning the final product from numerous impurities that can cause unpleasant side effects is quite a difficult task. Moreover, the chemical synthesis method also fails to achieve enantiomeric purity. The fact is that lysergic acid is represented by two stereoisomers, which are equally likely to be obtained during chemical synthesis, however, the D-isomer has a much higher potential for biological activity, while the L-isomer has practically no pharmacological value. These isomers are extremely difficult to separate during purification. Due to these problems, the chemical method cannot meet the demand for DLA in the pharmaceutical industry (about 20 tons per year).

Thus, the currently available methods for the industrial production of D-lysergic acid are quite complex and do not provide the desired level of technological control over the resulting product, which is required for the production of pharmacological preparations. Scientists from London and Singapore offer an alternative solution to this problem. They tried to "transplant" the genetic apparatus providing the pathway of biosynthesis of D-lysergic acid, ergot into a well-studied model organism that is easy to control during biosynthetic production. As this organism, they used the usual baker's yeast Saccharomyces cerevisiae.

Isolating the genetic apparatus that provides the enzyme pathway of D-lysergic acid biosynthesis and placing it in yeast cells allows solving a number of problems. Firstly, yeast from a technological point of view is a completely predictable organism that has already been used in hundreds (if not thousands) of biotechnological and bioengineering studies. Humanity has no other model organism so studied, the metabolic pathways of which would be described in more detail. Placing an isolated enzyme cascade inside the yeast eliminates the potential degeneration of the strain due to natural processes, and also greatly simplifies correction in case of malfunctions. Secondly, the reconstruction of the enzyme pathway in a heterologous system (i.e. in yeast) occurs using a selective pathway in the absence of potential "branching points" in the system that can direct synthesis in an undesirable direction, which theoretically guarantees the absence of variations in the resulting biosynthetic profile. In other words, if there are no enzymes in yeast that work with a similar substrate, the risk of obtaining a spectrum of similar by-products is eliminated, as it happens in Claviceps purpurea strains.

Biosynthesis of D-lysergic acid from L-tryptophan by the fungus involves the sequential operation of products of 8 genes called DmaW, EasF, EasC, EasE, EasD, EasA, EasG, and CloA. Unfortunately, in bioengineering it is not always possible to simply "copy and paste" — in complex biological systems, copied genes do not necessarily work the same way as in "home conditions". To solve this problem, researchers had to search for and select orthologs working in yeast — enzymes of other fungi homologous to the above-mentioned enzymes of Claviceps purpurea.

The initial stage of biosynthesis (green arrows in Fig. 3) the same for all strains of all fungi producing ergoalkaloids. The authors of the work under discussion took advantage of the fact that the pathway of biosynthesis from tryptophan (using tryptophan-dimethylallyltransferase 1, DmaW) to hanoclavin-I-aldehyde (using hanoclovin-I dehydrogenase, EASD) was well researched and described in previous works, and the necessary genes optimal for working inside yeast cells were available in a wide range diversity in databases. Scientists only needed to use the Enzyme Function Initiative-Enzyme Similarity Tool (EFI-EST) algorithm to find optimal homologues.

Fig. 3. The pathway of DLA biosynthesis from tryptophan to the final product. The natural branches of the biosynthesis pathway in the fungus Claviceps purpurea, leading to alternative products, are shown in pale colors. Colored arrows indicate the stages of the biosynthesis pathway. Green arrows: the initial stage of synthesis, common to all species. It consists of four stages in which five enzymes work (DmaW, EasF, EasC, EasE and EasD), and leads to the first major branching point — hanoclavin-I-aldehyde. Blue arrows: the final stage, which determines the variety of alkaloids in natural strains of Claviceps purpurea. This stage consists of two stages with three active enzymes (EasA, EasG and CloA). Orange arrows: branches of the biosynthesis pathway leading to alternative products. Figure from the article under discussion in Nature Communications

Thus, at the stage of conversion from 4-dimethylallyl-L-arabin (4DMA) to choanoclavin-I, the products of the EasC and EasE genes are needed. Moreover, EasE, taken directly from ergoalkaloid producing fungi (Claviceps purpurea and biochemically similar organisms), does not work in yeast cells. To replace the original EasE, the closest working analogue was taken — the easE_Aj gene of the fungus Aspergillus japonicus.

With the final stages of synthesis, the task was much more difficult — enzymes directly taken from Claviceps purpurea can not only work poorly in yeast cells, but also carry the risk of the formation of undesirable alternative products. Therefore, using the same EFI-EST algorithm, the researchers selected functional alternatives.

The EasA enzyme, which is key in the conversion of hanoclavin—I-aldehyde into alkaloids, has two possible activities: it can act as a reductase or as a cis-trans isomerase and is represented by many different isoforms, the differences between which cause a variety of alternative products of the lysergic pathway. To obtain D-lysergic acid, an orthologue with cis-trans-isomerase activity is required. The selection of an exact isoform among numerous analogues required the search for an isofunctional cluster, which was carried out using SSN (sequence similarity network) analysis. The search for the desired form of the enzyme was carried out with an eye to co—expression in strains of subsequent enzymes in the chain leading to DLA - EasD and EasG: evidence of their expression helped narrow the search to strains with the EasA isoform that had the necessary activity with greater frequency. Among all the isoforms of the enzyme, the one that had phenylalanine in the 176 position turned out to be the right one. In total, three isoform analogues have been identified that work in yeast cells: EasA_Ec (Epichloe coenophialia), EasA_Cp (Claviceps purpurea) and EasA_Nl (Neotyphodium lolii). The highest expression in yeast was observed when using the EasA_Ec form. And they began to use it further.

The last step in the synthesis of D-lysergic acid involves the conversion of agroclavin to DLA during the oxidation process. Oxidation occurs in two stages using cytochrome P450 and clavine oxidase (CloA). A single oxidation of agroclavin leads to the formation of either elimoclavin or lysergol, and a double one leads to the formation of either paspalic acid or the desired D—lysergic acid with possible isomerization of one acid into another. Among the whole variety of CloA orthologs, 15 responsible for double oxidation were found, five of which could work in yeast cells. Of these five, an orthologue from the fungus Claviceps purpurea was selected for the final "assembly" of the genetic "transplant", since it gave a more constant titer of the reaction product.

Assembling all the enzymes into one chain required a step-by-step assessment of the success of the enzymes. To do this, the researchers took several yeast lines. The initial chain from L-tryptophan to 4DMA was inserted into the first one. After the scientists were convinced that it worked well, they began to add one new gene to each subsequent yeast line, checking the stability of the enzyme chain at each stage. Finally, all the selected genes were successfully assembled and inserted into the genome of the final strain.

The final yeast line (strain DLAM33B) synthesized pure D-lysergic acid in an amount of 1.7 mg per liter. From the point of view of use in production, this result can be called quite mediocre. However, the researchers hope that with careful selection of optimal biotechnological parameters of cultivation, it will be possible to significantly increase productivity. The main value of the work done is that the enzyme chain assembled de novo gives biochemically and stoicheometrically pure D-lysergic acid with full product control. This greatly facilitates the testing of the quality and consistency of the properties of pharmacological preparations that will be produced using DLAM33B.

The technology presented in the work under discussion can be considered outstanding. Usually, researchers try to find the most suitable organism for this task, and then optimize it. This is orders of magnitude easier than recreating a chain of reactions in a new organism. Gene transfer is also used, but most often it is about "transplanting" only one or two genes. In this case, as a rule, the necessary end product is a protein — the product of the transferred gene (as, for example, in the case of the well-known fluorescent protein GDP). However, the successful transfer of a rather long chain of genes — and precisely in order to recreate the enzymatic pathway of biosynthesis, during which the target molecule gradually turns into the right one, which also has stereoisomers, and the reaction itself has several alternative products — is a highly outstanding result and, undoubtedly, an important step for biotechnology. At the same time, so far such a solution to biotechnological problems is costly and hardly suitable for ordinary problems. But it can be indispensable for tasks involving complex chains of transformations with a variety of alternative products.

A source: Wong et al., Reconstituting the complete biosynthesis of D-lysergic acid in yeast // Nature Communications. 2022.

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