How to Make Psilocybin with Yeast | Journal Club

Making Psilocybin in Yeast: An Overview of Research 00:00

"This is a fascinating work for a lot of reasons and a really good way to deliver a typical metabolic engineering story."

• The video reviews groundbreaking research where scientists successfully produced psilocybin in yeast, specifically using Saccharomyces cerevisiae, in significant quantities.

• Metabolic engineering offers a method to produce or degrade biochemical substances in microorganisms, and in this case, the focus is on creating psilocybin in brewers yeast.

• To produce any desired compound like psilocybin, it is essential to identify the necessary enzymes involved in the metabolic pathway.

Understanding the Metabolic Pathways 00:49

"If you want to find the metabolic pathway for producing psilocybin, you just type in KEGG psilocybin."

• Researchers utilize online databases like KEGG to explore metabolic pathways for compounds, including psilocybin. By inputting "psilocybin," one can access detailed pathways.

• Psilocybin is categorized as an indole alkaloid, and maps exist to illustrate how various chemicals transform through enzymatic processes.

• Key enzymes, such as psiK, play a crucial role—it converts psilocybin to psilocin and can reverse that process.

The Role of Tryptophan in Psilocybin Production 01:55

"The ubiquitous precursor for psilocybin is the amino acid tryptophan."

• Tryptophan, an essential amino acid, is vital for survival and is present in nearly all living organisms. It's a fundamental ingredient for synthesizing psilocybin.

• The process begins with tryptophan converting to tryptamine, which further converts to various derivatives up to psilocybin.

• Different organisms possess unique variants of these critical enzymes, which may influence the effectiveness of metabolic engineering efforts.

Study Context and Advances in Production Techniques 03:36

"This paper is about the metabolic engineering of Saccharomyces cerevisiae for the de novo production of psilocybin."

• The research examines the conversion of basic feedstocks into psilocybin by feeding yeast glucose, illustrating the concept of de novo production, where complex substances are synthesized without direct precursor input.

• The lab's previous work emphasizes yeast's potential for producing various bioproducts and demonstrates advancements in harnessing yeast for psilocybin synthesis efficiently.

• The paper indicates a titer—product concentration—of over half a gram per liter for psilocybin and psilocin, showcasing the effectiveness of their method.

Economic Implications of Yeast Production 05:45

"Imagine a liter of water. In a single liter, you're making the equivalent of 100 grams of dried mushrooms."

• The findings suggest that the yield achieved by the researchers could equate to a significant economic advantage: producing valuable psilocybin efficiently, akin to generating hundreds or thousands of dollars per liter.

• Given that psilocybin occurs in dried mushrooms at a concentration of about 1% and is contextually more viable to produce through engineered yeast than through traditional mushroom extraction, such advancements offer new commercial avenues.

Historical Context and Regulatory Landscape 07:35

"The LD50 of psilocybin in mice and rats is about 280 mg per kilogram."

• The discussion includes a historical overview of psilocybin’s discovery and its evolution in research, which has experienced a resurgence since the 1990s, particularly in regions with less regulatory pressure.

• Safety implications are discussed, with lethal dose figures reinforcing the relative safety margins in humans when consuming significant quantities of psilocybin.

• The comparison to mushroom extraction versus metabolic engineering frames the case for yeast production, as it is presented as a more feasible alternative for producing compounds like psilocybin commercially without excessive extraction costs.

Challenges in Psilocybin Production Using E. coli 11:02

"While some have successfully produced psilocybin in E. coli, it required adding other compounds to the media rather than achieving true de novo biosynthesis."

• Producing psilocybin in E. coli involves adding additional compounds to the growth media, which deviates from true de novo biosynthesis.

• The production levels reached over a gram per liter, highlighting the substantial potential for psilocybin production within microbial systems.

Advantages of Saccharomyces cerevisiae over E. coli 12:16

"Saccharomyces cerevisiae naturally possesses cytochrome P450 enzymes, making it more suitable for producing compounds like psilocybin."

• Saccharomyces cerevisiae, a eukaryotic microorganism, has the advantage of naturally possessing cytochrome P450 enzymes, which are essential for specific enzymatic functions in psilocybin biosynthesis.

• By comparison, E. coli, a prokaryote, lacks these enzymes, which limits its efficiency in producing complex compounds like psilocybin.

Psilocybin’s Conversion and Effects 13:03

"Psilocybin is not active on its own; rather, it is converted to psilocin, which is the active component contributing to therapeutic effects."

• Psilocybin itself is not pharmacologically active until it is converted to psilocin after consumption, which provides the desired therapeutic benefits.

• Understanding this conversion is crucial for anyone exploring metabolic pathways in yeast to produce psilocybin for therapeutic applications.

Glycolytic Pathway and Metabolic Flux 14:34

"Metabolic flux determines how much compound moves through a particular pathway, which is vital for optimizing psilocybin production."

• The concept of metabolic flux is central to optimizing the production of psilocybin, as it details how effectively carbon from glucose is converted into the target compound.

• Researchers aim to minimize side reactions that divert carbon away from the desired psilocybin synthesis pathway.

Genetic Engineering for Psilocybin Production 18:00

"Integrating five genes for psilocybin production could take two to three months, not including troubleshooting for compound production."

• Integrating multiple genes into a yeast strain is a complex and time-consuming process, potentially taking up to three months or more, depending on various factors like gene size and researcher skill.

• Researchers are likely to face challenges in achieving efficient psilocybin production even after successful genetic integration.

Utilizing the Periwinkle Gene in Tryptophan Conversion 19:41

"The Periwinkle gene has been identified as more effective than other options for converting tryptophan to 4-hydroxytryptamine."

• The Periwinkle gene has shown to be a valuable tool for converting tryptophan into 4-hydroxytryptamine, an essential precursor in the biosynthesis of psilocybin.

• This gene's effectiveness over other alternatives reflects the ongoing research in selecting the best biochemical pathways for enhanced production efficiency.

Liquid Chromatography-Mass Spectrometry (LC-MS) Techniques 20:55

"Using LC-MS allows researchers to precisely quantify the production of psilocybin based on the area under the peak on chromatograms."

• Liquid Chromatography-Mass Spectrometry (LC-MS) is instrumental in the analysis of psilocybin production, providing accurate quantification of compounds in metabolic engineering studies.

• The technique measures the time-dependent release of chemical compounds, allowing researchers to analyze production efficiency and make adjustments accordingly.

Psilocybin Production and the Rate Limiting Step 21:59

"They say they're making psilocybin and silin, and at this stage, just by putting in the basic enzymes, they're making a lot of tryptamine, but psilocybin and psin are just in the milligram per liter range, which isn't very good."

• The researchers observed that the production of psilocybin was limited during their experiments, noting that while a significant amount of tryptamine was produced, psilocybin and psilocin were only in low concentrations.

• They identified that the conversion of tryptamine to 4-hydroxytryptamine using cytochrome P450 was likely the rate-limiting step in the metabolic pathway.

Optimization Strategies for Psilocybin Production 23:14

"When you discover what the rate-limiting step is in metabolic engineering, you can try expressing that same enzyme a bunch of times."

• The researchers experimented with various strategies to optimize the conversion process, including overexpressing the cytochrome P450 enzyme and altering its promoter for increased activity.

• Specifically, they used a strong promoter (TEF1) to enhance the production of psilocybin and psilocin significantly, achieving 137 mg per liter of psilocybin and 82 mg per liter of psilocin.

Graph Analysis of Metabolic Engineering Success 24:38

"This strategy of adding the TEF1 promoted cytochrome P450 enzyme gave the highest titer of psilocybin and psilocin, whereas the other strategies didn't work nearly as well."

• The results were visualized in a bar graph, comparing the control strain to genetically engineered strains with different mutations.

• The data illustrated that without the biosynthesis genes for psilocybin, no psilocybin or psilocin could be produced. Hence, the addition of the relevant genes led to marked increases in these compounds.

Further Enhancing Tryptophan Availability 26:05

"They overexpressed Arrow one and Arrow two, and if you go into the literature, you can find that tryptophan has some natural levels in the cell and it's probably a limiting step as well."

• The team explored additional pathways to increase flux towards tryptophan by overexpressing specific genes (Arrow one and Arrow two) and knocking out a competing gene (Rick one).

• This manipulation aimed to redirect cellular resources to increase tryptophan levels, which would subsequently enhance psilocybin production.

Scaling Up to Real-World Applications 27:51

"They went from making somewhere on the order of 200 mg per liter to making nearly a gram per liter of psilocybin."

• Transitioning from small-scale experiments to larger production systems allowed researchers to increase yields significantly.

• By implementing a fed-batch fermentation process, they replicated conditions that maximized yeast growth and substrate utilization, ultimately surpassing 1 gram per liter of psilocybin and psilocin in production.

Exploring Promiscuous Enzymes and Novel Compounds 29:41

"The researchers were able to feed analogs to tryptophan or norbist and still have conversion to similar compounds, creating a new compound called Eurogen."

• Interestingly, the researchers discovered that some enzymes in the metabolic pathway exhibited promiscuity, enabling the conversion of different substrates.

• They successfully synthesized a novel compound, eurogen, which is similar to psilocybin, showcasing the potential for generating new products through heterologous expression in yeast.