Within the DBTL cycle, synthetic biology tool kits have had the greatest impact on the “build” phase. Rapid and precise diversity generation, including the construction and integration of expression assemblies into a platform, is a vital prerequisite to screening. Libraries of well characterized genetic parts provide metabolic engineers with a set of elements that can precisely control the expression of a pathway gene. To this end, vector sets, promoter sets, terminator sets, and signal peptide sets are the most common control elements used. A vector is a circular fragment of DNA that harbors pathway genes, a selection marker, and an origin of replication which dictates copy number and plasmid stability. Integration of synthetic biology constructs directly into the genome may obfuscate the use vectors, however shuttle vectors for cloning of constructs are generally still employed. Promoters are regulatory elements directly upstream of a gene of interest, which recruit transcriptional elements for gene expression. Promoters may be constitutive or inducible . The promoter “strength” correlates to the copy number of mRNA upon induction; promoters are often referred to as tight or leaky . Terminators are the regulatory elements downstream of the protein coding sequence, signaling transcriptional termination, and impact the half-life of mRNA. Signal peptides may be employed to direct expression to an organelle for localization or secretion. Prior to use, these genetic parts must be assembled into a single contiguous DNA fragment. Sequence independent cloning techniques such as Gibson assembly and yeast homologous recombination have replaced traditional methods such as digestion-ligation.Furthermore, gene fragments can now be affordably synthesized, circumventing strain procurement and DNA isolation.A once tedious and unpredictable process,rolling grow table heterologous gene expression has been streamlined using reliably functional elements; gene expression is now definitively “engineerable”.
As we gain a more comprehensive understanding of sophisticated cellular programs, we will be able to assemble even more robust and dynamic synthetic biology circuits. Once such systems are constructed, integration into the heterologous host is the final hurdle in the “build” phase. The recent discovery of CRISPR/Cas9 has ameliorated this challenge. Cas9, an RNA-guided DNA endonuclease, enables genomic modifications with unprecedented precision, greatly accelerating strain construction.Following the “build” phase, a screening approach is required in order to “test” the performance of synthetic constructs. Direct measurement of product titer using chromatography, mass spectrometry, and spectrophotometry and comparison to an authentic standard is the most accurate quantification method. Advancements in instrumentation have increased the throughput and accuracy while decreasing costs, however these methods are still considered low-to-medium throughput, requiring 1 minute – 1 hour per sample. Meanwhile, indirect measurements of product titer employing biological readouts have enabled high-throughput testing of strains. So called “biosensors” transduce chemical inputs into physiological outputs in order to establish a correlation between a titer and a selectable phenotype. Biosensors enable screening of constructs on the order of seconds or less per sample. In rare circumstances, a natural product is produced in sufficient quantities and has a unique enough absorbance spectrum to function directly as the selectable chromophore. More typically, a genetically-encoded biosensor must be engineered that robustly actuates a signal that can be correlated to the metabolite’s concentration. Biosensors consist of a sensor-actuator pair and are either RNA-based or protein-based. The sensor-input consists of binding of the biosensor to the secondary metabolite. Then, an actuator-output is generated resulting in modulation of transcription or translation of a selectable protein.
The genetic circuit may also encode Boolean logic in order to improve biosensor properties such as dynamic range or sensitivity.Selection is then performed either in situ or ex situ . For example, a cell viability screen can be established by tying a biosensor output to expression of an antibiotic resistance gene or complementation of an auxotroph. On the other hand, biosensor-dependent expression of a fluorescent protein enables high-throughput fluorescence-activated cell sorting for rapid analysis of entire populations of cells. Microbial opioid production has benefited greatly from the use of biosensors, as both RNA and protein based metabolite sensors have been reported for benzylisoquinoline alkaloid pathway intermediates.Adaptive laboratory evolution has also emerged as an efficient method to circumvent traditional DBTL strain construction. ALE employs natural selection and in vivo diversity generation for population-wide engineering, and has been primarily applied to primary metabolic products.Although several generalizable biosensor development platforms have been proposed, research towards rapid expansion of the variety of sensed metabolites is ongoing. Compared to organic synthesis and biochemical engineering, synthetic biology is a relatively nascent applied science. Despite this, immense progress has been made in the last 20 years, and a number of recent success stories illustrate the field’s potential. Research groups now routinely refactor pathways with more than 10 steps in A. nidulans and N. benthamiana, and pathways with more than 20 steps have been reconstituted using both S. cerevisiae and synthetic biochemistry. The ongoing challenge for these platforms is to improve titers and reduce costs sufficiently to compete with traditional production methods. General strategies range from improving flux through pathway bottlenecks to ameliorating growth defects from metabolic burden or toxicity, however a more nuanced engineering approach is often required. In depth discussions of the engineering strategies enabling benchmark production of the psychoactive natural products described in this review accompany the bio-synthetic pathway descriptions. Of all the psychoactive compounds that are either isolated as natural products or produced synthetically, hallucinogens may impart the most dramatic shifts in one’s psyche.
This broad class of substances can induce potent alterations to consciousness, mood, and perception resulting in vivid visual hallucinations, synesthesia, and a warped sense of time and space. The precise mixture of perceptual and somatic effects of hallucinogens is highly compound specific and thus has led to many debates on accurate nomenclature. There is yet to be a consensus with terms such as “psychedelic” and “entheogen” often used interchangeably with “hallucinogen” in different contexts. Natural sources of hallucinogens famously include “magic mushrooms” of the Psilocybe genus and other fungi such as ergot and fly agaric. Other well-known sources of hallucinogens are from the spineless cactus, peyote, the psychoactive brew, ayahuasca, and with a recent resurgence, nutmeg.Most natural hallucinogens are alkaloids derived from amino acids such a L-tryptophan, L-tyrosine , and L-glutamic acid, with one notable exception being the terpenoid salvinorin A. Numerous extensive reviews exist on the history, pharmacology, and potential as therapeutics of hallucinogens which we recommend.The serotonin or 5-hydroxytryptamine receptors, named for their native ligand, serotonin, have been implicated in the modulation of sensory perception, mood, cognition, memory,indoor plant table and more through the peripheral and central nervous systems There are many subtypes, and with the exception of 5-HT3 which is a ligand-gated ion channel, the rest are G-protein-coupled receptors, each with unique spatial distribution and localization in the brain.Phylogenetic analysis and low sequence identity demonstrates early divergence, implicating 5-HT receptors as one of the oldest receptor systems.The relationship between 5-HT receptors was first determined through testing of LSD. While hallucinogenic compounds like 3 have been shown to target multiple 5-HT receptors, the 5-HT2A receptor is most commonly associated with the majority of psychotropic effects.Previously, structure-activity relationship studies between 5-HT2A and numerous psychoactive compound scaffolds have demonstrated that hallucinogenic potency is not necessarily a function of affinity, likely due to more nuanced mechanisms of functional selectivity.However, a recent crystal structure of 3 complexed with 5-HT2B was reported and combined with molecular dynamic simulations, identified a molecular basis for the particular potency of 3. The authors demonstrate that the diethylamide side chain of 3 adopts a restrictive conformation when bound to 5-HT2B that increases residence time and improves β-arrestin translocation to the cell membrane. This enhanced β-arrestin translocation results in desensitization of the cell to stimuli by uncoupling G-proteins from receptors and could explain the long duration of action of 3.N,N-dimethyltryptamine is likely the most pervasive psychoactive compound across species and is found in dozens of plant and animal species, including humans.Root, bark, and leaf preparations from plants such as Psychotria viridis, containing DMT and its structural analogs have been used in shamanic ritual practices for at least 1000 years.Interestingly, in addition to plants, structural analogs 5- methoxy-N,N-dimethyltryptamine and bufotenin, are also found in the toxin of the Colorado River toad Incilius alvarius, formerly known as Bufo alvarius, whose remains have been found as a part of Olmec ritual ceremonies dating back to pre-Columbian Mesoamerica .Referred to colloquially as the “Psychedelic Toad of the Sonoran Desert,” exudates from the amphibian’s specialized glands may contain up to fifteen percentage dry weight , representing the most notable example of a psychoactive natural product of animal origin.
DMT was first isolated from the shrub Mimosa tenuiflora in 1946 by Oswaldo Gonçalves de Lima,but its hallucinogenic effects were not discovered for another decade., like all L-tryptophan derived hallucinogens, is a serotonin receptor agonist. While the functional selectivity of towards the 5HT2A receptor is believed to be necessary for its effects, can bind to many serotonin receptors that may also contribute to its psychoactivity.While the precise role of endogenous in humans has yet to be ascertained,one study speculates it may have a role in protecting from hypoxia.Further, has shown promise as a therapeutic anti-depressive agent and is known to promote neural plasticity.Interestingly, brominated forms of DMT such as, 5-bromo-N,N-dimethyltryptamine , have been isolated from the marine sponges and show particular promise as antidepressives.Finally, has limited neurotoxicity and only exhibits cardiovascular effects when taken intravenously in large doses, furthering its therapeutic potential.Psilocybin 1, one of the major natural products from hallucinogenic Psilocybe sp. , was first isolated from Psilocybe mexicana by Albert Hofmann in 1958 .The description of “magic mushrooms” in scientific literature and the subsequent isolation and characterization of their psychoactive metabolites was the culmination of decades of effort to identify the sacred mushroom that the South American Aztecs referred to as teonanacatl, meaning “god’s flesh.”Psilocybin 1 itself is not psychoactive, but rather exists as a prodrug. After ingestion, psilocybin 1 is metabolized through dephosphorylation and becomes psilocin, a potent psychotropic 5HT2A receptor agonist.In addition to its psychoactivity, has shown some promise as a therapeutic for treating depression, anxiety and tobacco addiction.A bio-synthetic pathway for psilocybin was proposed based on isotope feeding studies as early as 1968.Agurell et al. hypothesized that following decarboxylation, L-tryptophan, now tryptamine , would be methylated iteratively to form the psychoactive dimethyltryptamine . This was a reasonable hypothesis because indolethylamine-N-methyltransferases were a popular enzyme for study at the time following their discovery rat, rabbit, and human tissues.Recently, a psilocybin bio-synthetic cluster from Psilocybe cubensis and Psilocybe cyanescens was identified and characterized by Fricke et al. .The authors first sequenced the genomes of both Psilocybe sp. Then, using a combination of a methyltransferase, a hydroxylase, and a kinase as queries, a putative bio-synthetic cluster present in both species was identified and characterized. Fricke et al. determined that the iterative N-methylation was the terminal step of psilocybin biosynthesis by enzymatic action of PsiM whose sequence is unrelated to the well-characterized mammalian indolethylamineN-methyltransferases, and thus revised the hypothesis that DMT is an intermediate in psilocybin biosynthesis. Starting from L-tryptophan, PsiD catalyzes a decarboxylation reaction to yield. The amino acid sequence for PsiD diverges from the more common PLP-dependent aromatic amino acid decarboxylases and instead shares similarity with the PLP-independent phosphatidylserine decarboxylases. PsiH, a P450 monooxygenase, then hydroxylates the indole C4 to yield 4-hydroxytryptamine . Next PsiK, a predicted kinase, catalyzes the phosphorylation of 4-hydroxytryptamine into norbaeocystin using ATP as the phosphate donor. Phosphoryltransferase are relatively uncommon in natural product biosynthesis. Recent examples include the biosynthesis of calyculin protoxins and the lasso peptide paeninodin, in which phosphorylation plays a role in self-immunity which could highlight the importance of dedicated kinases.Lastly, PsiM methylates the terminal amine in in an iterative fashion using SAM as a methyl donor to give 1. PsiM only methylates phosphorylated tryptamine , indicating that psilocybin biosynthesis is nearly linear. In water, 1 undergoes spontaneous hydrolysis of the phosphate group to form , but PsiK accepts psilocin as a substrate and readily phosphorylates to reform psilocybin 1. As previously mentioned, this hydrolysis results in the psychoactive form, upon ingestion by vertebrates.