In many P450- catalyzed reactions in biosynthesis, the substrate radical can migrate to other atoms in the molecule through internal reactions and delocalization through π-bonds. This can lead to rearrangement of the carbon skeleton, as well as oxygen atom incorporation at distal positions from the initial abstraction site. In some cases, the Fe–OH can abstract a second hydrogen atom from the substrate to generate a second radical in the substrate that can recombine with the first one to terminate the reaction cycle. In this scenario, no oxygen atom is incorporated yet molecular oxygen is consumed. An additional feature of some bio-synthetic P450s is the ability to iteratively oxidize a substrate, either at a single carbon or at nearby atoms. For example, it is not uncommon to find a single P450 that can perform the six-electron oxidation of a methyl group into a carboxylic acid in both fungal and plant bio-synthetic pathways. One notable example of P450 catalysis in this review is the secologanin synthase found in the strictosidine bio-synthetic pathway that ultimately leads to ibogaine .The substrate is loganin which contains the iridoid core. SLS performs hydrogen abstraction followed by oxygen rebound at the methyl group on the cyclopentanol ring to give a primary hydroxyl group. This species then undergoes a Grob fragmentationlike reaction to cleave the C–C bond which reveals both an aldehyde and a terminal olefin in the product secologanin .This aldehyde then participates in the aforementioned Pictet-Spengler reaction with tryptamine to give strictosidine . Hence, although this example illustrates a “standard” P450 reaction, the hydroxylation modification triggers a significant skeletal rearrangement. A second example that illustrates oxidation without oxygen incorporation is found in the morphine bio-synthetic pathway, in which the salutaridine synthase catalyzes the phenyl coupling in R-reticuline to yield salutaridine.A radical addition mechanism is currently favored for this reaction: hydrogen abstraction from one of the phenol group generates an oxygen radical that is delocalized throughout the aromatic ring. The carbon radical then adds into the isoquinoline ring and recombines with the second radical that is generated by the P450 through the second hydrogen abstraction step. This forms a C–C bond that couples the two phenolic rings and gives rise to the rigidified morphinan scaffold of salutaridine that is found in morphine and related opioids.

In reality, psychoactive natural products are produced as complex mixtures of metabolites and frequently have partially undefined compositions.Variability in growth conditions, in addition to pests, disease, agrochemicals,vertical grow and climate may introduce further inconsistencies in product composition.In the event that a single psychoactive constituent is desired by the consumer and isolation from the native host is costly, total synthesis may be one strategy to establish a robust supply chain. In the last two decades, advances in DNA technologies have resulted in the development of an alternative production strategy: synthetic biology.Synthetic biologists use genetic tools to build designed biological systems with useful functionality. Whether or not synthetic biology can produce a viable process depends on the economic, environmental, and societal cost of alternative production strategies. However, as novel DNA-related technologies continue to arise, capabilities of molecular biologists are expected to expand. In 2010, Gibson assembly,DNA microarraysand zinc-finger nucleases were considered state-of-the-art. A PhD student that graduated in 2020, however, would have witnessed cost-efficient gene synthesis,66 RNA-seq,and CRISPR/ Cas968 emerge as routine. The substantial unrealized potential of synthetic biology is evidenced by continued investments across industry and academia. As these technologies expand, successful refactoring of a bio-synthetic pathway relies on the use of well-characterized “genetic parts” – these DNA-based elements permit coordinated expression of genes of interest in a heterologous host.Following the standardization of genetic engineering protocols and genetic parts, reliable metabolic engineering techniques have been established that enable improvements in engineered systems. The general methodology for synthetic biology-based heterologous production of natural products is outlined in Fig. 6. First, a bio-synthetic pathway must be elucidated such that a heterologous production strategy can be envisaged. Second, an appropriate bio-synthetic chassis must be selected. Finally, the engineer must iterate through the design, build, test, learn cycle until sufficiently high titers, production rates, and yields are reached.

Biocatalytic production methods benefit greatly from fully elucidated bio-synthetic pathways; a single missing bio-synthetic step may completely derail heterologous production efforts. Identification of natural product bio-synthetic logic is the primary focus of Sections 2 – 5. Early bio-synthetic investigations involved demonstrating that isotope labeled precursors could be site-specifically incorporated into final products, which provided connections between primary metabolism and natural product biogenesis. Now, genomic sequencing and synthetic biology tool kits permit gene knockouts in the native host or expression in a heterologous host for functional analysis. “Reconstitution” of the activity of a recombinantly expressed enzyme activity in vitro affords the most unequivocal evidence of a bio-synthetic sequence. It should be mentioned that availability of transcriptomics data has provided a quantum leap in the ability to identify candidate enzymes, particularly in unclustered plant pathways. Whereas bacterial and fungal bio-synthetic pathways are frequently colocalized in a “gene cluster,” examples of clustered plant pathways are scarce.Meanwhile, the differential abundance of RNA across plant tissues and cultivars gives metabolic engineers precise spatiotemporal gene expression data, which can be mined for information about bio-synthetic pathways. In recent years, RNA-Seq has been used to identify a wide range of plant natural product biosyntheses, including a number of key conversions in psychoactive natural product pathways.For instance, Facchini and coworkers utilized RNA-Seq to discover neopinone isomerase, which catalyzes a reaction previously believed to occur spontaneously in morphine biosynthesis.As an additional example, Luo et al. identified a functional prenyltransferase enabling cannabinoid production in S. cerevisiae by interrogating Cannabis sativa transcriptome data.In some cases, a bio-synthetic step from the native organism cannot be identified, or functional expression of a known pathway gene may not be feasible in a given organism. In this event, bioprospecting or mining the genomes of alternative organisms to identify functional proteins that carry out key reactions has been successfully applied. For example, incorporation of genes from Gallus gallus and Rattus norvegicus in place of missing or non-functional yeast metabolic steps was a crucial advancement in the development of MIA and BIA producing strains.

Alternatively, protein engineering strategies may be employed to alter the regiospecificity or substrate specificity of other wellcharacterized proteins in order to generate de novo suitable replacements for missing or nonfunctional steps. Dueber and coworkers employed this method to engineer a L-tyrosine hydroxylase, which normally requires a cofactor not produced in yeast, and used the evolved enzyme to produce a morphine precursor.The field of directed evolution is now well established,which can be implemented prior to DBTL or integrated into the DBTL pipeline. Following partial or complete pathway elucidation, a bio-synthetic strategy may be designed. For many psychoactive natural products, especially those which can be easily constructed from primary metabolites, de novo production from minimal media will provide the most cost-efficient route to a final product. Stephanopoulos and coworkers recently highlighted an alternative approach: the use of a late-stage pathway entry point to circumvent troublesome early bio-synthetic steps.Such “mixed carbon” feeding strategies may prove useful if an intermediate is commercially available or accessible via facile chemical synthesis. Efficient uptake of the late-stage entry point is another requirement, as transport limitations may prevent efficient substrate incorporation. The terms bio-transformation and bio-conversion are commonly used to refer to this type of hybrid synthetic approach,vertical outdoor farming which has been leveraged in the biosynthesis of psilocybin81 and an ibogaine precursor.Lastly, many in silico pathway design algorithms have been described in recent years, which perform automated retrobio-synthetic analyses to predict novel or optimized pathways.This approach has been successfully applied to primary metabolic products, highlighting the demand for continued investigation of secondary metabolic pathways. Machine-learning technologies linked to databases of reactions using automated DBTL are predicted to play a role in the future of natural product bio-manufacturing.A critical parameter in the successful refactoring of a natural product pathway is the selection of a suitable bio-synthetic chassis. Five representative bio-synthetic chasses are shown in Fig. 6. The model bacterium Escherichia coli has become a foundation of biotechnology as a DNA bearing model organism. E. coli laboratory strains have been customized for plasmid propagation and protein expression. Production of drugs with relatively short bio-synthetic pathways has been shown,with stepwise mixed-strain cultures leveraged for longer pathways.Saccharomyces cerevisiae was initially the subject of genetic studies, but has become a favorite organism in academia to demonstrate heterologous production of an impressive variety of plant or fungus-derived psychoactive drugs.The model ascomycete Aspergillus nidulans has also been used for the production of bio-active molecules due to its robust secondary metabolism and ability to splice fungal introns.Nicotiana benthamiana has proven useful in characterizing and reconstituting difficult plant pathways, and is particularly attractive due to the well-established and modular transient gene expression technologies.The fifth chassis is synthetic biochemistry, wherein long-lived “cell-free” enzymatic reactions have enabled high-titer flux through lengthy bio-synthetic pathways.One must carefully consider the features of a given pathway before deciding if a particular chassis meets the bio-synthetic requirements. Many natural product pathways evolved in the context of highly specialized organelles, cells, or tissues.In this case, pathway compartmentalization may be required in order to sequester reactive bio-synthetic intermediates from endogenous metabolism.

Currently, sub-cellular localization is possible through the use of organelle-targeting peptide signals fused to the N-terminus of pathway enzymes, or the use of intracellular protein scaffolds. The recent production of tropane alkaloids in yeast required extensive localization across six sub-cellular locations.Tissue specific pathway localization in multicellular model organisms has yet to be employed but will require the implementation of intercellular metabolite transport. Special attention must be given to enzymes that are membrane associated, including the cytochrome P450s.Even in the most appropriate chassis, functional expression of trafficked proteins may require extensive engineering. Galanie et al. employed a protein chimera strategy to ameliorate improper processing of a P450 for opioid biosynthesis in yeast.Solubilization of membrane anchored P450s has been successfully demonstrated, but a general strategy guaranteeing functional soluble expression of P450s is still a major technological hurdle.It is also important to consider the primary metabolite building blocks required for construction of the secondary metabolite to be produced. Individual organisms exhibit variable fluxes towards given metabolic pools, dictating initial maximum titers prior to strain engineering. To address this limitation, “metabolic chassis strains” – strains with increased flux towards dedicated natural product building blocks – have been developed. Microbial chasses for the production of N-methylpyrrolinium strictosidine -reticuline and a number of other psychoactive natural product precursors have been established in the last decade. The availability of a robust synthetic biology toolkit is another important factor to consider when selecting a production host. An ideal suite of molecular biology tools permits accurate and rapid genomic edits, precisely controlled gene expression, and diversity generation using libraries of genetic parts. More industrially “robust” organisms may also be utilized. These may be proprietary strains that outperform laboratory strains, but oftentimes lack the synthetic biology toolkit characteristic of the previously described model organisms. Proprietary methods may be developed for rational engineering, or random mutagenesis may be employed for non-rational diversity generation. Additional properties of robust chasses are faster growth, resistance to contamination, and a tailored metabolic profile. Predictable scalability and ease of downstream purification costs should also be considered when assessing platform commercialization.For academic purposes, however, it is most common to recapitulate bio-synthetic pathways in model organisms as a proof-of-concept. Iterative design methodologies are now commonplace in deploying synthetic biology-based engineering. In natural product production chasses, first generation strain prototypes almost never produce compounds in sufficient quantities to compete with alternative production strategies. As a result, many iterations of design, build, test, and learn are required before a process is cost competitive. The industrial feasibility of bio-process is often measured by titer , rate , and yield as these metrics relate to cost of goods sold .In addition to improving titers on the strain engineering front, large improvements in productivity can be made through bio-process engineering, which has benefitted immensely from automated design of experiment methodologies. The ability to iterate through the DBTL process is dependent on the bio-synthetic chassis, engineering strategy, and screening strategy, among other factors. Novel metabolic engineering approaches aim to reduce the cost or duration of some aspect of the DBTL cycle.As previously mentioned, “automated design” and “machine learning” technologies have only recently been deployed in metabolic engineering studies. Thus, we focus below on methodologies which streamline the “build” and “test” phases of iterative design.