Scalable Hybrid Synthetic/Biocatalytic Route to Psilocybin

Abstract Psilocybin, the principal indole alkaloid of Psilocybe mushrooms, is currently undergoing clinical trials as a medication against treatment‐resistant depression and major depressive disorder. The psilocybin supply for pharmaceutical purposes is met by synthetic chemistry. We replaced the problematic phosphorylation step during synthesis with the mushroom kinase PsiK. This enzyme was biochemically characterized and used to produce one gram of psilocybin from psilocin within 20 minutes. We also describe a pilot‐scale protocol for recombinant PsiK that yielded 150 mg enzyme in active and soluble form. Our work consolidates the simplicity of tryptamine chemistry with the specificity and selectivity of enzymatic catalysis and helps provide access to an important drug at potentially reasonable cost.


Introduction
The indole alkaloid psilocybin (1,S cheme 1) is the principal natural product of hallucinogenic Psilocybe mushrooms. [1] Pharmacologically,i tr epresents the stable prodrug of the actual active, yetu nstable 4-dephosphorylated analogue psilocin (2), which targets mainly the human 5-HT 2A -receptor as ap artial agonist. [2] Modern scientific therapeutics rediscovered 1 as a valuabledrug candidate. [3] Phase 2clinical trials have shown favorable results in the treatment of several indications, and 1 has received Breakthrough Therapy designation by the United States Food and Drug Administration (US FDA) in support of both treatment-resistantd epression and major depressive disorder.O ther indications include cancer-related existential distress and substance dependencies. [4] Given its status as ac andidate drug,t he demandf or medical-grade 1 is increasing. Traditionally,t his compound is accessible through chemical synthesis. [1,[5][6] Identification of the 1 biosynthetic genes and enzymes in Psilocybe mushrooms, in-cluding the kinase gene psiK,o pened up new avenues for the production of 1.H eterologously produced enzymes were used to biocatalytically synthesize 1 in vitro. [7] In vivo production in microbial hosts has also proven successful. [8] In addition, the in vivo approachi sa ttractive because of its scalability to pilotscale bioreactors and the availability of cofactors. However, in view of registrationa sapharmaceutical, the use of ac ellbased production system entails intensive procedures to remove cellular componentsa nd debris that are potentially allergenico rt oxic. Furthermore, unlike the in vitro or synthetic approaches, the targetedc ompound needs to be purified from complex matrices, whichmay involve ac ell lysate or broth.
Herein, we describe the first combined chemical/biocatalytic synthesis of 1.T his cell-freei nv itro procedure combines the facile synthetic access to 2 with side-product-free quantitative conversion through PsiK-mediated biocatalysis in as ubsequent step, thus eliminating heavy-metal-dependent hydrogenolysis and low yielding phosphorylation chemistry,t op roduce gramscale amountsof1.T ofacilitate future development of the biotechnological processes,w ep resent at horough biochemical characterization of P. cubensis PsiK and ap rotocol to heterologously produce and purify three-digit milligrama mounts of PsiK in soluble and active form.

Results and Discussion
First, we producedP siK in as mall scale as ah exahistidinetagged fusion using E. coli KRX pJF23 [6] and investigated its steady-state kinetic properties. Phosphate-buffered in vitro assays were analyzed for formationo f1 by UHPLC-MS. Previous work confirmed ad ual biosynthetic and protectiver ole for PsiK as it accepts both 4-hydroxytryptamine and 2 as substrates (Scheme 1). The former represents the phosphate acceptor during biosynthesis. The repair reaction, which is the relevant reactionf or our study,i ncludes phosphorylation of 2 back to 1,t hus removing instable and oligomerization-prone 2 from the cell. Oligomeric 2 causes the blueing reaction of injured "magic" mushrooms and binds to proteins, which would be fatal to the intact fungal mycelium. [9] To protect cell integrity,w ee xpectedt hat the velocityo ft he repair direction should at least equal that of the biosynthetic direction.
In its native form, P. cubensis PsiK is a4 0.4 kDa enzyme of 362 aa. Sequence alignmentw ith other characterized or putative small-molecule kinases (FigureS1) identified ac anonical ATPb inding domain that comprises ac onserved motif 57 KHA 59 . In the structureo ft he Arabidopsis thaliana (mouse-ear cress)5methylthioribose kinase (Protein Data Bank, accession #2 PYW), the equivalent lysiner esidue in the motif 63 KQA 66 ionically interacted with the a-phosphoryl group of ADP.I nt he Arabidopsis enzyme, the conserved aspartate residue D255 was shown to coordinate bivalent Mg 2 + cations in the active site. [10] The equivalent residue in PsiK is D224.
To verify its function, we engineered the cloned psiK cDNA to replace D224 with alanine. PsiK D224A was catalytically inactive when 2 was offered as substrate andn ot even traces of 1 were detected ( Figure 1). This result suggests as imilarc ation-coordinating role for D224 in the activesite.
Optimumt urnover was found at 40 8Ca nd at pH 7.0. Furthermore, PsiK activity depended on Mg 2 + .I nc ontrast, with Co 2 + as divalent cation,t he turnover dropped to 11.5 %a nd the enzyme remained nearly inactive (< 5%)w ith Ca 2 + ,C u 2 + , Ni 2 + ,M n 2 + ,o rZ n 2 + .P siK followed as igmoidk inetic response to increasing 2 and 4-hydroxytryptamine concentrations, with K 0.5 values of 72 mm and 67 mm,a nd a k cat of 16.1 s À1 and 4.5 s À1 for these substrates ( Figure S2).T his translates into catalytic efficiencies (k cat /K 0.5 )o f2 .24 10 5 and 6.67 10 4 m À1 s À1 , respectively,w ith 2 and 4-hydroxytryptamine as acceptor substrates. The K 0.5 value for ATPw as 89 mm. The above values identifiedt he repair function of PsiK as more than three times as efficient as the biosynthetic one. Size-exclusion chromatography showedt hat active PsiK is monomeric ( Figure 2). This situation is reminiscent of glucokinase, which has as ingle bind-ing site for its phosphate acceptor substrate (d-glucose), yet showsasigmoid kinetic, which is ac haracteristic of positive cooperativity. [11] With af uture large-scale process development in mind, a stirred-tank fermentation andp urification regime was established. E. coli KRX pJF23 was performed batch-wise in a7 5L reactor, filled with 30 Ly east-peptone-glucose medium. After harvest, cell disruption and FPLC-based Ni 2 + affinity chromatography,150.2 mg pure PsiK was obtained ( Figure S3).
[1] Various improved protocols have been developed since, [5] however,t he synthesis has still remainedp roblematic due in part to scalability in large-scale manufacturing, instability during the isolation of 2,i n-process impurities during their synthesis, hydrolysis of 1 during recrystallization, and the labilityo ft he O,O-dibenzyl ester intermediate. Regardless of the protocol, the final deprotection involves ah eavy metal (Pd/C), which is not preferred from ar egulatory affairs perspective for medical grade compounds. Furthermore, our approacha voids corrosivea nd pyrophoric n-butyllithium. Our route to provide upper milligram to gram amounts of 1 combines the simplicity of traditional tryptamine chemistry with the power of biocatalysis to eliminate heavy metals and the notoriously problematic phosphorylations tep, while also circumventing cell-based production. Demand for 1 is expected to increase. To translate from gram-to kilogram-scale, further optimization, such as reduced solventu se and an alternative to the preparative reversed-phase HPLC-based purification procedure, may be required. Also, selectivef iltration-precipitation-recrystallization may help make this process more amenable to scale-up. With these changes, and given the outstanding selectivity of the enzymatic phosphorylation step, we expect drastically reduced pricing for kilogram-scale 1 production.

Conclusion
We expect our results will motivate bioprocess research to further develop and scale up the presented protocol. Therefore, the current work is directed towards further upscaling, and optimizing the post-biocatalysis work-up. We concludet hat our research helps provide 1 at ar easonable cost, particularly to support non-profit endeavors that are geared towards re-introducingt his invaluable natural product to pharmaceutical and clinicaluse.
Pilot-scale PsiK production:T he inoculum was transferred to a2L shake flask, filled with 200 mL LB medium, for 12 h, at 37 8Ca nd 180 rpm. This seed culture was inoculated with an OD 600 of 0.05 and transferred to the main culture with af inal OD 600 of 3.5. The main batch culture (30 L, 37 8C) was carried out in a7 5L stirred tank reactor (C75, bbi, Berlin, Germany), using YPD medium (20 gL À1 glycerol, 20 gL À1 tryptone, 10 gL À1 yeast extract). The dissolved oxygen tension was controlled by the stirring rate at aminimum value of 20 %. The initial OD 600 was 0.023. PsiK production was induced after 10 hb ya dding 1gL À1 l-rhamnose. Upon induc- Figure 3. A) Progressoft he gram-scale synthetic/biocatalytic production of tion, the temperature was shifted to 16 8C. For fermentation details, see Figure S6. The fermentation continued for another 14 hb efore the broth was centrifuged at 14 000 gf or 30 min at 4 8C. Subsequently,t he biomass was suspended in 5L lysis buffer (50 mm sodium dihydrogen phosphate, 300 mm NaCl), amended with 20 mm imidazole, homogenized at 850 bar in ah igh-pressure Gaulin flow homogenizer which was rinsed with another 1L of lysis buffer,c entrifuged at 14 000 gf or 45 min at 4 8Ca nd finally filtered (0.45 mm). PsiK was further purified by immobilized metal affinityc hromatography on aF PLC system (¾ktaPilot, GE Healthcare), equipped with aF ineLINE Pilot 35 column filled with 200 mL Ni-Sepharose FF.T he flow rate was 20 mL min À1 .T he system was equilibrated in lysis buffer before 6L of supernatant were loaded. The column was washed step-wise with ag radient of 30 mm (2 column volumes), 40 mm (1 cv), and 50 mm imidazole (2 cv), using the same buffer.T he final elution was conducted with 500 mm imidazole, resulting in a2 00 mL fraction. The eluted enzyme was rebuffered on aA micon Ultra 15 centrifugal filter unit (Merck), equilibrated with phosphate buffer (50 mm,p H7.0), and stored at À80 8Cw ith 10 %g lycerol until used for production of 1.T he protein concentration was determined using Bradford'sm ethod. [16] The total yield was 150.2 mg pure PsiK. Prior to use for assays, PsiK was rebuffered by FPLC (below).
Size-exclusion chromatography:F PLC (¾kta Pure 25, GE Healthcare) fitted to aS uperdex 200 increase 10/300 GL column with 24 mL bed volume was used 1) to determine whether PsiK was active as am ono-or multimer,a nd 2) to rebufferP siK. Prior to sample loading, PsiK was concentrated on ac entrifugal filter unit (10 kDa cut off, Amicon Ultra 15, Merck). Elution was with phosphate buffer (10 mm sodium dihydrogen phosphate, 140 mm NaCl, pH 7.5) at af low of 0.5 mL min À1 .F or rebuffering and subsequent use in assays, PsiK was eluted with assay buffer (below).
In vitro PsiK assays:A ssays to detect activity or to record kinetics were set up in sodium dihydrogen phosphate buffer (50 mm, pH 7.0) and 3mm b-mercaptoethanol in af inal volume of 50 mL. ATPa nd MgCl 2 or chlorides of other bivalent cations (2 mm each) and either 4-hydroxytryptamine or 2 (1 mm)w ere added as substrate, PsiK was added at 1 mm.T he reactions were incubated for 10 min at 40 8C. Subsequently,t he assays were lyophilized, the residue was dissolved in 50 mLM eOH, centrifuged (11000 g, 10 min, 4 8C), filtered, and chromatographically analyzed (below). To determine optima, pH and temperature were varied between pH 6-8 and 20-50 8C. To investigate the storability of PsiK, aliquots were stored with or without additional glycerol (10 %, v/v) at 4, À20 and À80 8Ca nd analyzed by the above assay after 14 and 30 days.
Analytical liquid chromatography:U HPLC-ESIMS-analyses of in vitro assays were performed with an Agilent Infinity 1290 chromatograph with aL una Omega Polar C 18 column (100 2.1 mm, 1.6 mmp article size, Phenomenex), and coupled to an Agilent 6130 Single Quadrupole mass detector.T he flow was 0.5 mL min À1 . Diode array detection was between l = 200-600 nm. Chromatograms were extracted at l = 280 nm. Solvent Aw as 0.1 %( v/v) formic acid in H 2 O, solvent Bw as acetonitrile (ACN). The linear gradient was initially 1% B, increased to 10 %w ithin 3min, and to 100 %Bin 1min. LC-HRESIMS analysis in positive and negative mode with purified substances were performed with aT hermo Accela chromatograph coupled to an Exactive Orbitrap mass spectrometer,a sdescribed. [14] Gram-scale production of 1:S caled-up production was carried out in a1 .8 Lr eaction in sodium dihydrogen phosphate buffer (50 mm, pH 7.0). Before adding enzyme and substrates, the reaction buffer was pre-warmed to 40 8C. Subsequently,7 25 mg 2 (2 mm), 3.97 g ATPd isodium salt (4 mm), 1.46 gM gCl 2 ·6H 2 O( 4mm)a nd 422 mg b-mercaptoethanol (3 mm)w ere added. The reaction was initiated by adding 149.7 mg of purified PsiK (1.89 mm). Samples (50 mL) were taken in triplicates in 10 min intervals to monitor product formation by UHPLC-MS. After 90 min, the solvent was evaporated under reduced pressure, and the residue was dissolved in 500 mL MeOH, centrifuged (3 200 g, 15 min, 4 8C) also to remove precipitated protein and ADP,a nd filtered. The solvent was evaporated under reduced pressure. For chromatographic purification of 1,t he residue was dissolved in H 2 O/acetonitrile (ACN, 9:1, v/v) with 0.1 % trifluoroacetic acid (TFA), which yielded 893 mg (88.5 %) of pure 1. Preparative liquid chromatography:C ompound 1 was purified with an Agilent 1260 series instrument, equipped with aZ orbax Eclipse XDB C 18 column (250 21.2 mm, 10 mm). Solvent Aw as 0.1 %T FA in H 2 O, solvent Bw as ACN. Al inear gradient 5-100 %B within 20 min at af low of 20 mL min À1 was applied. Further workup included solvent/antisolvent precipitation in 20 mL acidic (pH 4) H 2 Oa nd 30 mL acetone. The precipitate was washed with cold acetone. To verify purity, 1 Ha nd 13 CNMR spectra of purified 1, dissolved in D 2 O, were recorded at 500 and 125 MHz, respectively, and 300 Kw ith aB ruker AvanceI II spectrometer.C hemical shifts were referenced to residual non-deuterated solvent (d H = 4.80 ppm).