Controlling the Regioselectivity of Baeyer–Villiger Monooxygenases by Mutation of Active‐Site Residues

Baeyer–Villiger monooxygenase (BVMO)‐mediated regiodivergent conversions of asymmetric ketones can lead to the formation of “normal” or “abnormal” lactones. In a previous study, we were able to change the regioselectivity of a BVMO by mutation of the active‐site residues to smaller amino acids, which thus created more space. In this study, we demonstrate that this method can also be used for other BVMO/substrate combinations. We investigated the regioselectivity of 2‐oxo‐Δ³‐4,5,5‐trimethylcyclopentenylacetyl‐CoA monooxygenase from Pseudomonas putida (OTEMO) for cis‐bicyclo[3.2.0]hept‐2‐en‐6‐one ( 1 ) and trans‐dihydrocarvone ( 2 ), and we were able to switch the regioselectivity of this enzyme for one of the substrate enantiomers. The OTEMO wild‐type enzyme converted (?)‐ 1 into an equal (50:50) mixture of the normal and abnormal products. The F255A/F443V variant produced 90 % of the normal product, whereas the W501V variant formed up to 98 % of the abnormal product. OTEMO F255A exclusively produced the normal lactone from (+)‐ 2 , whereas the wild‐type enzyme was selective for the production of the abnormal product. The positions of these amino acids were equivalent to those mutated in the cyclohexanone monooxygenases from Arthrobacter sp. and Acinetobacter sp. (CHMO_Arthro and CHMO_Acineto) to switch their regioselectivity towards (+)‐ 2 , which suggests that there are hot spots in the active site of BVMOs that can be targeted with the aim to change the regioselectivity.     

Direct Access to Medium‐Chain α,ω‐Dicarboxylic Acids by Using a Baeyer–Villiger Monooxygenase of Abnormal Regioselectivity

Baeyer–Villiger monooxygenases (BVMOs) are versatile biocatalysts in organic synthesis that can generate esters or lactones by inserting a single oxygen atom adjacent to a carbonyl moiety. The regioselectivity of BVMOs is essential in determining the ratio of two regioisomers for converting asymmetric ketones. Herein, we report a novel BVMO from Pseudomonas aeruginosa (PaBVMO); this has been exploited for the direct synthesis of medium‐chain α,ω‐dicarboxylic acids through a Baeyer–Villiger oxidation–hydrolysis cascade. PaBVMO displayed the highest abnormal regioselectivity toward a variety of long‐chain aliphatic keto acids (C₁₆–C₂₀) to date, affording dicarboxylic monoesters with a ratio of up to 95 %. Upon chemical hydrolysis, α,ω‐dicarboxylic acids and fatty alcohols are readily obtained without further treatment; this significantly reduces the synthetic steps of α,ω‐dicarboxylic acids from renewable oils and fats.     

Alkyl Formate Ester Synthesis by a Fungal Baeyer–Villiger Monooxygenase

We investigated Baeyer–Villiger monooxygenase (BVMO)‐mediated synthesis of alkyl formate esters, which are important flavor and fragrance products. A recombinant fungal BVMO from Aspergillus flavus was found to transform a selection of aliphatic aldehydes into alkyl formates with high regioselectivity. Near complete conversion of 10 mm octanal was achieved within 8 h with a regiomeric excess of ～80 %. Substrate concentration was found to affect specific activity and regioselectivity of the BVMO, as well as the rate of product autohydrolysis to the primary alcohol. More than 80 % conversion of 50 mm octanal was reached after 72 h (TTN nearly 20 000). Biotransformation on a 200 mL scale under unoptimized conditions gave a space‐time yield (STY) of 4.2 g L^?1 d^?1 (3.4 g L^?1 d^?1 extracted product).     

Discovery and Characterization of a Baeyer-Villiger Monooxygenase Using Sequence Similarity Network Analysis

Baeyer-Villiger monooxygenases (BVMOs) are important flavin-dependent enzymes which perform oxygen insertion reactions leading to valuable products. As reported in many studies, BVMOs are usually unstable during application, preventing a wider usage in biocatalysis. Here, we discovered a novel NADPH-dependent BVMO which originates from Halopolyspora algeriensis using sequence similarity networks (SSNs). The enzyme is stable at temperatures between 10 °C to 30 °C up to five days after the purification, and yields the normal ester product. In this study, the substrate scope was investigated for a broad range of aliphatic ketones and the enzyme was biochemically characterized to identify optimum reaction conditions. The best substrate (86 % conversion) was 2-dodecanone using purified enzyme. This novel BVMO could potentially be applied as part of an enzymatic cascade or in bioprocesses which utilize aliphatic alkanes as feedstock.     

Improving the Enantioselectivity of CHMOBrevi1 for Asymmetric Synthesis of Podophyllotoxin Precursor

(R)-β-piperonyl-γ-butyrolactones are key building blocks for the synthesis of podophyllotoxin, which have demonstrated remarkable potential in cancer treatment. Baeyer-Villiger monooxygenases (BVMOs)-mediated asymmetric oxidation is a green approach to produce chiral lactones. While several BVMOs were able to oxidize the corresponding cyclobutanone, most BVMOs gave the (S) enantiomer while Cyclohexanone monooxygenase (CHMO) from Brevibacterium sp. HCU1 gave (R) enantiomer, but with a low enantioselectivity (75 % ee). In this study, we use a strategy called “focused rational iterative site-specific mutagenesis” (FRISM) at residues ranging from 6 Å from substrate. The mutations by using a restricted set of rationally chosen amino acids allow the formation of a small mutant library. By generating and screening less than 60 variants, we achieved a high ee of 96.8 %. Coupled with the cofactor regeneration system, 9.3 mM substrate was converted completely in a 100-mL scale reaction. Therefore, our work reveals a promising synthetic method for (R)-β-piperonyl-γ-butyrolactone with the highest enantioselectivity, and provides a new opportunity for the chem-enzymatic synthesis of podophyllotoxin.     

Investigation of a New Type I Baeyer–Villiger Monooxygenase from Amycolatopsis thermoflava Revealed High Thermodynamic but Limited Kinetic Stability

Baeyer–Villiger monooxygenases (BVMOs) are remarkable biocatalysts, but, due to their low stability, their application in industry is hampered. Thus, there is a high demand to expand on the diversity and increase the stability of this class of enzyme. Starting from a known thermostable BVMO sequence from Thermocrispum municipale (TmCHMO), a novel BVMO from Amycolaptosis thermoflava (BVMO_Flava), which was successfully expressed in Escherichia coli BL21(DE3), was identified. The activity and stability of the purified enzyme was investigated and the substrate profile for structurally different cyclohexanones and cyclobutanones was assigned. The enzyme showed a lower activity than that of cyclohexanone monooxygenase (CHMO_Acineto) from Acinetobacter sp., as the prototype BVMO, but indicated higher kinetic stability by showing a twofold longer half-life at 30 °C. The thermodynamic stability, as represented by the melting temperature, resulted in a T_m value of 53.1 °C for BVMO_Flava, which was comparable to the T_m of TmCHMO (ΔT_m=1 °C) and significantly higher than the T_m value for CHMO_Acineto ((ΔT_m=14.6 °C)). A strong deviation between the thermodynamic and kinetic stabilities of BVMO_Flava was observed; this might have a major impact on future enzyme discovery for BVMOs and their synthetic applications.     

Recent developments in the application of Baeyer-Villiger monooxygenases as biocatalysts

Baeyer-Villiger monooxygenases (BVMOs) represent a specific class of monooxygenases that are capable of catalyzing a variety of oxidation reactions, including Baeyer-Villiger oxidations. The recently elucidated BVMO crystal structures have provided a more detailed insight into the complex mechanism of these flavin-containing enzymes. Biocatalytic studies on a number of newly discovered BVMOs have shown that they are very potent oxidative biocatalysts. In addition to catalyzing the regio- and enantioselective Baeyer-Villiger oxidations of a wide range of carbonylic compounds, epoxidations, and enantioselective sulfoxidations have also been shown to be part of their catalytic repertoire. This review provides an overview on the recent developments in BVMO-mediated biocatalytic processes, identification of the catalytic role of these enzymes in metabolic routes and prodrug activation, as well as the efforts in developing effective biocatalytic methodologies to apply BVMOs for the synthesis of high added value compounds.     

Production of 6,8-Dihydroxy Fatty Acids by Recombinant Escherichia coli Expressing T879A Variant 6,8-Linoleate Diol Synthase from Penicillium oxalicum

Recombinant Escherichia coli expressing T879A variant 6,8-linoleate diol synthase (LDS) from Penicillium oxalicum showed 2.1-fold higher activity than recombinant E. coli expressing wild-type 6,8-LDS for the production of 6,8-dihydroxy fatty acids (DiHFA) from linoleic acid. The optimal conditions for the production of 6,8-DiHFA by recombinant E. coli expressing T879A variant 6,8-LDS were pH 6.5, 35°C, 50 g L⁻¹ cells, 10 g L⁻¹ (35.7 mM) substrate, and 5% (v/v) dimethyl sulfoxide. Under these optimized conditions, 6.6 g L⁻¹ (22.1 mM) 6,8-dihydroxy-9,12(Z,Z)-octadecadienoic acid (DiHODE) and 7.1 g L⁻¹ (22.6 mM) 6,8-dihydroxy-9(Z)-octadecenoic acid (DiHOME) were produced from linoleic acid and oleic acid in 40 min, respectively. The volumetric productivities of 6,8-DiHODE and 6,8-DiHOME under these conditions were 9.9 and 10.7 mg L⁻¹ h⁻¹, respectively. The volumetric productivities of 6,8-DiHODE and 6,8-DiHOME were the highest values among those of all reported regiospecific DiHODE and DiHOME, respectively. To the best of our knowledge, this is the first quantitative biotechnological production of 6,8-DiHFA.     

Exploring the Substrate Specificity and Enantioselectivity of a Baeyer–Villiger Monooxygenase from Dietzia sp. D5: Oxidation of Sulfides and Aldehydes

Baeyer–Villiger monooxygenases (BVMOs) are valuable enzymes for specific oxyfunctionalization chemistry. They catalyze the oxidation of ketones to esters, but are also capable of oxidizing other chemical functions, namely aldehydes and heteroatoms such as sulfur, nitrogen, selenium and boron. The oxidation specificity and enantioselectivity of a newly characterized BVMO (BVMO4) from a strain of Dietzia towards sulfide- and aldehyde substrates have been studied. BVMO4 could react with sulfides containing an aromatic group. The presence of a substituent on the aromatic group was tolerated when they were in the meta- and para position and the oxidations yielded predominantly the (R)-sulfoxides. Similarly, BVMO4 displayed a higher activity for aldehydes containing a phenyl group, but long aliphatic aldehydes, namely octanal and decanal, were also accepted as substrate by this enzyme. The major oxidation products of the aldehyde substrates were the respective carboxylic acids in contrast to formate ester that was obtained in most of the previous reports. The Baeyer–Villiger oxidation of the substrate 2-phenylpropionaldehyde was studied in further detail and the corresponding acid product was obtained with good regio- and enantioselectivity. This is a unique feature for BVMO4 and is of great interest for further exploration of an alternative biocatalytic process.     

Structural evidence of a ketimine as the product of an amino acid with an aldehyde. An intermediate in the racemization and transamination of amino acids

The aldimine and ketimine forms of two Schiff base complexes formed by the condensation of two isomeric imidazole carboxaldehydes with an amino acid are reported. Reaction of L¹, the Schiff base condensate of 5-methyl-4-imidazolecarboxaldehyde (5Me4Im) and valine, with copper(II) perchlorate results in the isolation of [Cu(L¹)(5Me4Im)(ClO₄)] while the analogous reaction of L², the Schiff base condensate of 1-methyl-2-imidazolecarboxaldehyde (1Me2Im) with alanine, and nickel(II) results in the isolation of [Ni(L²)₂]. L¹ exhibits the expected aldimine form of the amino acid derived Schiff base, 5Me4Im-CH = N-CH(R)CO₂⁻, while L² exhibits the tautomeric ketimine form, 1Me2Im-CH₂–N = C(R’)CO₂⁻. Structural data clearly support the two tautomeric forms. The ketimine form, observed in [Ni(L²)₂], has been proposed as an intermediate in the racemization and transamination of amino acids.     

Application of the Stover–Kincannon kinetic model to nitrogen removal by Chlorella vulgaris in a continuously operated immobilized photobioreactor system

BACKGROUND: The aim of this study was to evaluate the ammonium nitrogen removal performance of algae culture Chlorella vulgaris in a novel immobilized photobioreactor system under different operating conditions and to determine the biokinetic coefficients using the Stover–Kincannon model. RESULTS: The photobioreactor was continuously operated at different initial ammonium nitrogen concentrations (NH₄‐N₀ = 10–48 mg L⁻¹), hydraulic retention times (HRT = 1.7–5.5 days) and nitrogen/phosphorus ratios (N/P = 4/1–13/1). Effluent NH₄‐N concentrations varied between 2.1 ± 0.5 mg L⁻¹ and 26 ± 1.2 mg L⁻¹ with increasing initial NH₄‐N concentrations from 10 ± 0.6 mg L⁻¹ to 48 ± 1.8 mg L⁻¹ at θ_H = 2.7 days. The maximum removal efficiency was obtained as 79 ± 4.5% at 10 mg L⁻¹ NH₄‐N concentration. Operating the system for longer HRT improved the effluent quality, and the percentage removal increased from 35 ± 2.4% to 93 ± 0.2% for 20 mg L⁻¹ initial NH₄‐N concentration. The N/P ratio had a substantial effect on removal and the optimum ratio was determined as N/P = 8/1. Saturation value constant, and maximum substrate utilization rate constant of the Stover–Kincannon model for ammonium nitrogen removal by C. vulgaris were determined as K_B = 10.3 mg L⁻¹ d⁻¹, U_max = 13.0 mg L⁻¹ day⁻¹, respectively. CONCLUSION: Results indicated that the algae‐immobilized photobioreactor system had an effective nitrogen removal capacity when the operating conditions were optimized. The optimal conditions for the immobilized photobioreactor system used in this study can be summarized as HRT = 5.5 days, N/P = 8 and NH₄‐N₀ = 20 mg L⁻¹ initial nitrogen concentration to obtain removal efficiency greater than 90%. Copyright © 2008 Society of Chemical Industry     

Influence of strain and fermentation time on the production,composition, and properties of xanthan gum

The objective of this study was to monitor the influence of Xanthomonas spp. and fermentation time on the production, properties, and composition of xanthan gum, verifying how these variables affect the biopolymer synthesis and properties. Three strains were monitored for 168 h of fermentation. Every 24 h, a sample was collected to determine cell growth, gum production, and substrate consumption. Fourier transform infrared, X-ray diffraction, rheology, molecular characterization, thermogravimetric analysis, sugars, and acids analyzes were performed to characterize the gums produced and to identify possible differences in these properties and composition. The results showed the influence of strain and the fermentation time in production (Pr₆₄₅ = 0.047 g L⁻¹ h⁻¹, Pr₂₉₀ = 0.039 g L⁻¹ h⁻¹, Pr₁₁₅₅ = 0.149 g L⁻¹ h⁻¹) and gum quality, since the viscosity was highest in the first 48 h for strains 290 (148.71 MPa s⁻¹) and 645 (135.28 MPa s⁻¹), while for Strain 1155, the maximum value identified for this property was 90.48 MPa s⁻¹. These results are of practical importance, since they allow obtaining better quality xanthan, enabling reduction of production costs. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2020 , 137, 48557.     

Coevolution of the Activity and Thermostability of an ϵ-Keto Ester Reductase for Better Synthesis of an (R)-α-Lipoic Acid Precursor

In this work, we have identified a significantly improved variant (S131Y/Q252I) of the natural ϵ-keto ester reductase CpAR2 from Candida parapsilosis for efficiently manufacturing (R)-8-chloro-6-hydroxyoctanoic acid [(R)-ECHO] through co-evolution of activity and thermostability. The activity of the variant CpAR2_S131Y/Q252I towards the ϵ-keto ester ethyl 8-chloro-6-oxooctanoate was improved to 214 U mg⁻¹—from 120 U mg⁻¹ in the case of the wild-type enzyme (CpAR2_WT)—and the half-deactivating temperature (T₅₀, for 15 min incubation) was simultaneously increased by 2.3 °C in relation to that of CpAR2_WT. Consequently, only 2 g L⁻¹ of lyophilized E. coli cells harboring CpAR2_S131Y/Q252I and a glucose dehydrogenase (GDH) were required in order to achieve productivity similar to that obtained in our previous work, under optimized reaction conditions (530 g L⁻¹ d⁻¹). This result demonstrated a more economical and efficient process for the production of the key (R)-α-lipoic acid intermediate ethyl 8-chloro-6-oxooctanoate.     

Artificial Maturation of the Highly Active Heterodimeric [FeFe] Hydrogenase from Desulfovibrio desulfuricans ATCC 7757

Hydrogenases catalyze the reduction of protons and oxidation of molecular hydrogen with high turnover frequencies and low overpotentials under ambient conditions. The heterodimeric [FeFe] hydrogenase from Desulfovibrio desulfuricans has an exceptionally high activity, and can be purified aerobically in an oxygen-stable inactive state. Recently, it was demonstrated that monomeric [FeFe] hydrogenases produced recombinantly in Escherichia coli can be artificially maturated by simply incubating the inactive “apo” enzymes with the synthetic [2Fe] cofactor mimic [Fe₂(adt)(CO)₄(CN)₂]²⁻. Here, we use the same technique to produce the heterodimeric “apo” hydrogenase from D. desulfuricans in E. coli with a high yield and purity, and maturate the “apo” enzyme with [Fe₂(adt)(CO)₄(CN)₂]²⁻ to generate fully active “holo” enzyme. Interestingly, the rate of the artificial maturation process with D. desulfuricans is significantly slower than that for all other hydrogenases tested so far. The artificially maturated enzyme is spectroscopically and electrochemically identical to the native enzyme and shows high rates of hydrogen production (3700 s⁻¹) and hydrogen oxidation (63,000 s⁻¹). We expect that our highly efficient production method will facilitate future studies of this enzyme and other related [FeFe] hydrogenases from Desulfovibrio species.     

Efficient Production of Epoxy-Norbornane from Norbornene by an Engineered P450 Peroxygenase

Epoxy-norbornane (EPO-NBE) is a crucial building block for the synthesis of various biologically active heterocyclic systems. To develop an efficient protocol for producing EPO-NBE using norbornene (NBE) as a substrate, cytochrome P450 enzyme from Pseudomonas putida (CYP238A1) was examined and its crystal structure (PDB code: 7X53) was resolved. Molecular mechanism analysis showed a high energy barrier related to iron-alkoxy radical complex formation. Therefore, a protein engineering strategy was developed and an optimal CYP238A1^NPV variant containing a local hydrophobic “fence” at the active site was obtained, which increased the H₂O₂-dependent epoxidation activity by 7.5-fold compared with that of CYP238A1^WT. Among the “fence”, Glu255 participates in an efficient proton transfer system. Whole-cell transformation using CYP238A1^NPV achieved an EPO-NBE yield of 77.6 g ⋅ L⁻¹ in a 30-L reactor with 66.3 % conversion. These results demonstrate the potential of this system for industrial production of EPO-NBE and provides a new biocatalytic platform for epoxidation chemistry.     

Creating new water-soluble Iso- and n-fatty acid arginate hydrochloride with antimicrobial properties

Typically, short- and long-chain lipids from oils exhibit different antimicrobial activities and therefore have been used in agriculture and aquaculture, biomedical therapeutic and antibacterial fields. However, these fatty acids have limitations in terms of bioactive efficacy, thermostability and aqueous solubility. In this study, water-soluble iso-fatty acid arginate hydrochloride derivatives with antimicrobial properties were produced by introducing branched (iso-) chain and other linear- (n-) chain fatty acids to the “arginine” amino acid molecule. The two-step synthetic route was straightforward and provided an efficient 88% and 76% product yields for ethyl n-oleoyl arginate hydrochloride and ethyl iso-oleoyl arginate hydrochloride, respectively. ATR-FT-IR, NMR, and LC-MS-Q-TOF techniques were used to thoroughly characterize and confirm the products. These arginate products had strong antimicrobial activities against Listeria innucua, a Gram-positive bacterium with minimum inhibitory concentrations and minimum bactericidal concentrations ranging from 1.8 µg mL⁻¹ to 29.1 µg mL⁻¹. Therefore, the study demonstrated the development of a novel class of antimicrobial compounds from iso-fatty acids and arginates.     

The Preparation of a Novel Corrosion Inhibitor and Its Corrosion Inhibition Behavior on 2024 Al-Cu-Mg Alloy in Acid Solution

A new corrosion inhibitor was synthesized from chloroacetyl chloride, 1,3-propanediamine, and dodecyldimethyl tertiary amine. The structure of the synthesized product was characterized using Fourier transform infrared(FTIR). The critical micelle concentration (CMC) was determined from surface tension measurements. The inhibition behavior of the corrosion inhibitor for 2024 Al-Cu-Mg alloy was studied using the weight-loss method and electrochemical measurements in hydrochloric acid solution. Experimental results show: The CMC is 7.767 × 10⁻⁴ mol L⁻¹ and IE (C _pro = 1 × 10⁻³ mol L⁻¹) ≈ 87.9%. The adsorption free energy is calculated to be −33.12 kJ mol⁻¹. Therefore, the adsorption mode is more inclined to physical adsorption. When the concentration of the synthesized product is lower than the CMC, it satisfies the Langmuir adsorption model; when the concentration is higher than the CMC, it no longer follows the Langmuir adsorption model.     

Membrane transport of organics. II. Permeation of some carboxylic acids through strongly basic polymer membrane

The permeability characteristics of the strongly basic polymer membrane Neosepta® AFN‐7, (Tokuyama Soda) have been studied for acetic, propionic, lactic, tartaric, oxalic, and citric acid. The results were interpreted by using the model of transport in reactive membranes. The specific constants, that is, the maximum flux J_max, the reactivity constant K, and the permeability coefficient (P), were calculated using the experimental quasi‐stationary fluxes and the equation derived as a sum of reaction–diffusion (Michaelis–Menten‐type), and the solution–diffusion transport equation. The constants K and J_max were found to range from 0.1 to 5 dm³ mol⁻¹ and from 0.4 × 10⁻⁷ to 2.5 × 10⁻⁷ mol cm⁻² s⁻¹ depending, on the acid properties. The values of K and J_max were correlated with the dissociation constants K_dis.acid, and the diffusion coefficients D_aq.acid in aqueous media, respectively. It was found that the reaction–diffusion flux is predominating for all acids, except for the lactic one, when the feed concentration is lower than 0.5 mol dm⁻³. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 71: 2179–2190, 1999     

Efficient Addition Reaction of Dibutylphosphane Oxide with Alkynes: New Mechanistic Proposal Involving a Duo of Palladium and Brønsted Acid

The addition reaction of dibutylphosphane oxide [Bu₂P(O)H] with alkynes proceeds efficiently in the presence of palladium‐chelating phosphane–Brønsted acid catalyst systems. Terminal alkynes afford branched‐structured products selectively. On the other hand, the same reaction using monodentate phosphane ligands or the reaction run in the absence of a Brønsted acid affords a much lower yield. A mechanistic study has revealed that Brønsted acids (XOH) interact with oxygen in M P(O)R₂ species (M=Pd, Pt) through hydrogen bonding to transform them to ionic M⁺←PR₂(OH⋅⋅⋅O⁻X) species, which was confirmed by NMR spectroscopy and X‐ray crystallography. The phosphane‐like PR₂(OH⋅⋅⋅O⁻X) moiety is coordinatively labile, as substantiated by the ligand exchange reaction with tert‐butyl isocyanide. A new mechanism that accommodates these observations has been proposed to rationalize the enhancement of catalytic activity and the regioselectivity induced by the Brønsted acid.     

Fermentative Production of Halogenated Tryptophan Derivatives with Corynebacterium glutamicum Overexpressing Tryptophanase or Decarboxylase Genes

The aromatic amino acid l -tryptophan serves as a precursor for many valuable compounds such as neuromodulators, indoleamines and indole alkaloids. In this work, tryptophan biosynthesis was extended by halogenation followed by decarboxylation to the respective tryptamines or cleavage to the respective indoles. Either the tryptophanase genes tnaAs from E. coli and Proteus vulgaris or the aromatic amino acid decarboxylase genes AADCs from Bacillus atrophaeus, Clostridium sporogenes, and Ruminococcus gnavus were expressed in Corynebacterium glutamicum strains producing (halogenated) tryptophan. Regarding indoles, final titers of 16 mg L⁻¹ 7-Cl-indole and 23 mg L⁻¹ 7-Br-indole were attained. Tryptamine production led to a much higher titer of 2.26 g L⁻¹ upon expression of AADC from B. atrophaeus. AADC enzymes were shown to be active with halogenated tryptophan in vitro and in vivo and supported production of 0.36 g L⁻¹ 7-Br-tryptamine with a volumetric productivity of 8.3 mg L⁻¹ h⁻¹ in a fed-batch fermentation.