Berberine Bridge Enzyme Research Articles

De novo production of protoberberine and benzophenanthridine alkaloids through metabolic engineering of yeast

Protoberberine alkaloids and benzophenanthridine alkaloids (BZDAs) are subgroups of benzylisoquinoline alkaloids (BIAs), which represent a diverse class of plant-specialized natural metabolites with many pharmacological properties. Microbial biosynthesis has been allowed for accessibility and scalable production of high-value BIAs. Here, we engineer Saccharomyces cerevisiae to de novo produce a series of protoberberines and BZDAs, including palmatine, berberine, chelerythrine, sanguinarine and chelirubine. An ER compartmentalization strategy is developed to improve vacuole protein berberine bridge enzyme (BBE) activity, resulting in >200% increase on the production of the key intermediate (S)-scoulerine. Another promiscuous vacuole protein dihydrobenzophenanthridine oxidase (DBOX) has been identified to catalyze two-electron oxidation on various tetrahydroprotoberberines at N7-C8 position and dihydrobenzophenanthridine alkaloids. Furthermore, cytosolically expressed DBOX can alleviate the limitation on BBE. This study highlights the potential of microbial cell factories for the biosynthesis of a diverse group of BIAs through engineering of heterologous plant enzymes.

A Secreted BBE-Like Enzyme Acting as a Drug-Binding Efflux Carrier Confers Microbial Self-Resistance to Mitomycin C.

The berberine bridge enzyme (BBE)-like flavoproteins have attracted continuous attention for their capability to catalyze various oxidative reactions. Here we demonstrate that MitR, a secreted BBE-like enzyme, functions as a special drug-binding efflux protein evolved from quinone reductase. Moreover, this protein provides self-resistance to its hosts toward the DNA-alkylating agent mitomycin C with a distinctive strategy, featured by independently performing drug binding and efflux.

Genome-Wide Characterization of Berberine Bridge Enzyme Gene Family in Wheat (Triticum aestivum L.) and the Positive Regulatory Role of TaBBE64 in Response to Wheat Stripe Rust.

Berberine bridge enzymes (BBEs), functioning as carbonate oxidases, enhance disease resistance in Arabidopsis and tobacco. However, the understanding of BBEs' role in monocots against pathogens remains limited. This study identified 81 TaBBEs with FAD_binding_4 and BBE domains. Phylogenetic analysis revealed a separation of the BBE gene family between monocots and dicots. Notably, RNA-seq showed TaBBE64's significant induction by both pathogen-associated molecular pattern treatment and Puccinia striiformis f. sp. tritici (Pst) infection at early stages. Subcellular localization revealed TaBBE64 at the cytoplasmic membrane. Knocking down TaBBE64 compromised wheat's Pst resistance, reducing reactive oxygen species and promoting fungal growth, confirming its positive role. Molecular docking and enzyme activity assays confirmed TaBBE64's glucose oxidation to produce H2O2. Since Pst relies on living wheat cells for carbohydrate absorption, TaBBE64's promotion of glucose oxidation limits fungal growth and resists pathogen infection. This study sheds light on BBEs' role in wheat resistance against biotrophic fungi.

“Stepwise or concerted or a hybrid mechanism for berberine bridge enzyme?”: A computational mechanistic study by QM-MM and QM methods

Berberine bridge enzyme (BBE) is a plant-based amine oxidase that catalyzes conversion of (S)-reticuline into (S)-scoulerine using flavin as cofactor in a stereospecific way in an alkaloid biosynthesis pathway. Based on the active site enzyme variants, a concerted mechanism was proposed involving hydride transfer, proton transfer, and substrate cyclization processes in a single step. In this mechanism, Glu417 residue acts as the catalytic base which deprotonates the phenolic proton of the substrate while a hydride ion transfers from the substrate to flavin and the substrate cyclizes. However, based on solvent and substrate deuterium kinetic isotope effect studies, it was proposed that the oxidation process occurs in a stepwise fashion in which a hydride ion transfer from substrate to flavin first, then cyclization of the substrate occurs together with the proton transfer process. In this study, we formulated computational models to elucidate the oxidation mechanism of (S)-reticuline into (S)-scoulerine using the crystal structure of enzyme complexed with (S)-reticuline. Both QM and QM-MM calculations revealed that a hybrid of concerted and stepwise mechanisms might be operative during the catalysis. It was found that a concerted hydride-proton transfer processes occurs forming a reactive intermediate which subsequently cyclize without an energy barrier as a decoupled step.

Cannabinoid Biosynthesis Using Noncanonical Cannabinoid Synthases.

We report enzymes from the berberine bridge enzyme (BBE) superfamily that catalyze the oxidative cyclization of the monoterpene moiety in cannabigerolic acid (CBGA) to form cannabielsoin (CBE). The enzymes are from a variety of organisms and are previously uncharacterized. Out of 232 homologues chosen from the enzyme superfamily, four orthologues were shown to accept CBGA as a substrate and catalyze the biosynthesis of CBE. The four enzymes discovered in this study were recombinantly expressed and purified in Pichia pastoris. These enzymes are the first report of heterologous expression of BBEs that did not originate from the Cannabis plant that catalyze the production of cannabinoids using CBGA as substrate. This study details a new avenue for discovering and producing natural and unnatural cannabinoids.

Effects of codon optimization, N-terminal truncation and gene dose on the heterologous expression of berberine bridge enzyme

Morphine, sanguinarine and chelerythrine are benzylisoquinoline alkaloids (BIAs), and these compounds possess strong biological activities. (S)-scoulerine is a commonly shared precursor of these compounds, and berberine bridge enzyme (BBE) is a key rate-limiting enzyme in the synthesis of (S)-scoulerine. We isolated the BBE gene from Macleaya cordata (McBBE) and used CEN.PK2-1C as a chassis strain. We compared the catalytic efficiency of five codon-optimized McBBE genes in Saccharomyces cerevisiae and finally obtained a yeast strain (YH03) that exhibited a 58-fold increase in yield (1.12mg/L). Then, we truncated the N-terminus of McBBE by 8 and 22 amino acids and found that with the increase in the number of N-terminal truncated amino acids, the production of (S)-scoulerine gradually decreased. Additionally, we used CRISPR-Cas9 to integrate the McBBE gene at the delta site of the S. cerevisiae genome to achieve stable genetic inheritance and found that the yield of (S)-scoulerine was not significantly increased in the integrated strain. In conclusion, our work achieved high-efficiency expression of McBBE in S. cerevisiae, explored the influence of N-terminal truncation on the yield of (S)-scoulerine, and obtained a genetically stable S. cerevisiae strain with high McBBE expression. This study provides a reference for further complex metabolic engineering optimization and lays a foundation for the efficient biosynthesis of BIAs.

Berberine bridge enzyme-like oxidase-catalysed double bond isomerization acts as the pathway switch in cytochalasin synthesis

Cytochalasans (CYTs), as well as their polycyclic (pcCYTs) and polymerized (meCYTs) derivatives, constitute one of the largest families of fungal polyketide-nonribosomal peptide (PK-NRP) hybrid natural products. However, the mechanism of chemical conversion from mono-CYTs (moCYTs) to both pcCYTs and meCYTs remains unknown. Here, we show the first successful example of the reconstitution of the CYT core backbone as well as the whole pathway in a heterologous host. Importantly, we also describe the berberine bridge enzyme (BBE)-like oxidase AspoA, which uses Glu538 as a general acid biocatalyst to catalyse an unusual protonation-driven double bond isomerization reaction and acts as a switch to alter the native (for moCYTs) and nonenzymatic (for pcCYTs and meCYTs) pathways to synthesize aspochalasin family compounds. Our results present an unprecedented function of BBE-like enzymes and highly suggest that the isolated pcCYTs and meCYTs are most likely artificially derived products.

Cloning and functional identification of PeWRKY41 from Populus × euramericana

Poplar is an important greening and economic tree species in China while damage caused by diseases and herbivorous insects seriously restricts its widespread planting. WRKYs play an important role in the response to biotic and abiotic stresses, but the defense mechanisms induced by poplar WRKY to protect the trees from herbivorous insects remain unclear. In this study, PeWRKY41 was cloned and its function was identified, aiming to provide guidance for producing salt-resistant and insect-resistant poplars. The results showed that the PeWRKY41 (1029 bp) encoded a nuclear-localized protein belonging to class III WRKY subfamily. The promoter region of PeWRKY41 contained several anti-stress-related cis-active elements that respond to the stress induced by ETH, MeJA, SA and NaCl. At the PeWRKY41-binding sites, the most significantly enriched motifs were W-box elements. Casein kinase interacts with PeWRKY41. Overexpression of PeWRKY41 improved the insect resistance and salt tolerance of transgenic Populus alba×Populus tremula var. glandulosa (84 K). A transcriptome analysis of showed that stress-resistance genes (protein kinase, berberine bridge enzyme, peroxidase, chitinase, etc.) were significantly upregulated, with the differentially expressed genes occurring in pathways related to biotic and abiotic stress defenses, such as flavonoid/flavonol biosynthesis, terpenoid backbone synthesis, phenylpropanoid biosynthesis, etc. The study provides references for the generation of stress-resistant poplars with potentially economic benefit.

Dissection of full-length transcriptome and metabolome of Dichocarpum (Ranunculaceae): implications in evolution of specialized metabolism of Ranunculales medicinal plants.

Several main families of Ranunculales are rich in alkaloids and other medicinal compounds; many species of these families are used in traditional and folk medicine. Dichocarpum is a representative medicinal genus of Ranunculaceae, but the genetic basis of its metabolic phenotype has not been investigated, which hinders its sustainable conservation and utilization. We use the third-generation high-throughput sequencing and metabolomic techniques to decipher the full-length transcriptomes and metabolomes of five Dichocarpum species endemic in China, and 71,598 non-redundant full-length transcripts were obtained, many of which are involved in defense, stress response and immunity, especially those participating in the biosynthesis of specialized metabolites such as benzylisoquinoline alkaloids (BIAs). Twenty-seven orthologs extracted from trancriptome datasets were concatenated to reconstruct the phylogenetic tree, which was verified by the clustering analysis based on the metabolomic profile and agreed with the Pearson correlation between gene expression patterns of Dichocarpum species. The phylogenomic analysis of phytometabolite biosynthesis genes, e.g., (S)-norcoclaurine synthase, methyltransferases, cytochrome p450 monooxygenases, berberine bridge enzyme and (S)-tetrahydroprotoberberine oxidase, revealed the evolutionary trajectories leading to the chemodiversity, especially that of protoberberine type, aporphine type and bis-BIA abundant in Dichocarpum and related genera. The biosynthesis pathways of these BIAs are proposed based on full-length transcriptomes and metabolomes of Dichocarpum. Within Ranunculales, the gene duplications are common, and a unique whole genome duplication is possible in Dichocarpum. The extensive correlations between metabolite content and gene expression support the co-evolution of various genes essential for the production of different specialized metabolites. Our study provides insights into the transcriptomic and metabolomic landscapes of Dichocarpum, which will assist further studies on genomics and application of Ranunculales plants.

Identification of proteins associated with Fusarium crown rot resistance in wheat using label-free quantification analysis

Fusarium crown rot (FCR), typically caused by Fusarium pseudograminearum, is a severe soil-borne disease that, in recent years, has become an emerging threat to Chinese wheat crops. For the first time in this study, we investigated and compared the proteomic characteristics of two Chinese wheat varieties (04 Zhong 36 and Xinmai 26) at 24, 48, and 72 h post-inoculation using label-free quantitative proteomic analysis. A total of 9 234 proteins were successfully quantified, of which 783 were differentially expressed after inoculation. These proteins were mainly involved in metabolic, single-organism, and cellular processes. Thirty-three proteins associated with defense, cell wall formation, photosynthesis, etc., showed consistently different expression between the two genotypes at multiple time points. In particular, chitinase, which degrades chitin in the fungal cell wall and limits fungal growth, was exclusively and consistently upregulated in 04 Zhong 36 across the three time points. Other proteins such as flavonoid O-methyltransferase, glycosyltransferase, and peroxidase were only upregulated in 04 Zhong 36, and proteins, including the berberine bridge enzyme and rubisco large subunit-binding protein, were specifically downregulated in Xinmai 26. The expression of transcripts encoding eight selected proteins through qRT-PCR analysis supported the proteomic profiles. Overall, the results of this study allow us to understand FCR resistance in wheat at the protein level. Some proteins and their corresponding genes may be useful resources for the genetic improvement of FCR resistance in wheat.

Discovery and Biosynthesis of Ascorbylated Securinega Alkaloids

Securinega alkaloids represent a class of plant secondary metabolites with intriguing bridged tetracyclic structures and promising biological activities. Despite extensive synthetic efforts and a number of innovative total syntheses, no relevant biosynthetic genes have been reported to date. In this study, fluesuffine A (1), a C-2 and C-3 functionalized derivative of Securinega alkaloids with a fused 5/5/5/6/5/6/5 congested heptacyclic ring system, was isolated from Flueggea suffruticosa. In the biogenetic study of 1, we found a berberine bridge enzyme (BBE)-like enzyme (FsBBE), which could efficaciously catalyze the condensation of allosecurinine and l-ascorbic acid (l-AA) or dehydroascorbic acid (DHA), resulting in the chemical skeleton of 1. FsBBE also could catalyze the condensation of allosecurinine and isoascorbic acid to form another unnatural Securinega alkaloid fluesuffine B (2), an epimer of 1. Moreover, the catalytic mechanism study of FsBBE revealed the existence of an enamine intermediate (3), which was previously proposed as a key intermediate in the biosynthesis of C-2 and C-3 functionalized derivatives of Securinega alkaloids. The formation of 3 suggested that the epimers were synthesized via an intermolecular enamine-type addition reaction. Hence, our findings provide further insight into the biosynthesis of C-2 and C-3 functionalized derivatives of Securinega alkaloids and expand the diversity of ascorbylated natural product.

A BBE-like Oxidase, AsmF, Dictates the Formation of Naphthalenic Hydroxyl Groups in Ansaseomycin Biosynthesis.

Ansaseomycins are ansamycin-type natural products produced through expression of the asm gene cluster in a heterologous host. A rare berberine bridge enzyme (BBE) like oxidase, AsmF, is encoded in the asm gene cluster. Deletion of asmF led to the accumulation of a series of structurally diverse compounds, all of which lacked the 23-hydroxyl group in naphthalenic motif. Our work demonstrated that AsmF dictated the formation of the naphthalenic hydroxyl group in ansaseomycin biosynthesis.

The catalytic machinery of the FAD-dependent AtBBE-like protein 15 for alcohol oxidation: Y193 and Y479 form a catalytic base, Q438 and R292 an alkoxide binding site

Monolignol oxidoreductases are members of the berberine bridge enzyme–like (BBE-like) protein family (pfam 08031) that oxidize monolignols to the corresponding aldehydes. They are FAD-dependent enzymes that exhibit the para-cresolmethylhydroxylase-topology, also known as vanillyl oxidase-topology. Recently, we have reported the structural and biochemical characterization of two monolignol oxidoreductases from Arabidopsis thaliana, AtBBE13 and AtBBE15. Now, we have conducted a comprehensive site directed mutagenesis study for AtBBE15, to expand our understanding of the catalytic mechanism of this enzyme class. Based on the kinetic properties of active site variants and molecular dynamics simulations, we propose a refined, structure-guided reaction mechanism for the family of monolignol oxidoreductases. Here, we propose that this reaction is facilitated stepwise by the deprotonation of the allylic alcohol and a subsequent hydride transfer from the Cα-atom of the alkoxide to the flavin. We describe an excessive hydrogen bond network that enables the catalytic mechanism of the enzyme. Within this network Tyr479 and Tyr193 act concertedly as active catalytic bases to facilitate the proton abstraction. Lys436 is indirectly involved in the deprotonation as this residue determines the position of Tyr193 via a cation-π interaction. The enzyme forms a hydrophilic cavity to accommodate the alkoxide intermediate and to stabilize the transition state from the alkoxide to the aldehyde. By means of molecular dynamics simulations, we have identified two different and distinct binding modes for the substrate in the alcohol and alkoxide state. The alcohol interacts with Tyr193 and Tyr479 while Arg292, Gln438 and Tyr193 form an alkoxide binding site to accommodate this intermediate. The pH-dependency of the activity of the active site variants revealed that the integrity of the alkoxide binding site is also crucial for the fine tuning of the pKa of Tyr193 and Tyr479. Sequence alignments showed that key residues for the mechanism are highly conserved, indicating that our proposed mechanism is not only relevant for AtBBE15 but for the majority of BBE-like proteins.

Altered gene expression and root thebaine production in polyploidized and methyl jasmonate-elicited Papaver bracteatum Lindl

Persian poppy (Papaver bracteatum Lindl.) is a perennial medicinal plant belonging to the Papaveraceae family that is endemic to the mountainous areas in Northern Iran. It is known for high amounts of the valuable benzylisoquinoline alkaloid thebaine. The effects of induced polyploidy as well as the effect of methyl Jasmonate (MeJA) elicitation on the root production of thebaine and on the expression of five alkaloid biosynthesis related genes were studied. The in vitro tetraploidy induction caused a significant increased expression of norcoclaurine synthase (NCS) and salutaridinol (SAT), and a significant decreased expression of berberine bridge enzyme (BBE) in the leaves. In the root tissues, the BBE, NCS, and SAT showed an increased expression in tetraploid plants, while codeinone reductase (COR) showed a decreased expression. A similar alteration pattern was found in mixoploid plants when compared to their diploid counterparts. MeJA at concentrations of 0.1 and 0.5 mM caused a remarkable increase in the thebaine content in the roots of treated plants, where the highest thebaine content was identified in plants elicited with 0.5 mM MeJA. Elicitation treatment caused a substantial increase in the expression of NCS and SAT in the leaves, while it had no major effect on BBE, codeine 3-O-demethylase (CODM) and COR. Expression analysis in the roots showed that MeJA caused a significant increase in the expression of only BBE and NCS, while expression of other studied genes remained unchanged. Our results may be exploited for improved thebaine production and the processing of Persian poppy.

In Silico Transcriptomic Analysis of Canadine Accumulation in Papaver Somniferum Cultivar

Papaver somniferum, colloquially known as opium poppy, currently remains as the only commercial source of pharmaceutically important benzylisoquinoline alkaloids, of which include canadine. Canadine is an important naturally occurring alkaloid in current demand for pharmaceutical studies. Understanding the biosynthesis in plantae will help in increasing production of this alkaloid. In the present study, a comparative transcriptomic analysis was performed between gene expression data procured from the normal cultivar and a high canadine cultivar. Results indicated differential expression of key enzymes in the phthalideisoquinoline pathway leading to canadine biosynthesis. Specifically, it was found that transcription of genes encoding berberine bridge enzyme canadine synthase, and scoulerine 9-O-methyltransferase were significantly increased in the high canadine cultivar compared to the normal cultivar. These findings provide basis for further elucidating the coordinated transcriptional processes underlying canadine accumulation in P. somniferum cultivars.

Biosynthesis of a Tricyclo[6.2.2.02,7 ]dodecane System by a Berberine Bridge Enzyme-Like Aldolase.

The aldol reaction is one of the most fundamental stereocontrolled carbon-carbon bond-forming reactions and is mainly catalyzed by aldolases in nature. Despite the fact that the aldol reaction has been widely proposed to be involved in fungal secondary metabolite biosynthesis, a dedicated aldolase that catalyzes stereoselective aldol reactions has only rarely been reported in fungi. Herein, we activated a cryptic polyketide biosynthetic gene cluster that was upregulated in the fungal wheat pathogen Parastagonospora nodorum during plant infection; this resulted in the production of the phytotoxic stemphyloxin II (1). Through heterologous reconstruction of the biosynthetic pathway and in vitro assay by using cell-free lysate from Aspergillus nidulans, we demonstrated that a berberine bridge enzyme (BBE)-like protein SthB catalyzes an intramolecular aldol reaction to establish the bridged tricyclo[6.2.2.02,7 ]dodecane skeleton in the post-assembly tailoring step. The characterization of SthB as an aldolase enriches the catalytic toolbox of classic reactions and the functional diversities of the BBE superfamily of enzymes.

Biocatalytic Enantioselective Oxidation of Sec-Allylic Alcohols with Flavin-Dependent Oxidases.

The oxidation of allylic alcohols is challenging to perform in a chemo‐ as well as stereo‐selective fashion at the expense of molecular oxygen using conventional chemical protocols. Here, we report the identification of a library of flavin‐dependent oxidases including variants of the berberine bridge enzyme (BBE) analogue from Arabidopsis thaliana (AtBBE15) and the 5‐(hydroxymethyl)furfural oxidase (HMFO) and its variants (V465T, V465S, V465T/W466H and V367R/W466F) for the enantioselective oxidation of sec‐allylic alcohols. While primary and benzylic alcohols as well as certain sugars are well known to be transformed by flavin‐dependent oxidases, sec‐allylic alcohols have not been studied yet except in a single report. The model substrates investigated were oxidized enantioselectively in a kinetic resolution with an E‐value of up to >200. For instance HMFO V465S/T oxidized the (S)‐enantiomer of (E)‐oct‐3‐en‐2‐ol (1 a) and (E)‐4‐phenylbut‐3‐en‐2‐ol with E>200 giving the remaining (R)‐alcohol with ee>99% at 50% conversion. The enantioselectivity could be decreased if required by medium engineering by the addition of cosolvents (e. g. dimethyl sulfoxide).

Modulation of benzylisoquinoline alkaloid biosynthesis by overexpression berberine bridge enzyme in Macleaya cordata

Macleaya cordata produces a variety of benzylisoquinoline alkaloids (BIAs), such as sanguinarine, protopine, and berberine, which are potential anticancer drugs and natural growth promoters. The genes encoding the berberine bridge enzyme (BBE) were isolated from M. cordata and Papaver somniferum, and then the two genes were overexpressed in M. cordata. Through liquid chromatography with triple-quadrupole mass spectrometry analysis, it was determined that McBBE-OX caused higher levels of (S)-norcoclaurine, (S)-coclaurine, (S)-N-cis-methylcoclaurine, (S)-reticuline, (S)-tetrahydrocolumbamine, (S)-tetrahydroberberine, (S)-cheilanthifoline, and (S)-scoulerine than PsBBE-OX, empty vector or control treatments. qRT-PCR analysis demonstrated that the introduced genes in the transgenic lines were all highly expressed. However, the levels of sanguinarine (SAN) and chelerythrine (CHE) in all the transgenic lines were slightly lower than those in the wild-type lines, possibly because the overexpression of McBBE causes feedback-inhibition. This is the first report on the overexpression of potential key genes in M. cordata, and the findings are important for the design of metabolic engineering strategies that target BIAs biosynthesis.

Oxidative cyclization of N-methyl-dopa by a fungal flavoenzyme of the amine oxidase family

Flavin-dependent enzymes catalyze many oxidations, including formation of ring structures in natural products. The gene cluster for biosynthesis of fumisoquins, secondary metabolites structurally related to isoquinolines, in the filamentous fungus Aspergillus fumigatus harbors a gene that encodes a flavoprotein of the amine oxidase family, termed fsqB (fumisoquin biosynthesis gene B). This enzyme catalyzes an oxidative ring closure reaction that leads to the formation of isoquinoline products. This reaction is reminiscent of the oxidative cyclization reported for berberine bridge enzyme and tetrahydrocannabinol synthase. Despite these similarities, amine oxidases and berberine bridge enzyme–like enzymes possess distinct structural properties, prompting us to investigate the structure–function relationships of FsqB. Here, we report the recombinant production and purification of FsqB, elucidation of its crystal structure, and kinetic analysis employing five putative substrates. The crystal structure at 2.6 Å resolution revealed that FsqB is a member of the amine oxidase family with a covalently bound FAD cofactor. N-methyl-dopa was the best substrate for FsqB and was completely converted to the cyclic isoquinoline product. The absence of the meta-hydroxyl group, as e.g. in l-N-methyl-tyrosine, resulted in a 25-fold lower rate of reduction and the formation of the demethylated product l-tyrosine, instead of a cyclic product. Surprisingly, FsqB did not accept the d-stereoisomer of N-methyltyrosine, in contrast to N-methyl-dopa, for which both stereoisomers were oxidized with similar rates. On the basis of the crystal structure and docking calculations, we postulate a substrate-dependent population of distinct binding modes that rationalizes stereospecific oxidation in the FsqB active site.

Biocatalyzed C−C Bond Formation for the Production of Alkaloids

AbstractTraditional methods of chemical synthesis of alkaloids exhibit various problems such as lack of enantioselectivity, the use of toxic chemical and intermediates, and multiple numbers of synthetic steps. Consequently, various enzymatic methods for the formation of C−C bonds in the alkaloid skeleton have been developed. Herein, we report advances achieved in the enzymatic or chemo‐enzymatic synthesis of pharmaceutically important alkaloids that employ three C−C bond forming enzymes: two Pictet‐Spenglerases and the oxidative C−C bond forming flavoenzyme Berberine Bridge Enzyme. Protein engineering studies, improving the substrate scope of these enzymes, and thereby leading to the synthesis of non‐natural alkaloids possessing higher or newer pharmacological activities, are also discussed. Furthermore, the integration of these biocatalysts with other enzymes, in multi‐enzymatic cascades for the enantioselective synthesis of alkaloids, is also reviewed. Current results suggest that these enzymes hold great promise for the generation of C−C bonds in the selective synthesis of alkaloid compounds possessing diverse pharmacological properties.