RESUMEN
Rivastigmine is one of the several pharmaceuticals widely prescribed for the treatment of Alzheimer's disease. However, its practical synthesis still faces many issues, such as the involvement of toxic metals and harsh reaction conditions. Herein, we report a chemo-enzymatic synthesis of Rivastigmine. The key chiral intermediate was synthesized by an engineered alcohol dehydrogenase from Lactobacillus brevis (LbADH). A semi-rational approach was employed to improve its catalytic activity and thermal stability. Several LbADH variants were obtained with a remarkable increase in activity and melting temperature. Exploration of the substrate scope of these variants demonstrated improved activities toward various ketones, especially acetophenone analogs. To further recycle and reuse the biocatalyst, one LbADH variant and glucose dehydrogenase were co-immobilized on nanoparticles. By integrating enzymatic and chemical steps, Rivastigmine was successfully synthesized with an overall yield of 66 %. This study offers an efficient chemo-enzymatic route for Rivastigmine and provides several efficient LbADH variants with a broad range of potential applications.
Asunto(s)
Alcohol Deshidrogenasa , Enzimas Inmovilizadas , Levilactobacillus brevis , Rivastigmina , Rivastigmina/química , Levilactobacillus brevis/enzimología , Alcohol Deshidrogenasa/metabolismo , Alcohol Deshidrogenasa/química , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Biocatálisis , Acetofenonas/química , Acetofenonas/metabolismo , Ingeniería de ProteínasRESUMEN
Production of commodity chemicals, such as benzene, toluene, ethylbenzene, and xylenes (BTEX), from renewable resources is key for a sustainable society. Biocatalysis enables one-pot multistep transformation of bioresources under mild conditions, yet it is often limited to biochemicals. Herein, we developed a non-natural three-enzyme cascade for one-pot conversion of biobased l-phenylalanine into ethylbenzene. The key rate-limiting photodecarboxylase was subjected to structure-guided semirational engineering, and a triple mutant CvFAP(Y466T/P460A/G462I) was obtained with a 6.3-fold higher productivity. With this improved photodecarboxylase, an optimized two-cell sequential process was developed to convert l-phenylalanine into ethylbenzene with 82 % conversion. The cascade reaction was integrated with fermentation to achieve the one-pot bioproduction of ethylbenzene from biobased glycerol, demonstrating the potential of cascade biocatalysis plus enzyme engineering for the production of biobased commodity chemicals.
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Derivados del Benceno , Tolueno , Biocatálisis , Derivados del Benceno/metabolismo , Tolueno/metabolismo , Benceno/metabolismo , Xilenos , Fenilalanina/metabolismoRESUMEN
Biocatalysis has undergone revolutionary progress in the past century. Benefited by the integration of multidisciplinary technologies, natural enzymatic reactions are constantly being explored. Protein engineering gives birth to robust biocatalysts that are widely used in industrial production. These research achievements have gradually constructed a network containing natural enzymatic synthesis pathways and artificially designed enzymatic cascades. Nowadays, the development of artificial intelligence, automation, and ultra-high-throughput technology provides infinite possibilities for the discovery of novel enzymes, enzymatic mechanisms and enzymatic cascades, and gradually complements the lack of remaining key steps in the pathway design of enzymatic total synthesis. Therefore, the research of biocatalysis is gradually moving towards the era of novel technology integration, intelligent manufacturing and enzymatic total synthesis.
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Biocatálisis , Animales , Inteligencia Artificial , Vías Biosintéticas , Enzimas/metabolismo , Humanos , Ingeniería de ProteínasRESUMEN
Apremilast is an important active pharmaceutical ingredient that relies on a resolution to produce the key chiral amine intermediate. To provide a new catalytic and enzymatic process for Apremilast, we performed the directed evolution of the amine transaminase fromVibriofluvialis. Six rounds of evolution resulted in the VF-8M-E variant with > 400-fold increase specific activity over the wildtype enzyme. A homology model of VF-8M-E was built and a molecular docking study was performed to explain the increase in activity. The purified VF-8M-E was successfully applied to produce the key chiral amine intermediate in enantiopure form and 49% conversion via a kinetic resolution, representing a new enzymatic access towards Apremilast.
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Aminas/metabolismo , Talidomida/análogos & derivados , Transaminasas/metabolismo , Aminas/química , Biocatálisis , Cinética , Estructura Molecular , Talidomida/química , Talidomida/metabolismo , Transaminasas/química , Vibrio/enzimologíaRESUMEN
Biocatalysis has found numerous applications in various fields as an alternative to chemical catalysis. The use of enzymes in organic synthesis, especially to make chiral compounds for pharmaceuticals as well for the flavors and fragrance industry, are the most prominent examples. In addition, biocatalysts are used on a large scale to make specialty and even bulk chemicals. This review intends to give illustrative examples in this field with a special focus on scalable chemical production using enzymes. It also discusses the opportunities and limitations of enzymatic syntheses using distinct examples and provides an outlook on emerging enzyme classes.
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Biocatálisis , Biotecnología/métodos , Enzimas/metabolismoRESUMEN
We engineered the cytochrome P450 monooxygenase CYP107D1 (OleP) from Streptomyces antibioticus for the stereo- and regioselective 7ß-hydroxylation of lithocholic acid (LCA) to yield ursodeoxycholic acid (UDCA). OleP was previously shown to hydroxylate testosterone at the 7ß-position but LCA is exclusively hydroxylated at the 6ß-position, forming murideoxycholic acid (MDCA). Structural and 3DM analysis, and molecular docking were used to identify amino acid residues F84, S240, and V291 as specificity-determining residues. Alanine scanning identified S240A as a UDCA-producing variant. A synthetic "small but smart" library based on these positions was screened using a colorimetric assay for UDCA. We identified a nearly perfectly regio- and stereoselective triple mutant (F84Q/S240A/V291G) that produces 10-fold higher levels of UDCA than the S240A variant. This biocatalyst opens up new possibilities for the environmentally friendly synthesis of UDCA from the biological waste product LCA.
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Proteínas Bacterianas/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Ácido Ursodesoxicólico/metabolismo , Proteínas Bacterianas/genética , Sitios de Unión , Dominio Catalítico , Sistema Enzimático del Citocromo P-450/genética , Ácido Desoxicólico/química , Ácido Desoxicólico/metabolismo , Hidroxilación , Ácido Litocólico/química , Ácido Litocólico/metabolismo , Simulación del Acoplamiento Molecular , Mutagénesis Sitio-Dirigida , Estereoisomerismo , Streptomyces/enzimología , Ácido Ursodesoxicólico/síntesis química , Ácido Ursodesoxicólico/químicaRESUMEN
Biocatalytic alkylations are important reactions to obtain chemo-, regio- and stereoselectively alkylated compounds. This can be achieved using S-adenosyl-l-methionine (SAM)-dependent methyltransferases and SAM analogs. It was recently shown that a halide methyltransferase (HMT) from Chloracidobacterium thermophilum can synthesize SAM from SAH and methyl iodide. We developed an iodide-based assay for the directed evolution of an HMT from Arabidopsis thaliana and used it to identify a V140T variant that can also accept ethyl-, propyl-, and allyl iodide to produce the corresponding SAM analogs (90, 50, and 70 % conversion of 15â mg SAH). The V140T AtHMT was used in one-pot cascades with O-methyltransferases (IeOMT or COMT) to achieve the regioselective ethylation of luteolin and allylation of 3,4-dihydroxybenzaldehyde. While a cascade for the propylation of 3,4-dihydroxybenzaldehyde gave low conversion, the propyl-SAH intermediate could be confirmed by NMR spectroscopy.
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Metiltransferasas/metabolismo , S-Adenosilmetionina/metabolismo , Biocatálisis , Humanos , Ingeniería de ProteínasRESUMEN
As an important bulk chemical, benzoic acid is currently manufactured from nonrenewable feedstocks under harsh conditions. Although there are natural pathways for biosynthesis of benzoic acid, they are often inefficient and subjected to complex regulation. Here we develop a nonnatural enzyme cascade to efficiently produce benzoic acid from styrene or biogenic L-phenylalanine under mild conditions. By using a modular approach, two whole-cell catalysts Escherichia coli LZ305 and LZ325 are engineered for coexpressing seven and nine enzymes for production of 133-146 mM benzoic acid (16.2-17.8 g/Laq ) with 88-97% conversion via seven- and nine-step cascade biotransformation of styrene and L-phenylalanine, respectively. The seven-step cascade represents a formal high-yielding biocatalytic oxidative cleavage of styrene, and the nine-step cascade showcases the high efficiency of extended nonnatural enzyme cascades. Moreover, to achieve benzoic acid production directly from low-cost renewable glycerol, a novel coupled fermentation-biotransformation process was developed by integration of fermentative production of L-phenylalanine with in situ biotransformation to give 63-70 mM benzoic acid (7.6-8.6 g/Laq ), which is around 20 times higher than the reported value via a natural pathway. The coupled fermentation-biotransformation process could be generally applicable to microbial production of growth-inhibitory or toxic chemicals in high concentrations.
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Ácido Benzoico/metabolismo , Ingeniería Metabólica/métodos , Biocatálisis , Biotransformación , Escherichia coli/citología , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentación , Oxidación-Reducción , Fenilalanina/metabolismo , Estireno/metabolismoRESUMEN
The biotin-streptavidin technology has been extensively exploited to engineer artificial metalloenzymes (ArMs) that catalyze a dozen different reactions. Despite its versatility, the homotetrameric nature of streptavidin (Sav) and the noncooperative binding of biotinylated cofactors impose two limitations on the genetic optimization of ArMs: (i) point mutations are reflected in all four subunits of Sav, and (ii) the noncooperative binding of biotinylated cofactors to Sav may lead to an erosion in the catalytic performance, depending on the cofactor:biotin-binding site ratio. To address these challenges, we report on our efforts to engineer a (monovalent) single-chain dimeric streptavidin (scdSav) as scaffold for Sav-based ArMs. The versatility of scdSav as host protein is highlighted for the asymmetric transfer hydrogenation of prochiral imines using [Cp*Ir(biot-p-L)Cl] as cofactor. By capitalizing on a more precise genetic fine-tuning of the biotin-binding vestibule, unrivaled levels of activity and selectivity were achieved for the reduction of challenging prochiral imines. Comparison of the saturation kinetic data and X-ray structures of [Cp*Ir(biot-p-L)Cl]·scdSav with a structurally related [Cp*Ir(biot-p-L)Cl]·monovalent scdSav highlights the advantages of the presence of a single biotinylated cofactor precisely localized within the biotin-binding vestibule of the monovalent scdSav. The practicality of scdSav-based ArMs was illustrated for the reduction of the salsolidine precursor (500 mM) to afford (R)-salsolidine in 90% ee and >17â¯000 TONs. Monovalent scdSav thus provides a versatile scaffold to evolve more efficient ArMs for in vivo catalysis and large-scale applications.
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Biotina/química , Ingeniería Química/métodos , Metaloproteínas/química , Compuestos Organometálicos/química , Estreptavidina/química , Sitios de Unión , Biotina/genética , Biotinilación , Catálisis , Cromatografía de Afinidad , Escherichia coli/genética , Hidrogenación , Iridio/química , Cinética , Metaloproteínas/genética , Estereoisomerismo , Estreptavidina/genéticaRESUMEN
Biotransformation is a green and useful tool for sustainable and selective chemical synthesis. However, it often suffers from the toxicity and inhibition from organic substrates or products. Here, we established a hollow fiber membrane bioreactor (HFMB)-based aqueous/organic biphasic system, for the first time, to enhance the productivity of a cascade biotransformation with strong substrate toxicity and inhibition. The enantioselective trans-dihydroxylation of styrene to (S)-1-phenyl-1,2-ethanediol, catalyzed by Escherichia coli (SSP1) coexpressing styrene monooxygenase and an epoxide hydrolase, was performed in HFMB with organic solvent in the shell side and aqueous cell suspension in the lumen side. Various organic solvents were investigated, and n-hexadecane was found as the best for the HFMB-based biphasic system. Comparing to other reported biphasic systems assisted by HFMB, our system not only shield much of the substrate toxicity but also deflate the product recovery burden in downstream processing as the majority of styrene stayed in organic phase while the diol product mostly remained in the aqueous phase. The established HFMB-based biphasic system enhanced the production titer to 143 mM, being 16-fold higher than the aqueous system and 1.6-fold higher than the traditional dispersive partitioning biphase system. Furthermore, the combination of biphasic system with HFMB prevents the foaming and emulsification, thus reducing the burden in downstream purification. HFMB-based biphasic system could serve as a suitable platform for enhancing the productivity of single-step or cascade biotransformation with toxic substrates to produce useful and valuable chemicals.
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Reactores Biológicos , Escherichia coli/metabolismo , Glicoles de Etileno/química , Oxigenasas/metabolismo , Estireno/metabolismo , Alcanos/química , Biocatálisis , Biotransformación , Epóxido Hidrolasas/metabolismo , Escherichia coli/enzimologíaRESUMEN
Sustainable synthesis of useful and valuable chiral fine chemicals from renewable feedstocks is highly desirable but remains challenging. Reported herein is a designed and engineered set of unique non-natural biocatalytic cascades to achieve the asymmetric synthesis of chiral epoxide, diols, hydroxy acid, and amino acid in high yield and with excellent eeâ values from the easily available biobased l-phenylalanine. Each of the cascades was efficiently performed in one pot by using the cells of a single recombinant strain over-expressing 4-10 different enzymes. The cascade biocatalysis approach is promising for upgrading biobased bulk chemicals to high-value chiral chemicals. In addition, combining the non-natural enzyme cascades with the natural metabolic pathway of the host strain enabled the fermentative production of the chiral fine chemicals from glucose.
RESUMEN
This work reports a new strategy for the detection and purification of levulinic acid (LA) and bio-compounds from the acid hydrolysis and enzymatic treatment of oil palm empty fruit bunch (OPEFB) through high-performance liquid chromatography (HPLC) techniques combined with ion/ligand chromatography. The detections of LA, biomass-saccharides, hydroxymethylfurfural (HMF), and furfural were successfully elucidated by optimizing the multiple reaction monitoring mode (MRM) and liquid chromatography conditions using a Pb2+ ligand exchange column in the liquid chromatography with tandem mass spectrometry (LC-MS/MS) approach. High-performance liquid chromatography with diode-array detection (HPLC-DAD) combined with an H+ ion exchange column also showed potency for detecting chromophoric compounds such as LA, HMF, furfural, and acid (by-products) but not biomass-saccharides. Both techniques showed acceptable validation in terms of linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy, precision, and stability in both quantitative and qualitative analysis. However, the LC-MS/MS approach showed higher sensitivity for detecting LA and HMF compared with HPLC-DAD. Samples comprised of cellobiose, glucose, HMF, and LA from the acid hydrolysis of cellulose to LA with a mineral acid, and the biocatalysis of cellulase and ß-glucosidase catalyzed cellulose (from OPEFB) to glucose were successfully monitored through the LC-MS/MS approach. In addition, using the optimal HPLC conditions obtained from LC-MS/MS, the purification of LA from other substances obtained from the hydrolysis reaction of cellulose (5 g) was successfully demonstrated by HPLC-DAD equipped with a fraction collector combined with an H+ ion exchange column at gram-scale of 1 g LA with a purification rate of 0.63 g ml-1 min-1.
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Plastic waste imposes a serious problem to the environment and society. Hence, strategies for a circular plastic economy are demanded. One strategy is the engineering of polyester hydrolases toward higher activity for the biotechnological recycling of polyethylene terephthalate (PET). To provide tools for the rapid characterization of PET hydrolases and the detection of degradation products like terephthalic acid (TPA), we coupled a carboxylic acid reductase (CAR) and the luciferase LuxAB. CAR converted TPA into the corresponding aldehydes in Escherichia coli, which yielded bioluminescence that not only semiquantitatively reflected amounts of TPA in hydrolysis samples but is suitable as a high-throughput screening assay to assess PET hydrolase activity. Furthermore, the CAR-catalyzed synthesis of terephthalaldehyde was combined with a reductive amination cascade in a one-pot setup yielding the corresponding diamine, suggesting a new strategy for the transformation of TPA as a product obtained from PET biodegradation.
RESUMEN
Synthetic biology, a course with a sound theoretical system and a wide application range, plays a role in the cultivation of innovative talents in the field of bioengineering. To this end, we have set up a synthetic biology course in our university. First, according to the concept of imparting basic knowledge, highlighting innovative practice, and keeping up with cutting-edge progress, we assembled a high-level teaching team for synthetic biology. The team constantly adjusted and optimized the course contents and achieved a novel and reasonable course system. Second, we introduced frontier cases of synthetic biology reported in high-level journals, as well as breaking news in this field in classroom teaching, which enriched the teaching contents and aroused students' interest. Third, taking these cases as the breakthrough point, we guided students to in-depth discussions through the learning-centered teaching mode to improve students' abilities of critical thinking and theoretical innovation. In summary, the course has achieved good teaching outcomes and improved the cultivation of innovative talents. Therefore, we share our work with peer teachers, aiming to give new insights into the teaching reform of synthetic biology and other related courses.
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Estudiantes , Biología Sintética , Bioingeniería , Humanos , Aprendizaje , UniversidadesRESUMEN
Fast screening of enzyme variants is crucial for tailoring biocatalysts for the asymmetric synthesis of non-natural chiral chemicals, such as amines. However, most existing screening methods either are limited by the throughput or require specialized equipment. Herein, we report a simple, high-throughput, low-equipment dependent, and generally applicable growth selection system for engineering amine-forming or converting enzymes and apply it to improve biocatalysts belonging to three different enzyme classes. This results in (i) an amine transaminase variant with 110-fold increased specific activity for the asymmetric synthesis of the chiral amine intermediate of Linagliptin; (ii) a 270-fold improved monoamine oxidase to prepare the chiral amine intermediate of Cinacalcet by deracemization; and (iii) an ammonia lyase variant with a 26-fold increased activity in the asymmetric synthesis of a non-natural amino acid. Our growth selection system is adaptable to different enzyme classes, varying levels of enzyme activities, and thus a flexible tool for various stages of an engineering campaign.
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Aminas , Aminoácidos , Monoaminooxidasa , Transaminasas/genética , CinacalcetRESUMEN
Developing (chemo)enzymatic cascades is very attractive for green synthesis, because they streamline multistep synthetic processes. In this Feature Article, we have summarized the recent advances in in vitro or whole-cell cascade reactions with a focus on the use of renewable bio-based resources as starting materials. This includes the synthesis of rare sugars (such as ketoses, L-ribulose, D-tagatose, myo-inositol or aminosugars) from readily available carbohydrate sources (cellulose, hemi-cellulose, starch), in vitro enzyme pathways to convert glucose to various biochemicals, cascades to convert 5-hydroxymethylfurfural and furfural obtained from lignin or xylose into novel precursors for polymer synthesis, the syntheses of phenolic compounds, cascade syntheses of aliphatic and highly reduced chemicals from plant oils and fatty acids, upgrading of glycerol or ethanol as well as cascades to transform natural L-amino acids into high-value (chiral) compounds. In several examples these processes have demonstrated their efficiency with respect to high space-time yields and low E-factors enabling mature green chemistry processes. Also, the strengths and limitations are discussed and an outlook is provided for improving the existing and developing new cascades.
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Carbohidratos/síntesis química , Animales , Catálisis , Etanol/química , Ácidos Grasos/química , Furaldehído/análogos & derivados , Furaldehído/química , Glicerol/química , Tecnología Química Verde , Humanos , Lignina/química , Aceites de Plantas/químicaRESUMEN
Alkenes are excellent starting materials for organic synthesis due to the versatile reactivity of C[double bond, length as m-dash]C bonds and the easy availability of many unfunctionalised alkenes. Direct regio- and/or enantioselective conversion of alkenes into functionalised (chiral) compounds has enormous potential for industrial applications, and thus has attracted the attention of researchers for extensive development using chemo-catalysis over the past few years. On the other hand, many enzymes have also been employed for conversion of alkenes in a highly selective and much greener manner to offer valuable products. Herein, we review recent advances in seven well-known types of biocatalytic conversion of alkenes. Remarkably, recent mechanism-guided directed evolution and enzyme cascades have enabled the development of seven novel types of single-step and one-pot multi-step functionalisation of alkenes, some of which are even unattainable via chemo-catalysis. These new reactions are particularly highlighted in this feature article. Overall, we present an ever-expanding enzyme toolbox for various alkene functionalisations inspiring further research in this fast-developing theme.
RESUMEN
Engineered enzyme cascades offer powerful tools to convert renewable resources into value-added products. Man-made catalysts give access to new-to-nature reactivities that may complement the enzyme's repertoire. Their mutual incompatibility, however, challenges their integration into concurrent chemo-enzymatic cascades. Herein we show that compartmentalization of complex enzyme cascades within E. coli whole cells enables the simultaneous use of a metathesis catalyst, thus allowing the sustainable one-pot production of cycloalkenes from oleic acid. Cycloheptene is produced from oleic acid via a concurrent enzymatic oxidative decarboxylation and ring-closing metathesis. Cyclohexene and cyclopentene are produced from oleic acid via either a six- or eight-step enzyme cascade involving hydration, oxidation, hydrolysis and decarboxylation, followed by ring-closing metathesis. Integration of an upstream hydrolase enables the usage of olive oil as the substrate for the production of cycloalkenes. This work highlights the potential of integrating organometallic catalysis with whole-cell enzyme cascades of high complexity to enable sustainable chemistry.
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Biocatálisis , Cicloparafinas/síntesis química , Ácidos Dicarboxílicos , Escherichia coli , Ácido Oléico , Aceite de Oliva , Ciclohexenos/síntesis química , Ciclopentanos/síntesis química , Descarboxilación , Hidrólisis , Compuestos Organometálicos , Oxidación-Reducción , Biología SintéticaRESUMEN
Enzymatic alcohol oxidation plays an important role in chemical synthesis. In the past two years, new alcohol oxidation enzymes were developed through genome-mining and protein engineering, such as new copper radical oxidases with broad substrate scope, alcohol dehydrogenases with altered cofactor preference and a flavin-dependent alcohol oxidase with enhanced oxygen coupling. New cofactor recycling methods were reported for alcohol dehydrogenase-catalyzed oxidation with photocatalyst and coupled glutaredoxin-glutathione reductase as promising examples. Different alcohol oxidation systems were used for the oxidation of primary and secondary alcohols, especially in the cascade conversion of alcohols to lactones, lactams, chiral amines, chiral alcohols and hydroxyketones. Among them, biocatalyst with low enantioselectivity demonstrated an interesting feature for complete conversion of racemic secondary alcohols through non-enantioselective oxidation.
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Alcoholes/metabolismo , Enzimas/metabolismo , Biocatálisis , Biotransformación , Biología Computacional , Enzimas/genética , Secuenciación de Nucleótidos de Alto Rendimiento , NADP/metabolismo , Oxidación-Reducción , Oxígeno/química , Ingeniería de Proteínas , Especificidad por SustratoRESUMEN
Production of chemicals from renewable feedstocks has been an important task for sustainable chemical industry. Although microbial fermentation has been widely employed to produce many biochemicals, it is still very challenging to access non-natural chemicals. Two methods (biotransformation and fermentation) have been developed for the first bio-derived synthesis of benzylamine, a commodity non-natural amine with broad applications. Firstly, a nine-step artificial enzyme cascade was designed by biocatalytic retrosynthetic analysis and engineered in recombinant E. coli LZ243. Biotransformation of l-phenylalanine (60â mm) with the E. coli cells produced benzylamine (42â mm) in 70 % conversion. Importantly, the cascade biotransformation was scaled up to 100â mL and benzylamine was successfully isolated in 57 % yield. Secondly, an artificial biosynthesis pathway to benzylamine from glucose was developed by combining the nine-step cascade with an enhanced l-phenylalanine synthesis pathway in cells. Fermentation with E. coli LZ249 gave benzylamine in 4.3â mm concentration from glucose. In addition, one-pot syntheses of several useful benzylamines from the easily available styrenes were achieved, representing a new type of alkene transformation by formal oxidative cleavage and reductive amination.