Recent trends in biocatalysis

Abstract

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.

Fig. 1. New trends in biocatalysis: integration of novel technologies, intelligent manufacturing, and enzymatic total synthesis.

Fig. 2. An overview of droplet microfluidic technologies relevant to ultrahigh-throughput screening. (A) A schematic representation of droplet formation using two aqueous components and subsequent fluorescence activated droplet sorting (FADS). (1) Single cells are injected simultaneously with (2) a mixture of assay components, usually containing a fluorogenic substrate and a lysis agent. (3) A fluorinated oil containing a surfactant is injected into the third inlet. This breaks up the aqueous mixture into monodisperse water-in-oil droplets. (4) The single-cell lysate contains both the genotype (plasmid) and (5) the phenotype (enzyme). (6) Active variants convert the substrate to a fluorescent product, which (7) can be detected using the appropriate lasers and photomultiplier tubes (not shown). (8) A fluorescence signal triggers an electric pulse, delivered through imbedded electrodes. The dielectrophoretic force pulls the droplet from the path of least resistance into the “sorted” channel. DNA is subsequently recovered from sorted droplets by plasmid isolation or PCR (not shown). (B) Droplets can be reinjected into a second chip to make double emulsions. In this setup, inlet 1 would contain the droplets, inlet 2 some oil to space the droplets, and inlet 3 the outer aqueous phase containing a surfactant like Tween. The resulting double emulsion droplets can be sorted using flow cytometers, similar to FACS. (C) As an alternative to fluorescence measurements, imbedded fibre optic cables have been used to measure the absorbance of droplets. An increased absorbance signal triggers an electric pulse that moves the droplet into the sorting channel. (D) Droplets can be split, evenly or unevenly, into smaller droplets. This is useful for example if a destructive detection method like mass spectrometry is used, so the first droplet is sacrificed, and the second droplet sorted. (E) Droplets can also be fused, which is useful, for example, for delivering larger volumes of substrate or lysis agents. The chip first aligns droplets and then uses an electric pulse to merge them. (F) Smaller volumes of liquid can also be added to droplets using a method called picoinjection. Droplets flow past an aqueous inlet and an electric field is used to combine the two aqueous phases. (G) Droplets can also be sorted into multiple channels based on (H) at least two fluorescence measurements. (I) An interesting and promising approach allows the label free detection of molecules in droplets using RNA or DNA aptamers. A unique feature of aptamers is that they allow different enantiomers to be detected using the l- and d-forms of the nucleic acid aptamers. In the example shown, fluorescently labelled DNA aptamers are hybridised with antisense oligonucleotides labelled with quenchers. Binding to the target molecule separates the fluorophores and quenchers, resulting in a fluorescence signal. This allows both the concentration and enantiopurity of the substance to be determined. Importantly, if an aptamer for one enantiomer is available, the other enantiomer is easily detectable by synthesising the opposite enantiomer of the aptamer. This figure was inspired by Kintses et al. and Neun et al.

Fig. 3. Genetically encoded biosensors. (A) (Allosteric) TFs which bind metabolites can act as transcriptional activators (shown in blue at the top) or repressors (shown in purple at the bottom). TFs can also recruit other activators or repressors to regulate the activity of RNA polymerase (RNAP; not shown). (B) Upon binding a ligand or metabolite, riboswitches can act on the levels of transcription and translation by the formation/resolution of a terminator hairpin (top) and the sequestration/release of the ribosome binding site (RBS, bottom, shown in yellow).

Scheme 1. Novel enzymes, enzymatic mechanisms and cascades in flavonoid synthesis. PAL: phenylalanine ammonia lyase; C4H: cinnamate 4-hydroxylase; PTAL: bifunctional phenylalanine/tyrosine ammonia lyase; CHS: chalcone synthase; CHIL: non-catalytic chalcone isomerase-like protein; CHI: chalcone isomerase; Fcr: flavanone- and flavanonol-cleaving reductase; OMT: O-methyltransferase; HMT: halide methyltransferase; PT: aromatic prenyltransferase; GT: glycosyltransferase.

Scheme 2. Novel enzymes, enzymatic mechanisms and cascades for the synthesis of benzylisoquinoline alkaloids and related tetrahydroisoquinolines. NCS: norcoclaurine synthase; BBE: berberine bridge enzyme; STORR: (S)- to (R)-reticuline epimerase; SalSyn: salutaridine synthase; TfNCS: norcoclaurine synthase from Thalictrum flavum; IR45: imine reductase from Streptomyces aurantiacus; CNMT: coclaurine N-methyltransferase; IRED: imine reductase; MAO-N: monoamine oxidase from Aspergillus niger; CHAO: cyclohexylamine oxidase; DAAO: d-amino acid oxidase; DpkA: piperidine-2-carboxylate reductase from Pseudomonas putida.

Scheme 3. Novel enzymes, enzymatic mechanisms and cascades for the synthesis of Colchicine. NCS: norcoclaurine synthase; P450: cytochrome P450 monooxygenase.

Scheme 4. Novel enzymes, enzymatic mechanisms and cascades for the synthesis of monoterpenoid indole alkaloids. STR: strictosidine synthase.

Scheme 5. Novel enzymes, enzymatic mechanisms and cascades for the synthesis of terpenoids. MEP: methylerythritol phosphate; MVA: mevalonate.

Scheme 6. Novel enzymes, enzymatic mechanisms and cascades for the synthesis of oxygenated plant triterpenes. tHMGR: feedback-insensitive version of HMG-CoA reductase; P450: cytochrome P450 monooxygenase.

Scheme 7. Novel enzymes, enzymatic mechanisms and cascades for the synthesis of Napyradiomycins. GPP: geranyl diphosphate; DMAPP: dimethylallyl diphosphate; NapH1, H3, and H9: vanadium-dependent haloperoxidases; NapT8 and T9: aromatic prenyltransferases.

Scheme 8. Steroidal precursors and APIs. (A) The biosynthesis of steroids through the MEV pathway leads to precursors like lanosterol, cycloartenol, and cholesterol, an important intermediate towards BAs and sex hormones. (B) Cortisol production from DCA involving fermentations with A. niger, R. arrhizus, and 11 chemical steps in total or from glucose through a heterologous pathway mimicking human steroid biosynthesis. DWF5: 7-dehydrocholesterol reductase. (C) Progesterone and testosterone are important APIs; the latter can be hydroxylated by P450BM3 variants in the positions 2, 7, and 16 (shades of blue), for example. Regiospecificity given in parentheses for KSA-1, KSA-14, and LG-23; stereospecificity for WIFI-WC and WWV-QRS. (D) OleP wild-type (OleP-WT) exclusively yields MDCA from LCA by 6β-hydroxylation. The triple mutant OleP-QAG yields the 7β-product UDCA, a reaction new to the biocatalytic toolbox of single enzymes. Important steroid substrates are numbered and modifications in target products indicated. Structures of BA precursors are highlighted in orange, products in blue, key enzymes in purple and accessory proteins in green.

Fig. 4. Traditional and updated assembly-line polyketide synthase module definitions. (A) Four different perspectives on the venemycin assembly-line PKS. (1) The synthase can be divided into two polypeptide chains called VemG (232 kDa) and VemH (140 kDa). (2) The PKS can also be viewed in terms of its domains. The adenylation domain (A) accepts the 3,5-dihydroxybenzoyl starter unit. The inactive ketoreductase domain (KR⁰) probably plays a structural role. The ketosynthase (KS), acyltransferase (AT) and acyl carrier protein (small circle) domains are the core components of PKSs. The thioesterase domain finally releases the polyketide from the synthase, often by cyclisation to form a macrolactone. (3) In the traditional view, the module boundaries are the N-terminus of the ketosynthase domain and the C-terminus of the acyl carrier protein domain. (4) In the updated definition, modules end at the C-termini of ketosynthase domains, reflecting the evolutionary co-migration of domains. (B) The venemycin and pikromycin assembly lines depicted using the new module definitions. The assembly-line steps and products are also shown. The pikromycin assembly line additionally includes dehydratase (DH) and enoylreductase (ER) domains. Note that modules can be split over different polypeptide chains. For example, Vem Mod2 is split between the VemG and VemH proteins. (C) The functions of the different catalytic domains exemplified by Pik Mod5, which has a full set of reductive domains. The domains are represented by spheres coloured by module as in (B). The process starts with a tetraketide intermediate attached to an acyl carrier protein, which is transferred to a cysteine residue on the ketosynthase domain. An acyltransferase domain loads the downstream ACP with an acyl-CoA-derived extender unit, in this case methylmalonyl-CoA. The ketosynthase catalyses decarboxylative condensation of the tetraketide intermediate and the extender unit to form an ACP-linked pentaketide intermediate. This intermediate is then subjected to reductive reactions by the ketoreductase, dehydratase, and enoylreductase domains (the carbonyl subjected to reduction is coloured green). All three reactions are optional so that other modules may stop at either the β-keto, β-hydroxy, or α,β-alkene intermediates. The resulting ACP-linked intermediate is substrate to either the next KS domain or the terminal thioesterase, which usually results in cyclisation. This figure was simplified and redrawn from the Miyazawa et al. and Smith et al.

Fig. 5. Hybrid polyketide synthases constructed using both the updated and the traditional module definitions. (A) The products of three hybrid assembly lines. (B) Three assembly lines constructed using the updated module definition are over an order of magnitude more productive (red numbers) than (C) assembly lines constructed using the traditional modules. Despite the success of assembly lines based on the new module definitions, the native VemG-VemH assembly line is still more than double as active (36 min⁻¹). The domain abbreviations are as for Fig. 4 and again the acyl carrier proteins are represented by small circles. This figure was simplified and redrawn from Miyazawa et al.

Scheme 9. De novo enzymatic synthesis of islatravir. GOase: galactose oxidase; PanK: pantothenate kinase; DERA: deoxyribose 5-phosphate aldolase; PPM: phosphopentomutase; PNP: purine nucleoside phosphorylase; HRP: horseradish peroxidase; AcK: acetate kinase; SP: sucrose phosphorylase. Starting materials are highlighted in orange. The product is highlighted in blue. Key enzymes in synthetic routes are highlighted in purple. Auxiliary enzymes are highlighted in green.

Scheme 10. Enzymatic total synthesis of cannabinoids in a cell-free system. ThiM: hydroxyethylthiazole kinase; IPK: isopentenyl kinase; IDI: isopentyl diphosphate isomerase; GPPS: geranyl pyrophosphate synhetase; AAE3: acyl activating enzyme 3; ADK: adenylate kinase; MdcA: malonate decarboxylase α subunit; PTA: phosphotransacetylase; AckA: acetate kinase; OLS: olivetol synthase; OAC: olivetolic acid cyclase; CBGA synthase: cannabigerolic acid synthase. Starting materials are highlighted in orange. The products are highlighted in blue. Key enzymes in this synthetic route are highlighted in purple. Auxiliary enzymes are highlighted in green.

Scheme 11. Enzymatic total synthesis of ikarugamycin. AckA: acetate kinase; Pta: phosphotransacetylase; MatB: malonyl-CoA synthetase. Starting materials are highlighted in orange. The product is highlighted in blue. Key enzymes in this synthetic route are highlighted in purple. Auxiliary enzymes are highlighted in green.

Scheme 12. Enzymatic total synthesis of kainic acid. KabA: N-prenyltransferase; KabC: α-ketoglutarate-dependent dioxygenase; αKG: α-ketoglutarate. Starting materials are highlighted in orange. The product is highlighted in blue. Key enzymes in this synthetic route are highlighted in purple.

Scheme 13. Enzymatic total synthesis of enterocin and wailupemycin. Starting materials are highlighted in orange. The products are highlighted in blue. Key enzymes in this synthetic route are highlighted in purple.

Scheme 14. Enzymatic total synthesis of the meroterpenoids merochlorin A and B. Mcl17: type III polyketide synthase; Mcl22: prenyl diphosphate synthase; Mcl23: aromatic prenyltransferase; Mcl24: vanadium-dependent chloroperoxidase. Starting materials are highlighted in orange. The products are highlighted in blue. Key enzymes in this synthetic route are highlighted in purple.

Scheme 15. Enzymatic total synthesis of hyoscyamine and scopolamine in yeast. AsADC: arginine decarboxylase; Car1: arginase; speB: agmatine ureohydrolase; Spe1: ornithine decarboxylase; Fms1: polyamine oxidase; AbPMT1 and DsPMT1: N-methyltransferases; DmMPO1^ΔC-PTS1: engineered N-methylputrescine oxidase; AbPYKS: pyrrolidine ketide synthase; AbCYP82M3: tropinone synthase; AtATR1: cytochrome P450 reductase; DsTR1: tropinone reductase 1; Aro8 and Aro9: aromatic aminotransferases; WfPPR: phenylpyruvate reductase; AbUGT: UDP-glucosyltransferase; DsRed-AbLS: littorine synthase; AbCYP82M3: tropinone synthase; AtATR1: cytochrome P450 reductase; AbCYP80F1: littorine mutase; DsHDH: hyoscyamine dehydrogenase; DsH6H: hyoscyamine 6β-hydroxylase/dioxygenase. Starting materials are highlighted in orange. The products are highlighted in blue. Key enzymes in this synthetic route are highlighted in purple.

Scheme 16. Light-induced natural and promiscuous enzymatic transformations. Fatty acid photodecarboxylase (A) is the only natural photoenzyme employed for biotransformations. The other examples (B–G) utilize the photophysical capabilities of nicotinamide and flavin cofactors to generate radical species (key intermediates are shown in the green boxes) as reactive intermediates.

Scheme 17. Cascade reactions combining photocatalytic and enzymatic steps. (A) Photocatalytic oxyfunctionalization generates the intermediate which is alkylated by a promiscuous lipase. (B) The photocatalyst generates a long-lived radical in the reaction solution, which is converted enzymatically. (C) A complex organo- and photocatalytic cycle allows racemization and thus to set two stereocentres in the enzymatic step.

Dong Yi

Thomas Bayer

Christoffel P. S. Badenhorst

Shuke Wu

Mark Doerr

Matthias Höhne

Uwe T. Bornscheuer