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| Open AccessProgrammable synthetic cell networks regulated by tuneable reaction rates
Our understanding of how compartmentalisation and intercellular communication can tune enzyme reactions is still in its infancy. Here, the authors show that multi-enzyme reactions within semi-permeable compartments have distinct properties compared to reactions in buffer solution.
- Adrian Zambrano
- , Giorgio Fracasso
- & T-Y. Dora Tang
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Article
| Open AccessHeat-fueled enzymatic cascade for selective oxyfunctionalization of hydrocarbons
Thermoelectric materials enable us to convert heat into electricity, but their application has been limited to high-temperature heat sources. Here, the authors show the direct conversion of low-grade waste heat into chemical energy via combining thermoelectric materials with biocatalysts below 100 °C.
- Jaeho Yoon
- , Hanhwi Jang
- & Chan Beum Park
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Article
| Open AccessEngineering substrate specificity of HAD phosphatases and multienzyme systems development for the thermodynamic-driven manufacturing sugars
Haloacid dehalogenase-like phosphatases are widespread across all domains of life and play a crucial role in the regulation of levels of sugar phosphate metabolites in cells. The authors report on the structure-guided engineering of phosphatases for dedicated substrate specificity for the conversion of sucrose and starch into fructose and mannose.
- Chaoyu Tian
- , Jiangang Yang
- & Yanhe Ma
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Article
| Open AccessComplete biosynthetic pathway to the antidiabetic drug acarbose
The market demand for acarbose, a drug used for treatment of patients affected by type-2 diabetes, has increased. In this article, the authors report the acarbose complete biosynthetic pathway, clarifying previously unknown steps and identifying a pseudoglycosyltransferase enzyme, AcbS, a homologue of AcbI that catalyzes the formation of a non-glycosidic C-N bond.
- Takeshi Tsunoda
- , Arash Samadi
- & Taifo Mahmud
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Article
| Open AccessControlling synthetic membraneless organelles by a red-light-dependent singlet oxygen-generating protein
Membraneless organelles play vital cellular roles, and control over their formation and state could have varied applications. Here, the authors develop photoresponsive synthetic condensates whose activity can be controlled through the use of light to trigger liquid-to-solid phase transition.
- Manjia Li
- , Byung Min Park
- & Fei Sun
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Article
| Open AccessArtificially sporulated Escherichia coli cells as a robust cell factory for interfacial biocatalysis
Whole-cell biocatalysis is of interest for the efficient production of a range of products but cell stability can be an issue. Here, the authors turn recombinant enzyme-producing E. coli into artificial spores to add protection from UV radiation, heating, and organic solvents by coating the cells with polydopamine to create robust cell factories.
- Zhiyong Sun
- , René Hübner
- & Changzhu Wu
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Article
| Open AccessOrder-of-magnitude enhancement in photocurrent generation of Synechocystis sp. PCC 6803 by outer membrane deprivation
The low extracellular electron transfer activity hampers the application of cyanobacteria in biophotovoltaics. Here, the authors report an order-of-magnitude enhancement in photocurrent generation of the cyanobacterium by deprivation of the outer cell membrane.
- Shoko Kusama
- , Seiji Kojima
- & Shuji Nakanishi
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Article
| Open AccessCell-free prototyping enables implementation of optimized reverse β-oxidation pathways in heterotrophic and autotrophic bacteria
An attractive route for carbon-negative synthesis of biochemical products is the reverse β-oxidation pathway coupled to the Wood-Ljungdahl pathway. Here the authors use a high-throughput in vitro prototyping workflow to screen 762 unique pathway combinations using cell-free extracts tailored for r-BOX to identify enzyme sets for enhanced product selectivity.
- Bastian Vögeli
- , Luca Schulz
- & Michael C. Jewett
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Article
| Open AccessKinetically restrained oxygen reduction to hydrogen peroxide with nearly 100% selectivity
The electrocatalytic oxygen reduction reaction has been investigated in recent years for the production of H2O2 from O2. In this article, the authors report a single-atom rhodium catalyst, based on the Flavin-dependent oxidase, for this transformation.
- Jinxing Chen
- , Qian Ma
- & Shaojun Dong
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Article
| Open AccessDiscovery and characterization of a terpene biosynthetic pathway featuring a norbornene-forming Diels-Alderase
Pericyclase enzymes are an expanding family of enzymes. Here, the authors identify the norbornene synthase SdnG, a pericyclase for the intramolecular Diels-Alder reaction between a cyclopentadiene and an olefinic dienophile to form the sordaricin norbornene structure, and reconstitute the sordaricin biosynthesis.
- Zuodong Sun
- , Cooper S. Jamieson
- & Yi Tang
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Article
| Open AccessRationally engineering santalene synthase to readjust the component ratio of sandalwood oil
Controlling the component ratios of plant essential oils is challenging in their heterologous bioproduction. Here, the authors combine metabolic and enzymatic engineering strategies to achieve the production of sandalwood oil with a desirable component ratio in baker’s yeast.
- Wenlong Zha
- , Fan Zhang
- & Jiachen Zi
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Article
| Open AccessCascade reaction networks within audible sound induced transient domains in a solution
It is difficult to achieve spatiotemporal control over chemical cascade reactions. Here, the authors report on the generation of transient domains in an aqueous medium using sound induced liquid vibrations allowing for control of chemical gradients and patterns and use this to pattern nanoparticles in hydrogels.
- Prabhu Dhasaiyan
- , Tanwistha Ghosh
- & Kimoon Kim
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Article
| Open AccessRadical SAM-dependent ether crosslink in daropeptide biosynthesis
Darobactin is a ribosomally synthesized and post-translationally modified peptide featuring a unique scaffold and potent activity against Gram-negative bacteria. Here, the authors identify darobactin synthase DarE as responsible for the bicyclic scaffold formation and propose the name daropeptide for this growing class of enzymes.
- Sijia Guo
- , Shu Wang
- & Qi Zhang
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Article
| Open AccessDeletion and tandem duplications of biosynthetic genes drive the diversity of triterpenoids in Aralia elata
Unlike Panax species, which can produce dammarane-type saponins, Aralia elata can only synthesize oleananetype saponins. Here, the authors reveal that the loss of the dammaranediol synthase-encoding gene and tandem duplication of triterpene biosynthetic genes drive structural divergences of saponins between the two genera.
- Yu Wang
- , He Zhang
- & Yuhua Li
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Article
| Open AccessCooperative catalysis by a single-atom enzyme-metal complex
Single atom catalysts have been described for efficient and selective metal catalysis, while enzymes have been known for their recognition and binding. In this manuscript, the authors develop a photochemical method to combine the two platforms in one, and demonstrate it by anchoring Pd atoms on Candida Antarctic lipase B, for highly efficient alkyl-alkyl cross-coupling reactions.
- Xiaoyang Li
- , Yufei Cao
- & Jun Ge
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Article
| Open AccessNanoemulsion-directed growth of MOFs with versatile architectures for the heterogeneous regeneration of coenzymes
Controlling the structure of hierarchical metal-organic frameworks via soft template remains a challenge. Here, the authors report a salt-assisted nanoemulsion-guided strategy to achieve continuous structure transition of hierarchical Zr-based MOFs.
- Ke Li
- , Yucheng Zhao
- & Jinlou Gu
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Article
| Open AccessMetal-responsive regulation of enzyme catalysis using genetically encoded chemical switches
Dynamic control over protein function is a central challenge in synthetic biology. Here the authors present an integrated computational and experimental workflow for engineering reversible protein switches; metal-chelating unnatural amino acids genetically encoded into two conformationally dynamic enzymes to yield robust switches.
- Yasmine S. Zubi
- , Kosuke Seki
- & Jared C. Lewis
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Article
| Open AccessHarnessing phosphonate antibiotics argolaphos biosynthesis enables a synthetic biology-based green synthesis of glyphosate
Current commercialized production processes of glyphosate are generally associated with manufacturing hazards and toxic wastes. Here, to address the problems, the authors report the bioproduction of aminomethylphosphonate (AMP) and a chemical process for the conversion of AMP to glyphosate.
- Leixia Chu
- , Xiaoxia Luo
- & Jiangtao Gao
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Article
| Open AccessSingle amino acid bionanozyme for environmental remediation
In the context of enviromental applications, refining enzymes into more minimalist structures could ease production costs, improve stability, and improve reusability. Here, the authors report a single amino acid bionanozyme that can catalyze the rapid oxidation of environmentally toxic phenolic contaminates and serves as a tool to detect biologically important neurotransmitters similar to the laccase enzyme.
- Pandeeswar Makam
- , Sharma S. R. K. C. Yamijala
- & Ehud Gazit
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Article
| Open AccessMechanism and biomass association of glucuronoyl esterase: an α/β hydrolase with potential in biomass conversion
Zong and coworkers combine computational and experimental methods to decipher in detail the mechanism of action of glucuronoyl esterases, enzymes with significant biotechnological potential for decoupling lignin from polysaccharides in biomass.
- Zhiyou Zong
- , Scott Mazurkewich
- & Leila Lo Leggio
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Article
| Open AccessA zirconium metal-organic framework with SOC topological net for catalytic peptide bond hydrolysis
Developing efficient and reusable artificial catalysts for peptide bond hydrolysis is challenging. This work presents the catalytic properties of a Zr-MOF, MIP-201, which features excellent catalytic activity and selectivity, good condition tolerance, and exceptional recycling ability.
- Sujing Wang
- , Hong Giang T. Ly
- & Christian Serre
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Article
| Open AccessBiocatalysed synthesis planning using data-driven learning
As of now, only rule-based systems support retrosynthetic planning using biocatalysis, while initial data-driven approaches are limited to forward predictions. Here, the authors extend the data-driven forward reaction as well as retrosynthetic pathway prediction models based on the Molecular Transformer architecture to biocatalysis.
- Daniel Probst
- , Matteo Manica
- & Teodoro Laino
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Article
| Open AccessData-informed discovery of hydrolytic nanozymes
Developing hydrolytic nanozymes remains challenging. Here the authors present a rational methodology to design hydrolytic nanozyme by developing a data-informed strategy to screen and identify potential scaffold and active sites of hydrolase-like nanozyme.
- Sirong Li
- , Zijun Zhou
- & Hui Wei
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Article
| Open AccessDroplet-based screening of phosphate transfer catalysis reveals how epistasis shapes MAP kinase interactions with substrates
Here, the authors use a droplet-based screen for phosphate transfer catalysis, testing variants of the human protein kinase MKK1 for its ability to activate its downstream target ERK2. Data reveal a flexible motif in the MKK1 docking domain that promotes efficient activation of ERK2, and suggest epistasis between the residues within that sequence.
- Remkes A. Scheele
- , Laurens H. Lindenburg
- & Florian Hollfelder
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Article
| Open AccessMerging enzymes with chemocatalysis for amide bond synthesis
Proteins, other metabolites and many valuable synthetic products contain amide bonds and there is a need for more sustainable amide synthesis routes. Here the authors show an integrated next generation multi-catalytic system, merging nitrile hydratase enzymes with a Cu-catalysed N-arylation reaction in a single reaction vessel, for the construction of ubiquitous amide bonds.
- Luis Bering
- , Elliott J. Craven
- & Jason Micklefield
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Article
| Open AccessAlgorithm-aided engineering of aliphatic halogenase WelO5* for the asymmetric late-stage functionalization of soraphens
The late-stage functionalization of unactivated carbon–hydrogen bonds is a difficult but important task, which has been met with promising but limited success through synthetic organic chemistry. Here the authors use machine learning to engineer WelO5* halogenase variants, which led to regioselective chlorination of inert C–H bonds on a representative polyketide that is a non-natural substrate for the enzyme.
- Johannes Büchler
- , Sumire Honda Malca
- & Rebecca Buller
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Article
| Open AccessHierarchically encapsulating enzymes with multi-shelled metal-organic frameworks for tandem biocatalytic reactions
Mimicking multi-enzyme catalytic cascades in natural systems with spatial organization in confined structures is gaining increasing attention in the emerging field of systems chemistry. Here, the authors demonstrate that multi-shelled metal-organic frameworks can be used as a hierarchical scaffold to spatially organize enzymes on nanoscale to enhance cascade catalytic efficiency.
- Tiantian Man
- , Caixia Xu
- & Li Li
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Article
| Open AccessCatalytic flexibility of rice glycosyltransferase OsUGT91C1 for the production of palatable steviol glycosides
Steviol glycosides from the plant Stevia rebaudiana are already used as lowcalorie sweeteners, but the most abundant naturally occurring compounds have a bitter aftertaste. Here, the authors characterize and engineer rice glycosyltransferase OsUGT91C1 to facilitate the large-scale production of naturally rare but palatable glycosides Reb D and Reb M
- Jinzhu Zhang
- , Minghai Tang
- & Wei Cheng
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Article
| Open AccessVersatile selective evolutionary pressure using synthetic defect in universal metabolism
Rational design of enzymes with new or improved properties is rarely straightforward, and artificial selection pressure approaches that link an improvement in the target to cell growth are an alternative. Here, the authors show that diverse enzymes sharing the ubiquitous cofactor NAD(P)+ can substitute for defective NAD+ regeneration, representing a very broadly-applicable artificial selection.
- Lara Sellés Vidal
- , James W. Murray
- & John T. Heap
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Article
| Open AccessEfficient biosynthesis of nucleoside cytokinin angustmycin A containing an unusual sugar system
Angustmycin A is a nucleoside antibiotic having anti-mycobacterial and cytokinin activities. Here, the authors report the whole pathway leading to angustmycin A biosynthesis in Streptomyces and achieve the heterologous production of angustmycin A in E. coli.
- Le Yu
- , Wenting Zhou
- & Wenqing Chen
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Article
| Open AccessA modular toolbox to generate complex polymeric ubiquitin architectures using orthogonal sortase enzymes
Ubiquitin (Ub) and Ub-like modifiers (Ubls) can form chains of various topologies, but preparing defined chains for functional studies remains challenging. Here, the authors develop chemoenzymatic tools to tailormake Ub/Ubl chains and study the involvement of specific Ub/SUMO chains in DNA repair.
- Maximilian Fottner
- , Maria Weyh
- & Kathrin Lang
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Article
| Open AccessDe novo biosynthesis of bioactive isoflavonoids by engineered yeast cell factories
Isoflavonoids are a class of industrially important plant natural products, but their low abundance and structural complexity limits their availability. Here, the authors engineer Saccharomyces cerevisiae metabolism to become a platform for efficient production of daidzein which is core chemical scaffold for isoflavonoid biosynthesis, and show its application for production of bioactive glucosides from glucose.
- Quanli Liu
- , Yi Liu
- & Jens Nielsen
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Article
| Open AccessThiocysteine lyases as polyketide synthase domains installing hydropersulfide into natural products and a hydropersulfide methyltransferase
Enzymes installing an intact hydropersulfide (-SSH) group into natural products have so far not been identified. Here, the authors report the characterization of an S-adenosyl methionine-dependent hydropersulfide methyltransferase (GnmP) for guangnanmycin biosynthesis, and identification of three SH domains within several NRPS-PKS assembly lines as thiocysteine lyases.
- Song Meng
- , Andrew D. Steele
- & Ben Shen
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Article
| Open AccessDirected evolution of prenylated FMN-dependent Fdc supports efficient in vivo isobutene production
Isobutene is a high value gaseous alkene that is widely used as fuel additive and a chemical building block. Here, the authors report an alternative pathway for isobutene bioproduction by directed evolution of prenylated FMN-dependent ferulic acid decarboxylase.
- Annica Saaret
- , Benoît Villiers
- & David Leys
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Article
| Open AccessA guanidine-degrading enzyme controls genomic stability of ethylene-producing cyanobacteria
The metabolic pathways used by microbes to degrade guanidine or mitigate its toxicity remain unclear. Here, the authors report a guanidine degrading enzyme that controls genomic stability of ethylene producing cyanobacterial strains.
- Bo Wang
- , Yao Xu
- & Jianping Yu
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Article
| Open AccessToward scalable biocatalytic conversion of 5-hydroxymethylfurfural by galactose oxidase using coordinated reaction and enzyme engineering
5-Hydroxymethylfurfural (HMF) can be transformed to a range of industrially useful derivatives, such as 2,5-diformylfuran (DFF), but the reactions needed for efficient industrial production are hindered by several issues. Here, the authors perform reaction and enzyme engineering resulting in a galactose oxidase variant with high activity towards HMF, improved oxygen binding and high productivity.
- William R. Birmingham
- , Asbjørn Toftgaard Pedersen
- & Nicholas J. Turner
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Article
| Open AccessIntegration of photocatalytic and dark-operating catalytic biomimetic transformations through DNA-based constitutional dynamic networks
Nucleic acid-based constitutional dynamic networks (CDNs) enable control of various catalytic processes, but it is challenging to achieve intercommunication between different CDNs and by that mimic complex cell biology networks. Here, the authors report two CDNs that control the integration of photochemical and dark-operating processes, and show their intercommunication afforded by environmental components.
- Chen Wang
- , Michael P. O’Hagan
- & Itamar Willner
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Article
| Open AccessLight-driven decarboxylative deuteration enabled by a divergently engineered photodecarboxylase
The chemical processes for the selective incorporation of deuterium into small molecules, of interest to organic and medicinal chemistry, are well established, while the enzymatic methods remain underdeveloped. Here, the authors use an enzymatic approach employing Chlorella variabilis NC64A photodecarboxylase that catalyses decarboxylative deuteration of various carboxylic acids with D2O, and identify enzyme variants that can employ substrates with different chain length acids.
- Jian Xu
- , Jiajie Fan
- & Qi Wu
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Article
| Open AccessPhysical constraints and functional plasticity of cellulases
Enzyme reactions at interfaces are common in both Nature and industrial applications but no general kinetic framework exists for interfacial enzymes. Here, the authors kinetically characterize 83 cellulases and identify a scaling relationship between ligand binding strength and maximal turnover, a so-called linear free energy relationship, which may help rationalize cellulolytic mechanisms and guide the selection of technical enzymes.
- Jeppe Kari
- , Gustavo A. Molina
- & Peter Westh
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Article
| Open AccessMachine learning differentiates enzymatic and non-enzymatic metals in proteins
The authors generate the largest structural dataset of enzymatic and non-enzymatic metalloprotein sites to date. They use this dataset to train a decision-tree ensemble machine learning algorithm that allows them to distinguish between catalytic and non-catalytic metal sites. The computational model described here could also be useful for the identification of new enzymatic mechanisms and de novo enzyme design.
- Ryan Feehan
- , Meghan W. Franklin
- & Joanna S. G. Slusky
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Article
| Open AccessFlavin-dependent halogenases catalyze enantioselective olefin halocyclization
Catalytic enantioselective halocyclization of alkenes is an important bond forming tool and a key step in natural product biosynthesis, but so far no examples of the enzymatic counterpart of this reaction on simple achiral olefins have been reported. Here, the authors describe examples of engineered flavin-dependent halogenases that catalyze halolactonization of olefins with high enantioselectivity and near-native catalytic activity.
- Dibyendu Mondal
- , Brian F. Fisher
- & Jared C. Lewis
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Article
| Open AccessBiased cytochrome P450-mediated metabolism via small-molecule ligands binding P450 oxidoreductase
P450 oxidoreductase (POR) selectively activates numerous cytochromes P450 (CYP), crucial for metabolism of drugs, steroids and xenobiotics and natural product biosynthesis. Here, the authors identify ligands that bind POR and bias its specificity towards CYP redox partners, activating distinct metabolic cascades in cells.
- Simon Bo Jensen
- , Sara Thodberg
- & Nikos S. Hatzakis
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Article
| Open AccessDirect 1,3-butadiene biosynthesis in Escherichia coli via a tailored ferulic acid decarboxylase mutant
Microorganisms cannot naturally produce 1,3-butadiene, an important monomer for synthetic rubber and plastic production, using glucose as carbon source. Here, the authors combine the cis,cis-muconic acid-production pathway and tailor ferulic acid decarboxylase mutations to achieve its production in E. coli.
- Yutaro Mori
- , Shuhei Noda
- & Akihiko Kondo
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Article
| Open AccessDiscovery of fungal oligosaccharide-oxidising flavo-enzymes with previously unknown substrates, redox-activity profiles and interplay with LPMOs
Microbial oxidoreductases are key in biomass breakdown. Here, the authors expand the specificity and redox scope within fungal auxiliary activity 7 family (AA7) enzymes and show that AA7 oligosaccharide dehydrogenases can directly fuel cellulose degradation by lytic polysaccharide monooxygenases.
- Majid Haddad Momeni
- , Folmer Fredslund
- & Maher Abou Hachem
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Article
| Open AccessCreating enzymes and self-sufficient cells for biosynthesis of the non-natural cofactor nicotinamide cytosine dinucleotide
Nicotinamide cytosine dinucleotide is an NAD mimic that can be used to mediate redox reactions. Here the authors design an NCD synthetase for the intracellular production of NCD.
- Xueying Wang
- , Yanbin Feng
- & Zongbao K. Zhao
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Article
| Open AccessA [6+4]-cycloaddition adduct is the biosynthetic intermediate in streptoseomycin biosynthesis
Streptoseomycin is a potent antibiotic that contains a pentacyclic 5/14/10/6/6 ring system. Here, the authors report the enzymatic and non-enzymatic steps of the downstream modification of streptoseomycin biosynthesis and show a [6 + 4]-cycloaddition adduct as an unexpected biosynthetic intermediate.
- Kai Biao Wang
- , Wen Wang
- & Hui Ming Ge
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Article
| Open AccessPervasive cooperative mutational effects on multiple catalytic enzyme traits emerge via long-range conformational dynamics
Connecting conformational dynamics and epistasis has so far been limited to a few proteins and a single fitness trait. Here, the authors provide evidence of positive epistasis on multiple catalytic traits in the evolution and dynamics of engineered cytochrome P450 monooxygenase, offering insights for in silico protein design.
- Carlos G. Acevedo-Rocha
- , Aitao Li
- & Manfred T. Reetz
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Article
| Open AccessVersatility in acyltransferase activity completes chicoric acid biosynthesis in purple coneflower
Biosynthetic pathway of chicoric acid in purple coneflower has not been fully elucidated, though the compound has been shown to have potential health benefits. Here, the authors report the involvement of both BAHD and SCPL acyltransferases in its biosynthesis and show the pathway is unique to Echinacea species.
- Rao Fu
- , Pingyu Zhang
- & Yang Zhang
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Article
| Open AccessActive-site loop variations adjust activity and selectivity of the cumene dioxygenase
Active-site loops are important for catalytic properties of enzymes, but challenging to engineer due to their high flexibility and diversity. Here, the authors identify and engineer hot-spots in the loops of cumene dioxygenase, obtain variants with changed activity, regio- and enantioselectivity, and present a Linker In Loop Insertion approach for loop modification.
- Peter M. Heinemann
- , Daniel Armbruster
- & Bernhard Hauer