Rubisco Function, Evolution, and Engineering.

Carbon fixation is the process by which CO2 is converted from a gas into biomass. The Calvin-Benson-Bassham cycle (CBB) is the dominant carbon-consuming pathway on Earth, driving >99.5% of the ∼120 billion tons of carbon that are converted to sugar by plants, algae, and cyanobacteria. The carboxylase enzyme in the CBB, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco), fixes one CO2 molecule per turn of the cycle into bioavailable sugars. Despite being critical to the assimilation of carbon, rubisco's kinetic rate is not very fast, limiting flux through the pathway. This bottleneck presents a paradox: Why has rubisco not evolved to be a better catalyst? Many hypothesize that the catalytic mechanism of rubisco is subject to one or more trade-offs and that rubisco variants have been optimized for their native physiological environment. Here, we review the evolution and biochemistry of rubisco through the lens of structure and mechanism in order to understand what trade-offs limit its improvement. We also review the many attempts to improve rubisco itself and thereby promote plant growth.

Dual film-like organelles enable spatial separation of orthogonal eukaryotic translation.

Engineering new functionality into living eukaryotic systems by enzyme evolution or de novo protein design is a formidable challenge. Cells do not rely exclusively on DNA-based evolution to generate new functionality but often utilize membrane encapsulation or formation of membraneless organelles to separate distinct molecular processes that execute complex operations. Applying this principle and the concept of two-dimensional phase separation, we develop film-like synthetic organelles that support protein translation on the surfaces of various cellular membranes. These sub-resolution synthetic films provide a path to make functionally distinct enzymes within the same cell. We use these film-like organelles to equip eukaryotic cells with dual orthogonal expanded genetic codes that enable the specific reprogramming of distinct translational machineries with single-residue precision. The ability to spatially tune the output of translation within tens of nanometers is not only important for synthetic biology but has implications for understanding the function of membrane-associated protein condensation in cells.

Selective chemical tracking of Dnmt1 catalytic activity in live cells.

Enzymatic methylation of cytosine to 5-methylcytosine in DNA is a fundamental epigenetic mechanism involved in mammalian development and disease. DNA methylation is brought about by collective action of three AdoMet-dependent DNA methyltransferases, whose catalytic interactions and temporal interplay are poorly understood. We used structure-guided engineering of the Dnmt1 methyltransferase to enable catalytic transfer of azide tags onto DNA from a synthetic cofactor analog, Ado-6-azide, in vitro. We then CRISPR-edited the Dnmt1 locus in mouse embryonic stem cells to install the engineered codon, which, following pulse internalization of the Ado-6-azide cofactor by electroporation, permitted selective azide tagging of Dnmt1-specific genomic targets in cellulo. The deposited covalent tags were exploited as "click" handles for reading adjoining sequences and precise genomic mapping of the methylation sites. The proposed approach, Dnmt-TOP-seq, enables high-resolution temporal tracking of the Dnmt1 catalysis in mammalian cells, paving the way to selective studies of other methylation pathways in eukaryotic systems.

Advancing sustainable biotechnology through protein engineering.

The push for industrial sustainability benefits from the use of enzymes as a replacement for traditional chemistry. Biological catalysts, especially those that have been engineered for increased activity, stability, or novel function, and are often greener than alternative chemical approaches. This Review highlights the role of engineered enzymes (and identifies directions for further engineering efforts) in the application areas of greenhouse gas sequestration, fuel production, bioremediation, and degradation of plastic wastes.

Film-like organelles equip cells with multiple genetic codes.

The simultaneous incorporation of distinct noncanonical amino acids into different proteins within eukaryotic cells remains challenging. This new study by Reinkemeierand Lemke demonstrates how 2D phase separation can be used to engineer spatially separated organelles. These film-like organelles translate proteins independently from each other and the canonical genetic code.

Fine-tuning an aromatic ring-hydroxylating oxygenase to degrade high molecular weight polycyclic aromatic hydrocarbon.

Rieske nonheme iron aromatic ring-hydroxylating oxygenases (RHOs) play pivotal roles in determining the substrate preferences of polycyclic aromatic hydrocarbon (PAH) degraders. However, their potential to degrade high molecular weight PAHs (HMW-PAHs) has been relatively unexplored. NarA2B2 is an RHO derived from a thermophilic Hydrogenibacillus sp. strain N12. In this study, we have identified four "hotspot" residues (V236, Y300, W316, and L375) that may hinder the catalytic capacity of NarA2B2 when it comes to HMW-PAHs. By employing structure-guided rational enzyme engineering, we successfully modified NarA2B2, resulting in NarA2B2 variants capable of catalyzing the degradation of six different types of HMW-PAHs, including pyrene, fluoranthene, chrysene, benzo[a]anthracene, benzo[b]fluoranthene, and benzo[a]pyrene. Three representative variants, NarA2B2W316I, NarA2B2Y300F-W316I, and NarA2B2V236A-W316I-L375F, not only maintain their abilities to degrade low-molecular-weight PAHs (LMW-PAHs) but also exhibited 2 to 4 times higher degradation efficiency for HMW-PAHs in comparison to another isozyme, NarAaAb. Computational analysis of the NarA2B2 variants predicts that these modifications alter the size and hydrophobicity of the active site pocket making it more suitable for HMW-PAHs. These findings provide a comprehensive understanding of the relationship between three-dimensional structure and functionality, thereby opening up possibilities for designing improved RHOs that can be more effectively used in the bioremediation of PAHs.

Leveraging intrinsic flexibility to engineer enhanced enzyme catalytic activity.

Dynamic motions of enzymes occurring on a broad range of timescales play a pivotal role in all steps of the reaction pathway, including substrate binding, catalysis, and product release. However, it is unknown whether structural information related to conformational flexibility can be exploited for the directed evolution of enzymes with higher catalytic activity. Here, we show that mutagenesis of residues exclusively located at flexible regions distal to the active site of Homo sapiens kynureninase (HsKYNase) resulted in the isolation of a variant (BF-HsKYNase) in which the rate of the chemical step toward kynurenine was increased by 45-fold. Mechanistic pre­steady-state kinetic analysis of the wild type and the evolved enzyme shed light on the underlying effects of distal mutations (>10 Å from the active site) on the rate-limiting step of the catalytic cycle. Hydrogen-deuterium exchange coupled to mass spectrometry and molecular dynamics simulations revealed that the amino acid substitutions in BF-HsKYNase allosterically affect the flexibility of the pyridoxal-5'-phosphate (PLP) binding pocket, thereby impacting the rate of chemistry, presumably by altering the conformational ensemble and sampling states more favorable to the catalyzed reaction.

Vanillyl alcohol oxidase from Diplodia corticola: Residues Ala420 and Glu466 allow for efficient catalysis of syringyl derivatives.

Vanillyl alcohol oxidases (VAOs) belong to the 4-phenol oxidases family and are found predominantly in lignin-degrading ascomycetes. Systematical investigation of the enzyme family at the sequence level resulted in discovery and characterization of the second recombinantly produced VAO member, DcVAO, from Diplodia corticola. Remarkably high activities for 2,6-substituted substrates like 4-allyl-2,6-dimethoxy-phenol (3.5 ± 0.02 U mg-1) or 4-(hydroxymethyl)-2,6-dimethoxyphenol (6.3 ± 0.5 U mg-1) were observed, which could be attributed to a Phe to Ala exchange in the catalytic center. In order to rationalize this rare substrate preference among VAOs, we resurrected and characterized three ancestral enzymes and performed mutagenesis analyses. The results indicate that a Cys/Glu exchange was required to retain activity for É£-hydroxylations and shifted the acceptance towards benzyl ethers (up to 4.0 ± 0.1 U mg-1). Our findings contribute to the understanding of the functionality of VAO enzyme group, and with DcVAO, we add a new enzyme to the repertoire of ether cleaving biocatalysts.

Efficient Transferase Engineering for SAM Analog Synthesis from Iodoalkanes.

S-Adenosyl-l-methionine (SAM) is an important cosubstrate in various biochemical processes, including selective methyl transfer reactions. Simple methods for the (re)generation of SAM analogs could expand the chemistry accessible with SAM-dependent transferases and go beyond methylation reactions. Here we present an efficient enzyme engineering strategy to synthesize different SAM analogs from "off-the-shelf" iodoalkanes through enzymatic alkylation of S-adenosyl-l-homocysteine (SAH). This was achieved by mutating multiple hydrophobic and structurally dynamic amino acids simultaneously. Combinatorial mutagenesis was guided by the natural amino acid diversity and generated a highly functional mutant library. This approach increased the speed as well as the scale of enzyme engineering by providing a panel of optimized enzymes with orders of magnitude higher activities for multiple substrates in just one round of enzyme engineering. The optimized enzymes exhibit catalytic efficiencies up to 31 M-1 s-1, convert various iodoalkanes, including substrates bearing cyclopropyl or aromatic moieties, and catalyze S-alkylation of SAH with very high stereoselectivities (>99 % de). We further report a high throughput chromatographic screening system for reliable and rapid SAM analog analysis. We believe that the methods and enzymes described herein will further advance the field of selective biocatalytic alkylation chemistry by enabling SAM analog regeneration with "off-the-shelf" reagents.

Engineering of a Malonyl-CoA Ligase for Production of Fluorinated Polyketide Extender Units.

Enzymatic platforms for producing malonyl-CoA-based extender units required for polyketide biosynthesis are often based on malonyl-CoA ligases such as MatB from Rhizobium trifolii and Rhodopseudomonas palustris. However, despite broad interest in the fluorination of polyketides and prior success with engineering MatB homologs, the suitability of MatB for accessing the tertiary substituted fluoromethylmalonyl-CoA needed to produce flurithromycin and solithromycin has not yet been reported. Herein, we report the structure-guided engineering of a MatB homolog to optimize the production of fluoromethylmalonyl-CoA, resulting in a variant with increased conversion and providing a platform to produce a suitable building block mixture for fluorinated macrolide production. Additionally, the mutant demonstrated broad utility for various substituted malonyl-CoAs. The MatB mutant sets the stage to access fluorinated macrolides by coupling it with altered PKS machinery to install fluorinated malonyl-CoA into macrolide scaffolds.

Click, Compute, Create: A Review of Web-based Tools for Enzyme Engineering.

Enzyme engineering, though pivotal across various biotechnological domains, is often plagued by its time-consuming and labor-intensive nature. This review aims to offer an overview of supportive in silico methodologies for this demanding endeavor. Starting from methods to predict protein structures, to classification of their activity and even the discovery of new enzymes we continue with describing tools used to increase thermostability and production yields of selected targets. Subsequently, we discuss computational methods to modulate both, the activity as well as selectivity of enzymes. Last, we present recent approaches based on cutting-edge machine learning methods to redesign enzymes. With exception of the last chapter, there is a strong focus on methods easily accessible via web-interfaces or simple Python-scripts, therefore readily useable for a diverse and broad community.

Biosensor-Guided Engineering of a Baeyer-Villiger Monooxygenase for Aliphatic Ester Production.

Esters are valuable aroma compounds and can be produced enzymatically by Baeyer-Villiger monooxygenases (BVMOs) from (aliphatic) ketone precursors. However, a genetically encoded biosensor system for the assessment of BVMO activity and the detection of reaction products is missing. In this work, we assembled a synthetic enzyme cascade - featuring an esterase, an alcohol dehydrogenase, and LuxAB - in the heterologous host Escherichia coli. Target esters are produced by a BVMO, subsequently cleaved, and the corresponding alcohol oxidized through the artificial pathway. Ultimately, aldehyde products are detected in vivo by LuxAB, a luciferase from Photorhabdus luminescens that emits bioluminescence upon the oxidation of aldehydes to the corresponding carboxylates. This biosensor system greatly accelerated the screening and selection of active BVMO variants from a focused library, omitting commonly used low-throughput chromatographic analysis. Engineered enzymes accepted linear aliphatic ketones such as 2-undecanone and 2-dodecanone and exhibited improved ester formation.

Selective microbial production of lacto-N-fucopentaose I in Escherichia coli using engineered α-1,2-fucosyltransferases.

Lacto-N-fucopentaose I (LNFP I) is the second most abundant fucosylated human milk oligosaccharide (HMO) in breast milk after 2'-fucosyllactose (2'-FL). Studies have reported that LNFP I exhibits antimicrobial activity against group B Streptococcus and antiviral effects against Enterovirus and Norovirus. Microbial production of HMOs by engineered Escherichia coli is an attractive, low-cost process, but few studies have investigated production of long-chain HMOs, including the pentasaccharide LNFP I. LNFP I is synthesized by α1,2-fucosyltransfer reaction to the N-acetylglucosamine moiety of the lacto-N-tetraose skeleton, which is catalyzed by α1,2-fucosyltransferase (α1,2-FucT). However, α1,2-FucTs competitively transfer fucose to lactose, resulting in formation of the byproduct 2'-FL. In this study, we constructed LNFP I-producing strains of E. coli with various α1,2-fucTs, and observed undesired 2'-FL accumulation during fed-batch fermentation, although, in test tube assays, some strains produced LNFP I without 2'-FL. We hypothesized that promiscuous substrate selectivity of α1,2-FucT was responsible for 2'-FL production. Therefore, to decrease the formation of byproduct 2'-FL, we designed 15 variants of FsFucT from Francisella sp. FSC1006 by rational and semi-rational design approaches. Five of these variants of FsFucT surpassed a twofold reduction in 2'-FL production compared with wild-type FsFucT while maintaining comparable levels of LNFP I production. These designs encompassed substitutions in either a loop region of the enzyme (residues 154-171), or in specific residues (Q7, H162, and L164) that influence substrate binding either directly or indirectly. In particular, the E. coli strain that expressed FsFucT_S3 variants, with a substituted loop region (residues 154-171) forming an α-helix structure, achieved an accumulation of 19.6 g/L of LNFP I and 0.04 g/L of 2'-FL, while the E. coli strain expressing the wild-type FsFucT accumulated 12.2 g/L of LNFP I and 5.85 g/L of 2'-FL during Fed-bach fermentation. Therefore, we have successfully demonstrated the selective and efficient production of the pentasaccharide LNFP I without the byproduct 2'-FL by combining protein engineering of α1,2-FucT designed through in silico structural modeling of an α1,2-FucT and docking simulation with various ligands, with metabolic engineering of the host cell.

A growth-based screening strategy for engineering the catalytic activity of an oxygen-sensitive formate dehydrogenase.

Enzyme engineering is a powerful tool for improving or altering the properties of biocatalysts for industrial, research, and therapeutic applications. Fast and accurate screening of variant libraries is often the bottleneck of enzyme engineering and may be overcome by growth-based screening strategies with simple processes to enable high throughput. The currently available growth-based screening strategies have been widely employed for enzymes but not yet for catalytically potent and oxygen-sensitive metalloenzymes. Here, we present a screening system that couples the activity of an oxygen-sensitive formate dehydrogenase to the growth of Escherichia coli. This system relies on the complementation of the E. coli formate hydrogenlyase (FHL) complex by Mo-dependent formate dehydrogenase H (EcFDH-H). Using an EcFDH-H-deficient strain, we demonstrate that growth inhibition by acidic glucose fermentation products can be alleviated by FHL complementation. This allows the identification of catalytically active EcFDH-H variants at a readily measurable cell density readout, reduced handling efforts, and a low risk of oxygen contamination. Furthermore, a good correlation between cell density and formate oxidation activity was established using EcFDH-H variants with variable catalytic activities. As proof of concept, the growth assay was employed to screen a library of 1,032 EcFDH-H variants and reduced the library size to 96 clones. During the subsequent colorimetric screening of these clones, the variant A12G exhibiting an 82.4% enhanced formate oxidation rate was identified. Since many metal-dependent formate dehydrogenases and hydrogenases form functional complexes resembling E. coli FHL, the demonstrated growth-based screening strategy may be adapted to components of such electron-transferring complexes.IMPORTANCEOxygen-sensitive metalloenzymes are highly potent catalysts that allow the reduction of chemically inert substrates such as CO2 and N2 at ambient pressure and temperature and have, therefore, been considered for the sustainable production of biofuels and commodity chemicals such as ammonia, formic acid, and glycine. A proven method to optimize natural enzymes for such applications is enzyme engineering using high-throughput variant library screening. However, most screening methods are incompatible with the oxygen sensitivity of these metalloenzymes and thereby limit their relevance for the development of biosynthetic production processes. A microtiter plate-based assay was developed for the screening of metal-dependent formate dehydrogenase that links the activity of the tested enzyme variant to the growth of the anaerobically grown host cell. The presented work extends the application range of growth-based screening to metalloenzymes and is thereby expected to advance their adoption to biosynthesis applications.

Flexible active-site loops fine-tune substrate specificity of hyperthermophilic metallo-oxidases.

Hyperthermophilic ('superheat-loving') archaea found in high-temperature environments such as Pyrobaculum aerophilum contain multicopper oxidases (MCOs) with remarkable efficiency for oxidizing cuprous and ferrous ions. In this work, directed evolution was used to expand the substrate specificity of P. aerophilum McoP for organic substrates. Six rounds of error-prone PCR and DNA shuffling followed by high-throughput screening lead to the identification of a hit variant with a 220-fold increased efficiency (kcat/Km) than the wild-type for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) without compromising its intrinsic activity for metal ions. The analysis of the X-ray crystal structure reveals four proximal mutations close to the T1Cu active site. One of these mutations is within the 23-residues loop that occludes this site, a distinctive feature of prokaryotic MCOs. The increased flexibility of this loop results in an enlarged tunnel and one additional pocket that facilitates bulky substrate-enzyme interactions. These findings underscore the synergy between mutations that modulate the dynamics of the active-site loop enabling enhanced catalytic function. This study highlights the potential of targeting loops close to the T1Cu for engineering improvements suitable for biotechnological applications.

Discovery and directed evolution of C-C bond formation enzymes for the biosynthesis of ß-hydroxy-α-amino acids and derivatives.

ß-Hydroxy-α-amino acids (ß-HAAs) have extensive applications in the pharmaceutical, chemical synthesis, and food industries. The development of synthetic methodologies aimed at producing optically pure ß-HAAs has been driven by practical applications. Among the various synthetic methods, biocatalytic asymmetric synthesis is considered a sustainable approach due to its capacity to generate two stereogenic centers from simple prochiral precursors in a single step. Therefore, extensive efforts have been made in recent years to search for effective enzymes which enable such biotransformation. This review provides an overview on the discovery and engineering of C-C bond formation enzymes for the biocatalytic synthesis of ß-HAAs. We highlight examples where the use of threonine aldolases, threonine transaldolases, serine hydroxymethyltransferases, α-methylserine aldolases, α-methylserine hydroxymethyltransferases, and engineered alanine racemases facilitated the synthesis of ß-HAAs. Additionally, we discuss the potential future advancements and persistent obstacles in the enzymatic synthesis of ß-HAAs.

Directed Evolution and Immobilization of Lactobacillus brevis Alcohol Dehydrogenase for Chemo-Enzymatic Synthesis of Rivastigmine.

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.

Hotspot Wizard-informed engineering of a hyperthermophilic ß-glucosidase for enhanced enzyme activity at low temperatures.

Hyperthermophilic enzymes serve as an important source of industrial enzymes due to their high thermostability. Unfortunately, most hyperthermophilic enzymes suffer from reduced activity at low temperatures (e.g., ambient temperature), limiting their applicability. In addition, evolving hyperthermophilic enzymes to increase low temperature activity without compromising other desired properties is generally difficult. In the current study, a variant of ß-glucosidase from Pyrococcus furiosus (PfBGL) was engineered to enhance enzyme activity at low temperatures through the construction of a saturation mutagenesis library guided by the HotSpot Wizard analysis, followed by its screening for activity and thermostability. From this library construction and screening, one PfBGL mutant, PfBGL-A4 containing Q214S/A264S/F344I mutations, showed an over twofold increase in ß-glucosidase activity at 25 and 50°C compared to the wild type, without compromising high-temperature activity, thermostability and substrate specificity. Our experimental and computational characterizations suggest that the findings with PfBGL-A4 may be due to the elevation of local conformational flexibility around the active site, while slightly compacting the global protein structure. This study showcases the potential of HotSpot Wizard-informed engineering of hyperthermophilic enzymes and underscores the interplays among temperature, enzyme activity, and conformational flexibility in these enzymes.

Engineering of Rieske dioxygenase variants with improved cis-dihydroxylation activity for benzoates.

Rieske dioxygenases have a long history of being utilized as green chemical tools in the organic synthesis of high-value compounds, due to their capacity to perform the cis-dihydroxylation of a wide variety of aromatic substrates. The practical utility of these enzymes has been hampered however by steric and electronic constraints on their substrate scopes, resulting in limited reactivity with certain substrate classes. Herein, we report the engineering of a widely used member of the Rieske dioxygenase class of enzymes, toluene dioxygenase (TDO), to produce improved variants with greatly increased activity for the cis-dihydroxylation of benzoates. Through rational mutagenesis and screening, TDO variants with substantially improved activity over the wild-type enzyme were identified. Homology modeling, docking studies, molecular dynamics simulations, and substrate tunnel analysis were applied in an effort to elucidate how the identified mutations resulted in improved activity for this polar substrate class. These analyses revealed modification of the substrate tunnel as the likely cause of the improved activity observed with the best-performing enzyme variants.

Combinatorial metabolic engineering of Bacillus subtilis for menaquinone-7 biosynthesis.

Menaquinone-7 (MK-7), a form of vitamin K2, supports bone health and prevents arterial calcification. Microbial fermentation for MK-7 production has attracted widespread attention because of its low cost and short production cycles. However, insufficient substrate supply, unbalanced precursor synthesis, and low catalytic efficiency of key enzymes severely limited the efficiency of MK-7 synthesis. In this study, utilizing Bacillus subtilis BSAT01 (with an initial MK-7 titer of 231.0 mg/L) obtained in our previous study, the glycerol metabolism pathway was first enhanced to increase the 3-deoxy-arabino-heptulonate 7-phosphate (DHAP) supply, which led to an increase in MK-7 titer to 259.7 mg/L. Subsequently, a combination of knockout strategies predicted by the genome-scale metabolic model etiBsu1209 was employed to optimize the central carbon metabolism pathway, and the resulting strain showed an increase in MK-7 production from 259.7 to 318.3 mg/L. Finally, model predictions revealed the methylerythritol phosphate pathway as the major restriction pathway, and the pathway flux was increased by heterologous introduction (Introduction of Dxs derived from Escherichia coli) and fusion expression (End-to-end fusion of two enzymes by a linker peptide), resulting in a strain with a titer of 451.0 mg/L in a shake flask and 474.0 mg/L in a 50-L bioreactor. This study achieved efficient MK-7 synthesis in B. subtilis, laying the foundation for large-scale MK-7 bioproduction.