LongLarf

We demonstrate a novel nCAS9-mutagenic polymerase-based continuous evolution platform for improvement of enzymatic activity that functions at ultra-high throughput. By cycling cells between growth, mutagenesis, and microfluidics-based sorting, we mimic natural evolution but at a pace that is orders of magnitude faster, yielding an alditol oxidase variant with 10.5-fold improved catalytic efficiency for waste product, glycerol as a substrate.

Abstract

Enzymes are highly specific catalysts delivering improved drugs and greener industrial processes. Naturally occurring enzymes must typically be optimized which is often accomplished through directed evolution; however, this is still a labor- and capital-intensive process, due in part to multiple molecular biology steps including DNA extraction, in vitro library generation, transformation, and limited screening throughput. We present an effective and broadly applicable continuous evolution platform that enables controlled exploration of fitness landscape to evolve enzymes at ultrahigh throughput based on direct measurement of enzymatic activity. This drop-based microfluidics platform cycles cells between growth and mutagenesis followed by screening with minimal human intervention, relying on the nCas9 chimera with mutagenesis polymerase to produce in vivo gene diversification using sgRNAs tiled along the gene. We evolve alditol oxidase to change its substrate specificity towards glycerol, turning a waste product into a valuable feedstock. We identify a variant with a 10.5-fold catalytic efficiency.

Publication date: 29 June 2023

Source: Tetrahedron, Volume 140

Author(s): Jixia Fan, Yifan Du, Rongrong Zhao, Qixing Liu, Haifeng Zhou

Publication date: 30 May 2023

Source: Tetrahedron, Volume 138

Author(s): Sanjay Subhash Gaikwad, Shankesh Chandrakant Zyate, Suresh B. Waghmode, Amardeep Ramprasad Jadhao

Nature Reviews Chemistry, Published online: 25 April 2023; doi:10.1038/s41570-023-00497-8

Highly selective and efficient synthesis of primary alcohols from epoxides could be achieved under mild conditions by iron catalysis, this process used formic acid as hydrogen source and was suitable for both alkyl- and aryl-substituted epoxides.

Abstract

A highly selective, efficient and practical method for synthesizing primary alcohols was presented. By using cheap Fe(BF₄)₂ ⋅ 6H₂O and /tris[2-(diphenylphosphino)phenyl]phosphine (L1) as catalysts with formic acid as hydrogen source, a variety of primary alcohols bearing versatile functional groups could be obtained from both alkyl- and aryl-substituted epoxides under mild conditions.

Crystal clear: We evaluate the significance of poly(ethylene terephthalate) (PET) crystallinity on enzymatic degradation rates of six PET hydrolysing enzymes, present a new quantitative assessment of PET crystallinity tolerance of the enzymes, and provide structural comparisons and molecular dynamics simulations of the enzymes to explain their differences in attack mode, lag phase, and enzymatic rate responses to increased PET substrate crystallinity.

Abstract

The rate response of poly(ethylene terephthalate) (PET)-hydrolases to increased substrate crystallinity (X _C) of PET manifests as a rate-lowering effect that varies significantly for different enzymes. Herein, we report the influence of X _C on the product release rate of six thermostable PET-hydrolases. All enzyme reactions displayed a distinctive lag phase until measurable product formation occurred. The duration of the lag phase increased with X _C. The recently discovered PET-hydrolase PHL7 worked efficiently on “amorphous” PET disks (X _C≈10 %), but this enzyme was extremely sensitive to increased X _C, whereas the enzymes LCC_ICCG, LCC, and DuraPETase had higher tolerance to increases in X _C and had activity on PET disks having X _C of 24.4 %. Microscopy revealed that the X _C-tolerant hydrolases generated smooth and more uniform substrate surface erosion than PHL7 during reaction. Structural and molecular dynamics analysis of the PET-hydrolyzing enzymes disclosed that surface electrostatics and enzyme flexibility may account for the observed differences.

Pincer metal organometallic complexes have been widely used as catalysts for a many organictransformations in both industrial and social applications. Pincer metal complexes have extra thermal stability and easy functionalization capability, they were extensively utilized for synthesis of novel catalytic moieties. Recently, the catalysts exhibited excellent behaviour towards various immerging systems, such as the hydrogenation of CO₂, nitriles, amides, esters, olefins and alkynes, which are valuable for industrial production, academic research, and green environment. The main goal of this review was to highlight how pincer metal complexes function in these catalytic hydrogenation reactions, as well as their mechanistic studies.

Abstract

In the last two decades, pincer metal complexes have been highly utilized as catalysts for a variety of organic transformations in both industrial and social applications. Owing to the extraordinary chemical characteristics, such as extra thermal stability and easy functionalization, they were extensively utilized for generating novel catalytic species. Besides, the catalytic activity of these pincer metal complexes was achieved with high atom economy and eco-friendly routes. Recently, the catalysts exhibited excellent behaviour towards various immerging systems, such as the hydrogenation of CO₂, nitriles, amides, esters, olefins and alkynes, which are valuable for industrial production, academic research, and green environment. The main goal of this reviewwas to highlight how pincer metal complexes function in these catalytic hydrogenation reactions, as well as their mechanistic studies. Specifically, we focus on the most updated advancements in hydrogenation in the last few years. Some outlooks and future suggestions for further related work conclude the paper.

Asymmetric phase transfer catalysis constitutes an ever increasingly useful tool to construct chiral skeletons under mild and practical conditions. This Review describes new concise pathways for the synthesis of chiral motifs made possible by the design of new chiral PTC catalysts such as chiral onium salts, chiral phosphate anions and chiral bis-urea hydrogen bond donors.

Abstract

Phase Transfer Catalysis (PTC) is a powerful tool to perform reactions in a practical fashion, both in laboratory and industrial scale. Significant cost savings and major process improvements can be achieved in reactions performed under PTC conditions. In the last few years remarkable results in stereoselective reactions were achieved using chiral, non-racemic quaternary ammonium salts. Moreover, the use of bulky, chiral phosphate anions paired with achiral cations to generate lipophilic ion pairs allowed to design new avenues for the stereoselective construction of important building blocks. Hydrogen bond interactions were also shown to provide new pathways for asymmetric nucleophilic substitutions using insoluble reagents under PTC conditions. This Review will focus on recent advances in developing practical synthetic routes to construct molecules in a stereoselective fashion under PTC conditions.

Publication date: 10 August 2023

Source: Chem, Volume 9, Issue 8

Author(s): Adam D. Moorhouse, Joshua A. Homer, John E. Moses

A bioinspired approach using enzyme electrocatalysis for the efficient direct reduction of CO₂ at low concentrations to formate using Carbonic Anhydrase co-immobilized with Formate Dehydrogenase in a mesoporous indium tin oxide electrode is described.

Abstract

The electrolysis of dilute CO₂ streams suffers from low concentrations of dissolved substrate and its rapid depletion at the electrolyte-electrocatalyst interface. These limitations require first energy-intensive CO₂ capture and concentration, before electrolyzers can achieve acceptable performances. For direct electrocatalytic CO₂ reduction from low-concentration sources, we introduce a strategy that mimics the carboxysome in cyanobacteria by utilizing microcompartments with nanoconfined enzymes in a porous electrode. A carbonic anhydrase accelerates CO₂ hydration kinetics and minimizes substrate depletion by making all dissolved carbon available for utilization, while a highly efficient formate dehydrogenase reduces CO₂ cleanly to formate; down to even atmospheric concentrations of CO₂. This bio-inspired concept demonstrates that the carboxysome provides a viable blueprint for the reduction of low-concentration CO₂ streams to chemicals by using all forms of dissolved carbon.

The electronic nature of the Pd catalyst changes from π-philicity to oxophilicity during the catalytic cycle, and this change plays an essential role in controlling the chemoselectivity of the cyclization reactions.

Abstract

It is well documented in the literature that 1,6-enynes are cyclized using PhI(OAc)₂ (PIDA) in the presence of Pd(OAc)₂ as a catalyst to yield cyclopropyl ketones. In contrast, it has been reported that when 1,6-enynes are substituted by a hydroxy group at the α-position to the alkyne, the chemoselectivity of the cyclization reaction is altered, and polycyclic oxa-heterocycles are formed. This suggests that the hydroxy substituent plays a crucial role in changing the mechanism of the reaction. The aim of this study is to use density functional theory (DFT) calculations at the SMD/M06-D3/def2TZVP//SMD/M06/SDD,6-31G(d) level of theory to shed light on the reason for this change by investigating the detailed mechanistic aspects of these transformations. This study demonstrates that the electronic nature of the Pd catalyst changes from π-philicity to oxophilicity during the catalytic cycle, and this change plays an essential role in controlling the chemoselectivity of the cyclization reactions. In addition, it was found that (1) the hypervalent iodine reagent PIDA serves not only as an oxidant for the oxidation of Pd(II) to Pd(IV), but also as a nucleophile that drives the acetoxypalladation step of the reaction, (2) the oxidation of Pd(II) to Pd(IV) by the iodonium ion [PhIOAc]⁺ occurs via an interesting mechanism involving coordination of [PhIOAc]⁺ to the Pd(II) centre, followed by a twist in the hypervalent iodine, and (3) Pd π-complexes are not very susceptible to oxidation. (4) A Pd(II) complex can be six coordinate if the Pd centre is partially oxidized.

The epoxide hydrolase from Alphaproteobacteria bacterium was identified to convert meso-3,4-epoxytetrahydrofuran (1) to (3R,4R)-tetrahydrofurandiol [(RR)-2] predominantly. The enzyme was engineered via directed evolution to revert its original enantioselectivity to generate (3S,4S)-tetrahydrofurandiol [(SS)-2] in quantitative yield and to tolerate an extremely high substrate concentration desired for industrial processes.

Abstract

Chiral vicinal diols are important intermediates in the synthesis of pharmaceuticals. Epoxide hydrolases catalyze hydrolytic ring opening of epoxides to produce the corresponding vicinal diols, providing an attractive way to access these building blocks under mild conditions in a stereoselective and atom-efficient manner. In this study, an epoxide hydrolase is identified and engineered to form (3S,4S)-tetrahydrofurandiol in high optical purity via the desymmetrization of meso-3,4-epoxytetrahydrofuran. In nine rounds of directed evolution, the enzyme's native (3R,4R)-stereopreference was reversed and its activity was dramatically improved to achieve quantitative yield under remarkably high 500 g/L substrate concentration and low enzyme loading. Computational modelling provides insights on the changes in enzyme-substrate interaction that result in divergent enantioselectivities afforded by evolved variants.

This review looks at how, as gelators, cyclic peptides can self-assemble to form nanotubes or even higher-order assemblies. They can also form cylindrical structures by conjugation with polymers. The assembled structures represent important platforms for drug delivery or function as nanomedicine themselves when triggered by certain stimuli.

Abstract

Cyclic peptides are important building blocks for forming functional structures and have been applied in various fields. Considering the significant structural and functional roles of cyclic peptides in materials science and the attributed biophysical advantages, we provide an overview of cyclic peptide types that can self-assemble to form nanotubes, recent progress in stimuli-triggered cyclic peptide assembly, and methods to construct peptide and polymer conjugates based on cyclic peptides with alternative chirality. Specifically, we highlight the roles that stimuli-triggered cyclic peptides and their conjugates play in biomedical applications. Recent progress in other cyclic peptides acting as gelators in drug delivery and biomedicine are also summarized. These cyclic peptides with self-assembly properties are expected to act as adaptive systems for drug delivery and selective disease targeting.

Abstract

Although optical pure amino alcohols are in high demand due to their widespread applicability, they still remain challenging to synthesize, since commonly elaborated protection strategies are required. Here, a multi-enzymatic methodology is presented that circumvents this obstacle furnishing enantioenriched 1,3-amino alcohols out of commodity chemicals. A Type I aldolase forged the carbon backbone with an enantioenriched aldol motif, which was subsequently subjected to enzymatic transamination. A panel of 194 TAs was tested on diverse nine aldol products prepared through different nucleophiles and electrophiles. Due to the availability of (R)- and (S)-selective TAs, both diastereomers of the 1,3-amino alcohol motif were accessible. A two-step process enabled the synthesis of the desired amino alcohols with up to three chiral centers with de up to >97 in the final products.

Publication date: 13 July 2023

Source: Chem, Volume 9, Issue 7

Author(s): Yu Fu, Xiaohong Liu, Yan Xia, Xuzhen Guo, Juan Guo, Junshuai Zhang, Weining Zhao, Yuzhou Wu, Jiangyun Wang, Fangrui Zhong