Rational Design of Lipase ROL to Increase Its Thermostability for Production of Structured Tags.

1,3-regiospecific lipases are important enzymes that are heavily utilized in the food industries to produce structured triacylglycerols (TAGs). The lipase (ROL) has recently gained interest because this enzyme possesses high selectivity and catalytic efficiency. However, its low thermostability limits its use towards reactions that work at lower temperature.

A high-throughput pipeline for scalable kit-free RNA extraction.

An overreliance on commercial, kit-based RNA extraction in the molecular diagnoses of infectious disease presents a challenge in the event of supply chain disruptions and can potentially hinder testing capacity in times of need. In this study, we adapted a well-established, robust TRIzol-based RNA extraction protocol into a high-throughput format through miniaturization and automation. The workflow was validated by RT-qPCR assay for SARS-CoV-2 detection to illustrate its scalability without interference to downstream diagnostic sensitivity and accuracy.

Crystal Structure of Cucumene Synthase, a Terpenoid Cyclase That Generates a Linear Triquinane Sesquiterpene.

Linear triquinanes are sesquiterpene natural products with hydrocarbon skeletons consisting of three fused five-membered rings. Importantly, several of these compounds exhibit useful anticancer, anti-inflammatory, and antibiotic properties. However, linear triquinanes pose significant challenges to organic synthesis because of the structural and stereochemical complexity of their hydrocarbon skeletons.

Directed Evolution of Quorum-Quenching Enzymes: A Method for the Construction of a Directed Evolution Platform and Characterization of a Quorum-Quenching Lactonase from Geobacillus kaustophilus.

A thermostable quorum-quenching lactonase from Geobacillus kaustophilus (GKL) was used as a template for in vitro directed evolution experiments. Here we describe the overexpression and purification of wild-type GKL, the construction of a quorum-quenching directed evolution platform using bioluminescence as a reporter, and the in vitro kinetic assay for the determination of kinetic parameters of wild-type GKL and its mutants.

Anti-virulent Disruption of Pathogenic Biofilms using Engineered Quorum-quenching Lactonases.

The rapid emergence of multi-drug resistant bacteria has accelerated the need for novel therapeutic approaches to counter life-threatening infections. The persistence of bacterial infection is often associated with quorum-sensing-mediated biofilm formation. Thus, the disruption of this signaling circuit presents an attractive anti-virulence strategy.

Computational-guided discovery and characterization of a sesquiterpene synthase from Streptomyces clavuligerus.

Terpenoids are a large structurally diverse group of natural products with an array of functions in their hosts. The large amount of genomic information from recent sequencing efforts provides opportunities and challenges for the functional assignment of terpene synthases that construct the carbon skeletons of these compounds. Inferring function from the sequence and/or structure of these enzymes is not trivial because of the large number of possible reaction channels and products.

Predicting the functions and specificity of triterpenoid synthases: a mechanism-based multi-intermediate docking approach.

Terpenoid synthases construct the carbon skeletons of tens of thousands of natural products. To predict functions and specificity of triterpenoid synthases, a mechanism-based, multi-intermediate docking approach is proposed. In addition to enzyme function prediction, other potential applications of the current approach, such as enzyme mechanistic studies and enzyme redesign by mutagenesis, are discussed.

Disruption of biofilm formation by the human pathogen Acinetobacter baumannii using engineered quorum-quenching lactonases.

Acinetobacter baumannii is a major human pathogen associated with multidrug-resistant nosocomial infections; its virulence is attributed to quorum-sensing-mediated biofilm formation, and disruption of biofilm formation is an attractive antivirulence strategy. Here, we report the first successful demonstration of biofilm disruption in a clinical isolate of A. baumannii S1, using a quorum-quenching lactonase obtained by directed evolution; this engineered lactonase significantly reduced the biomass of A.

Structural evidence of a productive active site architecture for an evolved quorum-quenching GKL lactonase.

The in vitro evolution and engineering of quorum-quenching lactonases with enhanced reactivities was achieved using a thermostable GKL enzyme as a template, yielding the E101G/R230C GKL mutant with increased catalytic activity and a broadened substrate range [Chow, J. Y., Xue, B.

Establishing a toolkit for precursor-directed polyketide biosynthesis: exploring substrate promiscuities of acid-CoA ligases.

Polyketides are chemically diverse and medicinally important biochemicals that are biosynthesized from acyl-CoA precursors by polyketide synthases. One of the limitations to combinatorial biosynthesis of polyketides has been the lack of a toolkit that describes the means of delivering novel acyl-CoA precursors necessary for polyketide biosynthesis. Using five acid-CoA ligases obtained from various plants and microorganisms, we biosynthesized an initial library of 79 acyl-CoA thioesters by screening each of the acid-CoA ligases against a library of 123 carboxylic acids.

Directed evolution of a thermostable quorum-quenching lactonase from the amidohydrolase superfamily.

A thermostable quorum-quenching lactonase from Geobacillus kaustophilus HTA426 (GI: 56420041) was used as an initial template for in vitro directed evolution experiments. This enzyme belongs to the phosphotriesterase-like lactonase (PLL) group of enzymes within the amidohydrolase superfamily that hydrolyze N-acylhomoserine lactones (AHLs) that are involved in virulence pathways of quorum-sensing pathogenic bacteria. Here we have determined the N-butyryl-L-homoserine lactone-liganded structure of the catalytically inactive D266N mutant of this enzyme to a resolution of 1.

Directed evolution of a quorum-quenching lactonase from Mycobacterium avium subsp. paratuberculosis K-10 in the amidohydrolase superfamily.

The PLL(PTE-like lactonase)-group of enzymes within the amidohydrolase superfamily hydrolyze N-acyl-homoserine lactones (AHLs) that are involved in bacterial quorum-sensing pathways. These enzymes possess the (beta/alpha)(8)-barrel fold and serve as attractive templates for in vitro evolution and engineering of quorum-quenching biological molecules that can serve as antivirulence therapeutic agents. Using a quorum-quenching lactonase from Mycobacterium avium subsp.

The meiosis-specific Sid2p-related protein Slk1p regulates forespore membrane assembly in fission yeast.

Cytokinesis in all organisms involves the creation of membranous barriers that demarcate individual daughter cells. In fission yeast, a signaling module termed the septation initiation network (SIN) plays an essential role in the assembly of new membranes and cell wall during cytokinesis. In this study, we have characterized Slk1p, a protein-kinase related to the SIN component Sid2p.