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Significance This paper describes a novel strategy for predicting the function of terpene synthases. Functional assignment of terpene synthases is a daunting task because product selectivity is not high in many terpene synthases, and mutations in and near the active sites of selective enzyme can result in synthesis of different products. Using a homology model of an unknown terpene synthase, we developed an algorithm that predicted the enzyme synthesizes a linear triquinane. We confirmed this prediction; specifically, the enzyme converts farnesyl diphosphate to a linear triquinine sesquiterpene: (5 S ,7 S ,10 R ,11 S )-cucumene. The findings highlight the potential for using computational approaches to assist in the discovery and characterization of unknown terpene synthases. 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. We tackle this problem by developing an algorithm to enumerate possible carbocations derived from the farnesyl cation, the first reactive intermediate of the substrate, and evaluating their steric and electrostatic compatibility with the active site. The homology model of a putative pentalenene synthase (Uniprot: B5GLM7) from Streptomyces clavuligerus was used in an automated computational workflow for product prediction. Surprisingly, the workflow predicted a linear triquinane scaffold as the top product skeleton for B5GLM7. Biochemical characterization of B5GLM7 reveals the major product as (5 S ,7 S ,10 R ,11 S )-cucumene, a sesquiterpene with a linear triquinane scaffold. To our knowledge, this is the first documentation of a terpene synthase involved in the synthesis of a linear triquinane. The success of our prediction for B5GLM7 suggests that this approach can be used to facilitate the functional assignment of novel terpene synthases.

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.

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. This semi-automated, kit-free approach offers a versatile alternative to prevailing integrated solid-phase RNA extraction proprietary systems, with the added advantage of improved cost-effectiveness for high volume acquisition of quality RNA whether for use in clinical diagnoses or for diverse molecular applications.

1,3-regiospecific lipases are important enzymes that are heavily utilized in the food industries to produce structured triacylglycerols (TAGs). The Rhizopus oryzae 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. Most importantly, the enzyme cannot be used for the production of 1,3-dioleoyl-2-palmitoylglycerol (OPO) and 1,3-stearoyl-2-oleoyl-glycerol (SOS) due to the high melting points of the substrates used for the reaction. Despite various engineering efforts used to improve the thermostability of ROL, the enzyme is unable to function at temperatures above 60 °C. Here, we describe the rational design of ROL to identify variants that can retain their activity at temperatures higher than 60 °C. After two rounds of mutagenesis and screening, we were able to identify a mutant ROL_10x that can retain most of its activity at 70 °C. We further demonstrated that this mutant is useful for the synthesis of SOS while minimal product formation was observed with ROL_WT. Our engineered enzyme provides a promising solution for the industrial synthesis of structured lipids at high temperature.

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. We tackle this problem by developing an algorithm to enumerate possible carbocations derived from the farnesyl cation, the first reactive intermediate of the substrate, and evaluating their steric and electrostatic compatibility with the active site. The homology model of a putative pentalenene synthase (Uniprot: B5GLM7) fromStreptomyces clavuligeruswas used in an automated computational workflow for product prediction. Surprisingly, the workflow predicted a linear triquinane scaffold as the top product skeleton for B5GLM7. Biochemical characterization of B5GLM7 reveals the major product as (5S,7S,10R,11S)-cucumene, a sesquiterpene with a linear triquinane scaffold. To our knowledge, this is the first documentation of a terpene synthase involved in the synthesis of a linear triquinane. The success of our prediction for B5GLM7 suggests that this approach can be used to facilitate the functional assignment of novel terpene synthases.

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.