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Methods in Enzymology

Nigel Scrutton
No abstract text is available yet for this article.
2018: Methods in Enzymology
Guangkai Bian, Tian Ma, Tiangang Liu
Terpenoids represent a highly diverse group of natural products with wide applications. Engineering approaches have been used to increase titers of many value-added terpenoids, such as farnesene, taxadiene, lycopene, and astaxanthin. In this chapter, we review the in vitro reconstitution-based targeted engineering of terpenoids, as well as approaches for the mining of terpene cyclases and for increasing the chemical diversity. Information gained from in vitro reconstitution extends our understanding of the mechanisms underlying terpenoid biosynthesis, the contributions of enzymes and cofactors, and key enzymes and rate-limiting steps for the development of an ideal biosynthetic production system...
2018: Methods in Enzymology
Florence Huynh, David J Miller, Rudolf K Allemann
Sesquiterpene synthases catalyze the conversion of farnesyl diphosphate to more than 300 different hydrocarbon and alcohol natural products which often contain multiple fused rings and stereocenters. Recent work has taken advantage of the exquisite stereospecificity of these enzymes to synthesize complex novel sesquiterpenoids using nonnatural substrates. In this chapter, we describe the expression, purification, and use of one such synthase to convert a nonnatural substrate to a novel cyclic ether, thereby expanding the terpenome...
2018: Methods in Enzymology
Joanna C Sadler, Lucy Green, Neil Swainston, Douglas B Kell, Andrew Currin
Directed evolution (DE) is a powerful tool for optimizing an enzyme's properties toward a particular objective, such as broader substrate scope, greater thermostability, or increased kcat . A successful DE project requires the generation of genetic diversity and subsequent screening or selection to identify variants with improved fitness. In contrast to random methods (error-prone PCR or DNA shuffling), site-directed mutagenesis enables the rational design of variant libraries and provides control over the nature and frequency of the encoded mutations...
2018: Methods in Enzymology
Quanli Liu, Tao Yu, Kate Campbell, Jens Nielsen, Yun Chen
Amino acids find various applications in biotechnology in view of their importance in the food, feed, pharmaceutical, and personal care industries as nutrients, additives, and drugs, respectively. For the large-scale production of amino acids, microbial cell factories are widely used and the development of amino acid-producing strains has mainly focused on prokaryotes Corynebacterium glutamicum and Escherichia coli. However, the eukaryote Saccharomyces cerevisiae is becoming an even more appealing microbial host for production of amino acids and derivatives because of its superior molecular and physiological features, such as amenable to genetic engineering and high tolerance to harsh conditions...
2018: Methods in Enzymology
Satoshi Yuzawa, Amin Zargar, Bo Pang, Leonard Katz, Jay D Keasling
Reduced polyketides are a subclass of natural products that have a variety of medical, veterinary, and agricultural applications and are well known for their structural diversity. Although these compounds do not resemble each other, they are all made by a class of enzymes known as modular polyketide synthases (PKSs). The commonality of PKS domains/modules that compose PKSs and the understanding of the relationship between the sequence of the PKS and the structure of the compound it produces render modular PKSs as excellent targets for engineering to produce novel compounds with predicted structures...
2018: Methods in Enzymology
Christopher J Robinson, Mark S Dunstan, Neil Swainston, James Titchmarsh, Eriko Takano, Nigel S Scrutton, Adrian J Jervis
The microbial production of commodity, fine, and specialty chemicals is a driving force in biotechnology. An essential requirement is to introduce biosynthetic pathways to the target compound(s) into chassis organisms. First suitable enzymes must be selected and characterized, and then genetic pathways must be designed and assembled into suitable expression vectors. The design of these pathways is crucial for balancing the pathway for efficient in vivo activity. This can be achieved through optimization of the pathway regulation by altering transcription and translation rates...
2018: Methods in Enzymology
Sebastian Wenk, Oren Yishai, Steffen N Lindner, Arren Bar-Even
The introduction of synthetic pathways into microbes often requires substantial modifications of the host metabolism. Here, we present and discuss key experimental aspects required for modifying microbial central metabolism. We introduce the concept of dividing pathways into metabolic modules, the activity of which can be selected for and optimized in dedicated gene-deletion strains. We provide a comprehensive methodology for systematic pathway implementation in vivo, ranging from gene-deletion methods for the creation of selection strains to cloning strategies that allow fine-tuned expression of individual pathway enzymes in synthetic operons...
2018: Methods in Enzymology
Ashty S Karim, Michael C Jewett
Engineering biological systems for the production of biofuels and bioproducts holds great potential to transform the bioeconomy, but often requires laborious, time-consuming design-build-test cycles. For decades cell-free systems have offered quick and facile approaches to study enzymes with hopes of informing cellular processes, mainly in the form of purified single-enzyme activity assays. Over the past 20 years, cell-free systems have grown to include multienzymatic systems, both purified and crude. By decoupling cellular growth objectives from enzyme pathway engineering objectives, cell-free systems provide a controllable environment to direct substrates toward a single, desired product...
2018: Methods in Enzymology
Jiang Xiao-Ran, Yin Jin, Chen Xiangbin, Chen Guo-Qiang
Traditional microbial chassis, including Escherichia coli, Bacillus subtilis, Ralstonia eutropha, and Pseudomonas putida, are grown under neutral pH and mild osmotic pressure for production of chemicals and materials. They tend to be contaminated easily by many microorganisms. To address this issue, next-generation industrial biotechnology employing halophilic Halomonas spp. has been developed for production of bioplastics polyhydroxyalkanoates (PHAs) and other chemicals. Halomonas spp. that can be grown contamination free under open and unsterile condition at alkali pH and high NaCl have been engineered to produce several PHA polymers in elongated or enlarged cells...
2018: Methods in Enzymology
Pablo Carbonell, Mathilde Koch, Thomas Duigou, Jean-Loup Faulon
In this protocol, we describe in silico design methods that can assist in the engineering of production pathways that are based on enzymatic transformations. The described protocols are the basis for automated processes to be integrated into an iterative Design-Build-Test-Learn cycle in synthetic biology for chemical production. Selecting the right enzyme sequence for a desired biocatalytic activity from the extensive catalogue of sequences available in databases is challenging and can dramatically influence the success of bioproducing chemical compounds...
2018: Methods in Enzymology
Eva Garcia-Ruiz, Jamie Auxillos, Tianyi Li, Junbiao Dai, Yizhi Cai
For many years, researchers have devised elegant techniques to assemble genetic parts into larger constructs. Recently, increasing needs for complex DNA constructs has driven countless attempts to optimize DNA assembly methods for improved efficiency, fidelity, and modularity. These efforts have resulted in simple, robust, standardized, and fast protocols that enable the implementation of high-throughput DNA assembly projects for the fabrication of large synthetic genetic constructs. Recently our groups have developed the YeastFab assembly, a highly efficient method for the design and construction of DNA-building blocks based on the native elements from Saccharomyces cerevisiae...
2018: Methods in Enzymology
Carl Schultz, Jiazhang Lian, Huimin Zhao
Design and construction of an optimal microbial cell factory typically requires overexpression, knockdown, and knockout of multiple gene targets. In this chapter, we describe a combinatorial metabolic engineering strategy utilizing an orthogonal trifunctional CRISPR system that combines transcriptional activation, transcriptional interference, and gene deletion (CRISPR-AID) in the yeast Saccharomyces cerevisiae. This strategy enables multiplexed perturbation of the metabolic and regulatory networks in a modular, parallel, and high-throughput manner...
2018: Methods in Enzymology
Laura N Jeffreys, Hazel M Girvan, Kirsty J McLean, Andrew W Munro
The cytochrome P450 monooxygenase enzymes (P450s) catalyze a diverse array of chemical transformations, most originating from the insertion of an oxygen atom into a substrate that binds close to the P450 heme. The oxygen is delivered by a highly reactive heme iron-oxo species (compound I) and, according to the chemical nature of the substrate and its position in the active site, the P450 can catalyze a wide range of reactions including, e.g., hydroxylation, reduction, decarboxylation, sulfoxidation, N- and O-demethylation, epoxidation, deamination, CC bond formation and breakage, nitration, and dehalogenation...
2018: Methods in Enzymology
Max J L J Fürst, Caterina Martin, Nikola Lončar, Marco W Fraaije
Many proteins are rapidly deactivated when exposed to high or even ambient temperatures. This cannot only impede the study of a particular protein, but also is one of the major reasons why enzyme catalysis is still widely unable to compete with established chemical processes. Furthermore, differences in protein stability are a challenge in synthetic biology, when individual modules prove to be incompatible. The targeted stabilization of proteins can overcome these hurdles, and protein engineering techniques are more and more reliably supported by computational chemistry tools...
2018: Methods in Enzymology
Sebastian C Cosgrove, Agata Brzezniak, Scott P France, Jeremy I Ramsden, Juan Mangas-Sanchez, Sarah L Montgomery, Rachel S Heath, Nicholas J Turner
Synthesis of the chiral amine moiety is a key challenge for synthetic organic chemistry due to its prevalence in many biologically active molecules. Imine reductase and amine oxidase enzymes have enabled the biocatalytic synthesis of a host of chiral amine compounds. In this chapter, procedures for the synthesis of chiral amines using imine reductases (IREDs), the recently discovered IRED homologues reductive aminases, and amine oxidases (AOs) are described. Amine oxidases have been the subject of mutagenesis approaches for improvement of substrate scope...
2018: Methods in Enzymology
Karen N Allen
No abstract text is available yet for this article.
2018: Methods in Enzymology
Steven P D Harborne, Jannik Strauss, Ainoleena Turku, Matthew A Watson, Roman Tuma, Sarah A Harris, Adrian Goldman
Membrane-bound pyrophosphatases couple the hydrolysis of inorganic pyrophosphate to the pumping of ions (sodium or protons) across a membrane in order to generate an electrochemical gradient. This class of membrane protein is widely conserved across plants, fungi, archaea, and bacteria, but absent in multicellular animals, making them a viable target for drug design against protozoan parasites such as Plasmodium falciparum. An excellent understanding of many of the catalytic states throughout the enzymatic cycle has already been afforded by crystallography...
2018: Methods in Enzymology
Daniel Roston, Xiya Lu, Dong Fang, Darren Demapan, Qiang Cui
We discuss the application of quantum mechanics/molecular mechanics (QM/MM) free energy simulations to the analysis of phosphoryl transfers catalyzed by two enzymes: alkaline phosphatase and myosin. We focus on the nature of the transition state and the issue of mechanochemical coupling, respectively, in the two enzymes. The results illustrate unique insights that emerged from the QM/MM simulations, especially concerning the interpretation of experimental data regarding the nature of enzymatic transition states and coupling between global structural transition and catalysis in the active site...
2018: Methods in Enzymology
Daniel R Dempsey, Philip A Cole
Since the discovery of C-tail phosphorylation of PTEN almost 20 years ago, much progress has been made in understanding its regulatory influences on the cellular function of PTEN. Phosphorylation of Ser380, Thr382, Thr383, and Ser385 drives a PTEN conformational change from an open to closed state where catalytic function is impaired, plasma membrane binding is reduced, and cellular stability is enhanced. Despite these advances, a detailed structural and mechanistic model of how these phosphorylations impact PTEN function is lacking...
2018: Methods in Enzymology
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