scholarly journals Improvement of an Escherichia coli whole-cell biocatalyst for geranyl glucoside production using directed evolution

2021 ◽  
Author(s):  
Julian Ruediger ◽  
Wilfried Schwab
Keyword(s):  
Directed Evolution ◽  
Production Systems ◽  
Low Cost ◽  
Continuous Production ◽  
Expression System ◽  
Screening Method ◽  
Fold Increase ◽  
Small Scale ◽  
Biotechnological Production ◽  
Whole Cell

The biotechnological production of glycosides is an economically competitive manufacturing alternative to classical chemical synthesis. Through continuous production improvement, glycosides can now be used in low-cost products by various industries. However, many production systems still suffer from low yields. Directed evolution, coupled with a suitable screening method, can tackle this challenge. We generated glycosyltransferase mutants through error-prone-PCR and screened the library using a small-scale whole-cell biotransformation system. The screening of only 176 colonies yielded three putative candidates. Detailed investigations revealed that the reason for the increase in product titer was mainly due to different expression effects of the mutagenized genes rather than improved enzyme kinetics. In total, a 60-fold increase in product formation was achieved. Therefore, in addition to the quality of the mutant library, an efficient and stable expression system is crucial to achieve high concentrations of active enzyme and product, as formation of inclusion bodies and other inactive forms of the biocatalyst reduces productivity.

scholarly journals A Generic, Whole-Cell–Based Screening Method for Baeyer-Villiger Monooxygenases

2013 ◽  
Vol 18 (6) ◽  
pp. 678-687 ◽  
Author(s):  
Hanna M. Dudek ◽  
Petra Popken ◽  
Edwin van Bloois ◽  
Wouter A. Duetz ◽  
Marco W. Fraaije
Keyword(s):  
Detection Method ◽  
Catalytic Properties ◽  
Expression System ◽  
Screening Method ◽  
Whole Cell ◽  
E Coli ◽  
Coenzyme Regeneration ◽  
Escherichia Coli Cells ◽  
Phosphite Dehydrogenase ◽  
Screening Site

Baeyer-Villiger monooxygenases (BVMOs) have been receiving increasing attention as enzymes useful for biocatalytic applications. Industrial requirements call for rapid and extensive redesign of these enzymes. In response to the need for screening large libraries of BVMO mutants, we established a generic screening method that allows screening of Escherichia coli cells expressing active BVMOs in 96-well plate format. For this, we first developed an expression system for production of phenylacetone monooxygenase (PAMO) in the periplasm of E. coli. This allows probing the enzyme for any target substrate while it is also compatible with extracellular coenzyme regeneration. For coenzyme regeneration, we used phosphite dehydrogenase, which forms phosphate upon NADPH recycling. This allowed the use of a chromogenic molybdate-based phosphate determination assay. The screening procedure was supplemented with a detection method for identification of mutant enzymes that act as NADPH oxidases, thereby excluding false-positives. The whole-cell–based screening method was validated by screening site–saturation libraries of PAMO and resulted in the identification of PAMO mutants with altered catalytic properties. This new method can be used for screening libraries of BVMOs for activity with any desired substrate and therefore is a powerful tool for engineering of these enzymes.

scholarly journals A Survey on Intelligent Agricultural Information Handling Methodologies

2019 ◽  
Vol 11 (12) ◽  
pp. 3278 ◽  
Author(s):  
Yorghos Voutos ◽  
Phivos Mylonas ◽  
John Katheniotis ◽  
Anastasia Sofou
Keyword(s):  
Information Technologies ◽  
Production Systems ◽  
Low Cost ◽  
Small Scale ◽  
Research Knowledge ◽  
Current State ◽  
Smart Farming ◽  
Efficient Data ◽  
Art Research ◽  
The Internet Of Things

The term intelligent agriculture, or smart farming, typically involves the incorporation of computer science and information technologies into the traditional notion of farming. The latter utilizes plain machinery and equipment used for many decades and the only significant improvement made over the years has been the introduction of automation in the process. Still, at the beginning of the new century, there are ways and room for further vast improvements. More specifically, the low cost of rather advanced sensors and small-scale devices, now even connected to the Internet of Things (IoT), allowed them to be introduced in the process and used within agricultural production systems. New and emerging technologies and methodologies, like the utilization of cheap network storage, are expected to advance this development. In this sense, the main goals of this paper may be summarized as follows: (a) To identify, group, and acknowledge the current state-of-the-art research knowledge about intelligent agriculture approaches, (b) to categorize them according to meaningful data sources categories, and (c) to describe current efficient data processing and utilization aspects from the perspective of the main trends in the field.

scholarly journals Functional Expression of a Fungal Laccase in Saccharomyces cerevisiae by Directed Evolution

2003 ◽  
Vol 69 (2) ◽  
pp. 987-995 ◽  
Author(s):  
Thomas Bulter ◽  
Miguel Alcalde ◽  
Volker Sieber ◽  
Peter Meinhold ◽  
Christian Schlachtbauer ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Directed Evolution ◽  
Enzyme Stability ◽  
Expression System ◽  
Fold Increase ◽  
Total Activity ◽  
Functional Expression ◽  
Glycosylation Site ◽  
C Terminus ◽  
Improved Stability

ABSTRACT Laccase from Myceliophthora thermophila (MtL) was expressed in functional form in Saccharomyces cerevisiae. Directed evolution improved expression eightfold to the highest yet reported for a laccase in yeast (18 mg/liter). Together with a 22-fold increase in k cat, the total activity was enhanced 170-fold. Specific activities of MtL mutants toward 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and syringaldazine indicate that substrate specificity was not changed by the introduced mutations. The most effective mutation (10-fold increase in total activity) introduced a Kex2 protease recognition site at the C-terminal processing site of the protein, adjusting the protein sequence to the different protease specificities of the heterologous host. The C terminus is shown to be important for laccase activity, since removing it by a truncation of the gene reduces activity sixfold. Mutations accumulated during nine generations of evolution for higher activity decreased enzyme stability. Screening for improved stability in one generation produced a mutant more stable than the heterologous wild type and retaining the improved activity. The molecular mass of MtL expressed in S. cerevisiae is 30% higher than that of the same enzyme expressed in M. thermophila (110 kDa versus 85 kDa). Hyperglycosylation, corresponding to a 120-monomer glycan on one N-glycosylation site, is responsible for this increase. This S. cerevisiae expression system makes MtL available for functional tailoring by directed evolution.

scholarly journals Directed evolution of the 3C protease from coxsackievirus using a novel fluorescence-assisted intracellular method

2019 ◽  
Vol 400 (3) ◽  
pp. 405-415
Author(s):  
Sebastian W. Meister ◽  
Natalie M. Hendrikse ◽  
John Löfblom
Keyword(s):  
Directed Evolution ◽  
Proteolytic Activity ◽  
Fluorescent Protein ◽  
Screening Method ◽  
Fold Increase ◽  
Label Free ◽  
Peptide Bonds ◽  
3C Protease ◽  
High Throughput Method ◽  
Protease Substrate

Abstract Proteases are crucial for regulating biological processes in organisms through hydrolysis of peptide bonds. Recombinant proteases have moreover become important tools in biotechnological, and biomedical research and as therapeutics. We have developed a label-free high-throughput method for quantitative assessment of proteolytic activity in Escherichia coli. The screening method is based on co-expression of a protease of interest and a reporter complex. This reporter consists of an aggregation-prone peptide fused to a fluorescent protein via a linker that contains the corresponding substrate sequence. Cleavage of the substrate rescues the fluorescent protein from aggregation, resulting in increased fluorescence that correlates to proteolytic activity, which can be monitored using flow cytometry. In one round of flow-cytometric cell sorting, we isolated an efficiently cleaved tobacco etch virus (TEV) substrate from a 1:100 000 background of non-cleavable sequences, with around 6000-fold enrichment. We then engineered the 3C protease from coxsackievirus B3 (CVB3 3Cpro) towards improved proteolytic activity on the substrate LEVLFQ↓GP. We isolated highly proteolytic active variants from a randomly mutated CVB3 3Cpro library with up to 4-fold increase in activity. The method enables simultaneous measurement of proteolytic activity and protease expression levels and can therefore be applied for protease substrate profiling, as well as directed evolution of proteases.

scholarly journals Engineering Platforms for Directed Evolution of Laccase from Pycnoporus cinnabarinus

2011 ◽  
Vol 78 (5) ◽  
pp. 1370-1384 ◽  
Author(s):  
S. Camarero ◽  
I. Pardo ◽  
A. I. Cañas ◽  
P. Molina ◽  
E. Record ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Fusion Gene ◽  
Expression System ◽  
Fold Increase ◽  
Total Activity ◽  
Functional Expression ◽  
Redox Mediators ◽  
Pycnoporus Cinnabarinus ◽  
Content Type ◽  
Potential Use

ABSTRACTWhile thePycnoporus cinnabarinuslaccase (PcL) is one of the most promising high-redox-potential enzymes for environmental biocatalysis, its practical use has to date remained limited due to the lack of directed evolution platforms with which to improve its features. Here, we describe the construction of a PcL fusion gene and the optimization of conditions to induce its functional expression inSaccharomyces cerevisiae, facilitating its directed evolution and semirational engineering. The native PcL signal peptide was replaced by the α-factor preproleader, and this construct was subjected to six rounds of evolution coupled to a multiscreening assay based on the oxidation of natural and synthetic redox mediators at more neutral pHs. The laccase total activity was enhanced 8,000-fold: the evolved α-factor preproleader improved secretion levels 40-fold, and several mutations in mature laccase provided a 13.7-fold increase inkcat. While the pH activity profile was shifted to more neutral values, the thermostability and the broad substrate specificity of PcL were retained. Evolved variants were highly secreted byAspergillus niger(∼23 mg/liter), which addresses the potential use of this combined-expression system for protein engineering. The mapping of mutations onto the PcL crystal structure shed new light on the oxidation of phenolic and nonphenolic substrates. Furthermore, some mutations arising in the evolved preproleader highlighted its potential for heterologous expression of fungal laccases in yeast (S. cerevisiae).

scholarly journals Controlled Autolysis and Enzyme Release in a Recombinant Lactococcal Strain Expressing the Metalloendopeptidase Enterolysin A

2004 ◽  
Vol 70 (3) ◽  
pp. 1744-1748 ◽  
Author(s):  
Rita M. Hickey ◽  
R. Paul Ross ◽  
Colin Hill
Keyword(s):  
Large Scale ◽  
Production Systems ◽  
Expression System ◽  
Fold Increase ◽  
Lytic Enzyme ◽  
Enzyme Release ◽  
Gram Positive Bacteria ◽  
Wide Range ◽  
Potential Applications ◽  
A Cell

ABSTRACT This study concerns the exploitation of the lytic enzyme enterolysin A (EntL), produced by Enterococcus faecalis strain DPC5280, to elicit the controlled autolysis of starter lactococci. EntL, a cell wall metalloendopeptidase secreted by some E. faecalis strains, can kill a wide range of gram-positive bacteria, including lactococci. The controlled expression of entL, which encodes EntL, was achieved using a nisin-inducible expression system in a lactococcal host. Zymographic analysis of EntL activity demonstrated that active enzyme is produced by the recombinant lactococcal host. Indeed, expression of EntL resulted in almost complete autolysis of the host strain 2 h after induction with nisin. Model cheese experiments using a starter strain in addition to the inducible enterolysin-producing strain showed a 27-fold increase in activity with respect to the release of lactate dehydrogenase in the strain overexpressing EntL, demonstrating the potential of EntL production in large-scale cheese production systems. Indeed, the observation that a wide range of lactic bacteria are sensitive to EntL suggests that EntL-induced autolysis has potential applications with a variety of lactic acid bacteria and could be a basis for probiotic delivery systems.

Directed Evolution of P450 Fatty Acid Decarboxylases via High-Throughput Screening Towards Improved Catalytic Activity

2019 ◽  
Author(s):  
Huifang Xu ◽  
Weinan Liang ◽  
Linlin Ning ◽  
Yuanyuan Jiang ◽  
Wenxia Yang ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Fatty Acid ◽  
Directed Evolution ◽  
High Throughput ◽  
High Throughput Screening ◽  
Colorimetric Detection ◽  
Screening Method ◽  
Random Mutagenesis ◽  
Direct Production ◽  
Consumption Activity

P450 fatty acid decarboxylases (FADCs) have recently been attracting considerable attention owing to their one-step direct production of industrially important 1-alkenes from biologically abundant feedstock free fatty acids under mild conditions. However, attempts to improve the catalytic activity of FADCs have met with little success. Protein engineering has been limited to selected residues and small mutant libraries due to lack of an effective high-throughput screening (HTS) method. Here, we devise a catalase-deficient <i>Escherichia coli</i> host strain and report an HTS approach based on colorimetric detection of H<sub>2</sub>O<sub>2</sub>-consumption activity of FADCs. Directed evolution enabled by this method has led to effective identification for the first time of improved FADC variants for medium-chain 1-alkene production from both DNA shuffling and random mutagenesis libraries. Advantageously, this screening method can be extended to other enzymes that stoichiometrically utilize H<sub>2</sub>O<sub>2</sub> as co-substrate.

Causes of gear pump fails and methods for their elimination

2020 ◽  
pp. 98-107
Author(s):  
Vitaliy A. Zuyevskiy ◽  
Daniil O. Klimyuk ◽  
Ivan A. Shemberev
Keyword(s):  
Adhesion Strength ◽  
Production Systems ◽  
Low Cost ◽  
High Strength ◽  
Strength Properties ◽  
Research Purpose ◽  
Working Fluid ◽  
Gear Pump ◽  
Repair Service ◽  
Recovery Method

Gear pumps are an important element of many production systems and their replacement in case of failure can be quite expensive, so it is important to have a modern and well-tuned technology for their recovery. There are many methods for restoring the pump's performance, depending on the reason that led to its failure. (Research purpose) The research purpose is in determining what causes most often lead to loss of pump performance, and developing a recovery method that provides the greatest post-repair service life of the pump and low cost of repair. (Materials and methods) Authors took into account that the applied coatings must have sufficient adhesion strength and resistance to mechanical, thermal and corrosion loads during operation. It was found that most often significant leaks of the working fluid, leading to failure, occur due to an increase in the gap between the inner surface of the housing and the gears due to active wear of the housing wells. Authors determined that the method of electric spark treatment of worn-out housing wells is best suited to perform the task (a large post-repair resource and low costs). (Results and discussion) It was found by laboratory studies of the adhesion strength of electric spark coatings with various electrodes that the best transfer of the material to the substrate is provided by bronze electrodes BrMKts3-1. It was noted that the coatings applied using the BrMKts3-1 electrode have high strength properties. (Conclusions) Research conducted in the center for collective use "Nano-Center" VIM confirmed the possibility of effective recovery of the gear pump by electric spark treatment.

Tools of Transformation: Appropriate Technology in U.S. Countercultural Literature

2012 ◽  
Vol 44 (2) ◽  
pp. 75-93
Author(s):  
Peter Mortensen
Keyword(s):  
Low Cost ◽  
Appropriate Technology ◽  
Small Scale ◽  
Environmental Transformation ◽  
Buckminster Fuller ◽  
1960S And 1970S ◽  
The 1960S ◽  
Environmentally Sound ◽  
User Friendly ◽  
Second Wave

This essay takes its cue from second-wave ecocriticism and from recent scholarly interest in the “appropriate technology” movement that evolved during the 1960s and 1970s in California and elsewhere. “Appropriate technology” (or AT) refers to a loosely-knit group of writers, engineers and designers active in the years around 1970, and more generally to the counterculture’s promotion, development and application of technologies that were small-scale, low-cost, user-friendly, human-empowering and environmentally sound. Focusing on two roughly contemporary but now largely forgotten American texts Sidney Goldfarb’s lyric poem “Solar-Heated-Rhombic-Dodecahedron” (1969) and Gurney Norman’s novel Divine Right’s Trip (1971)—I consider how “hip” literary writers contributed to eco-technological discourse and argue for the 1960s counterculture’s relevance to present-day ecological concerns. Goldfarb’s and Norman’s texts interest me because they conceptualize iconic 1960s technologies—especially the Buckminster Fuller-inspired geodesic dome and the Volkswagen van—not as inherently alienating machines but as tools of profound individual, social and environmental transformation. Synthesizing antimodernist back-to-nature desires with modernist enthusiasm for (certain kinds of) machinery, these texts adumbrate a humanity- and modernity-centered post-wilderness model of environmentalism that resonates with the dilemmas that we face in our increasingly resource-impoverished, rapidly warming and densely populated world.

scholarly journals Development of a Novel High-Throughput Screen and Identification of Small-Molecule Inhibitors of the Gα–RGS17 Protein–Protein Interaction Using AlphaScreen

2011 ◽  
Vol 16 (8) ◽  
pp. 869-877 ◽  
Author(s):  
Duncan I. Mackie ◽  
David L. Roman
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
Protein Interaction ◽  
High Throughput Screening ◽  
Drug Targets ◽  
Screening Method ◽  
Fold Increase ◽  
Rgs Proteins ◽  
High Throughput Screen ◽  
Protein Protein Interaction

In this study, the authors used AlphaScreen technology to develop a high-throughput screening method for interrogating small-molecule libraries for inhibitors of the Gαo–RGS17 interaction. RGS17 is implicated in the growth, proliferation, metastasis, and the migration of prostate and lung cancers. RGS17 is upregulated in lung and prostate tumors up to a 13-fold increase over patient-matched normal tissues. Studies show RGS17 knockdown inhibits colony formation and decreases tumorigenesis in nude mice. The screen in this study uses a measurement of the Gαo–RGS17 protein–protein interaction, with an excellent Z score exceeding 0.73, a signal-to-noise ratio >70, and a screening time of 1100 compounds per hour. The authors screened the NCI Diversity Set II and determined 35 initial hits, of which 16 were confirmed after screening against controls. The 16 compounds exhibited IC50 <10 µM in dose–response experiments. Four exhibited IC50 values <6 µM while inhibiting the Gαo–RGS17 interaction >50% when compared to a biotinylated glutathione-S-transferase control. This report describes the first high-throughput screen for RGS17 inhibitors, as well as a novel paradigm adaptable to many other RGS proteins, which are emerging as attractive drug targets for modulating G-protein-coupled receptor signaling.

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