Integrating the Protein and Metabolic Engineering Toolkits for Next-Generation Chemical Biosynthesis
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Through microbial engineering, biosynthesis has the potential to produce thousands of chemicals used in everyday life. Metabolic engineering and synthetic biology are fields driven by the manipulation of genes, genetic regulatory systems, and enzymatic pathways for developing highly productive microbial strains. Fundamentally, it is the biochemical characteristics of the enzymes themselves that dictate flux through a biosynthetic pathway toward the product of interest. As metabolic engineers target sophisticated secondary metabolites, there has been little recognition of the reduced catalytic activity and increased substrate/product promiscuity of the corresponding enzymes compared to those of central metabolism. Thus, fine-tuning these enzymatic characteristics through protein engineering is paramount for developing high-productivity microbial strains for secondary metabolites. Here, we describe the importance of protein engineering for advancing metabolic engineering of secondary metabolism pathways. This pathway integrated enzyme optimization can enhance the collective toolkit of microbial engineering to shape the future of chemical manufacturing.Keywords:
Metabolic Engineering
Synthetic Biology
Protein Engineering
Metabolic pathway
Secondary metabolism
Microbial Metabolism
Protein Engineering
Protein design
Biocatalysis
Directed Molecular Evolution
Site-directed mutagenesis
Folding (DSP implementation)
Molecular engineering
Denaturation (fissile materials)
Metabolic Engineering
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Protein Engineering
Directed Molecular Evolution
Site-directed mutagenesis
Complement
Synthetic Biology
Protein design
Directed mutagenesis
Sequence (biology)
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Protein consensus-based surface engineering (ProCoS) is a simple and efficient method for directed protein evolution combining computational analysis and molecular biology tools to engineer protein surfaces. ProCoS is based on the hypothesis that conserved residues originated from a common ancestor and that these residues are crucial for the function of a protein, whereas highly variable regions (situated on the surface of a protein) can be targeted for surface engineering to maximize performance. ProCoS comprises four main steps: (i) identification of conserved and highly variable regions; (ii) protein sequence design by substituting residues in the highly variable regions, and gene synthesis; (iii) in vitro DNA recombination of synthetic genes; and (iv) screening for active variants. ProCoS is a simple method for surface mutagenesis in which multiple sequence alignment is used for selection of surface residues based on a structural model. To demonstrate the technique's utility for directed evolution, the surface of a phytase enzyme from Yersinia mollaretii (Ymphytase) was subjected to ProCoS. Screening just 1050 clones from ProCoS engineering—guided mutant libraries yielded an enzyme with 34 amino acid substitutions. The surface-engineered Ymphytase exhibited 3.8-fold higher pH stability (at pH 2.8 for 3 h) and retained 40% of the enzyme's specific activity (400 U/mg) compared with the wild-type Ymphytase. The pH stability might be attributed to a significantly increased (20 percentage points; from 9% to 29%) number of negatively charged amino acids on the surface of the engineered phytase.
Protein Engineering
Directed Molecular Evolution
Protein design
DNA shuffling
Synthetic Biology
Protein sequencing
Site-directed mutagenesis
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Protein engineering has been the most attractive strategy for biologists to redesign enzymes. As the simplest technique of protein engineering, directed evolution has been applied to many fields, such as industry, agriculture and medicine. An experiment of directed evolution comprises mutant libraries creation and screening or selection for enzyme variants with desired properties. Therefore, a successful application of directed evolution depends on whether or not one can generate a quality library and perform effective screening to find the desired properties. Directed evolution is already increasingly used in many laboratories to improve protein stability and activity, alter enzyme substrate specificity, or design new activities. Meanwhile, many more effective novel strategies of mutant library generation and screening or selection have emerged in recent years, and will continue to be developed. Combining computational/rational design with directed evolution has been developed as more available means to redesign enzymes. Keywords: Protein engineering, directed evolution, sequence diversity creation, novel strategy, computational design, rational design
Directed Molecular Evolution
Protein Engineering
Rational design
Synthetic Biology
Protein design
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Protein engineering in the context of metabolic engineering is increasingly important to the field of industrial biotechnology. As the demand for biologically produced food, fuels, chemicals, food additives, and pharmaceuticals continues to grow, the ability to design and modify proteins to accomplish new functions will be required to meet the high productivity demands for the metabolism of engineered organisms. We review advances in selecting, modeling, and engineering proteins to improve or alter their activity. Some of the methods have only recently been developed for general use and are just beginning to find greater application in the metabolic engineering community. We also discuss methods of generating random and targeted diversity in proteins to generate mutant libraries for analysis. Recent uses of these techniques to alter cofactor use; produce non-natural amino acids, alcohols, and carboxylic acids; and alter organism phenotypes are presented and discussed as examples of the successful engineering of proteins for metabolic engineering purposes.
Metabolic Engineering
Protein Engineering
Synthetic Biology
Industrial microbiology
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Proteins are one of the most multifaceted macromolecules in living systems. Proteins have evolved to function under physiological conditions and, therefore, are not usually tolerant of harsh experimental and environmental conditions. The growing use of proteins in industrial processes as a greener alternative to chemical catalysts often demands constant innovation to improve their performance. Protein engineering aims to design new proteins or modify the sequence of a protein to create proteins with new or desirable functions. With the emergence of structural and functional genomics, protein engineering has been invigorated in the post-genomic era. The three-dimensional structures of proteins with known functions facilitate protein engineering approaches to design variants with desired properties. There are three major approaches of protein engineering research, namely, directed evolution, rational design, and de novo design. Rational design is an effective method of protein engineering when the threedimensional structure and mechanism of the protein is well known. In contrast, directed evolution does not require extensive information and a three-dimensional structure of the protein of interest. Instead, it involves random mutagenesis and selection to screen enzymes with desired properties. De novo design uses computational protein design algorithms to tailor synthetic proteins by using the three-dimensional structures of natural proteins and their folding rules. The present review highlights and summarizes recent protein engineering approaches, and their challenges and limitations in the post-genomic era. Keywords: De novo design, directed evolution, genomics, protein engineering, random mutagenesis, rational design.
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The generation of high levels of new catalytic activities on natural and artificial protein scaffolds is a major goal of enzyme engineering. Here, we used random mutagenesis and selection in vivo to establish a sugar isomerisation reaction on both a natural (beta alpha)(8)-barrel enzyme and a catalytically inert chimeric (beta alpha)(8)-barrel scaffold, which was generated by the recombination of 2 (beta alpha)(4)-half barrels. The best evolved variants show turnover numbers and substrate affinities that are similar to those of wild-type enzymes catalyzing the same reaction. The determination of the crystal structure of the most proficient variant allowed us to model the substrate sugar in the novel active site and to elucidate the mechanistic basis of the newly established activity. The results demonstrate that natural and inert artificial protein scaffolds can be converted into highly proficient enzymes in the laboratory, and provide insights into the mechanisms of enzyme evolution.
Protein Engineering
DNA shuffling
Protein design
Directed Molecular Evolution
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Directed evolution and rational design are the two main approaches in protein engineering, which were developed in the quest to solve the limitations of enzymes regarding insufficient catalytic properties. Directed enzyme evolution utilizes random introduction of mutations or focused mutations of the type combinatorial active-site saturation test (CAST)/iterative saturation mutagenesis (ISM), while rational design exploits structural and mechanistic information with computational aids. A panoramic view of directed enzyme evolution and rational enzyme design is given in this chapter, including the methods, aims, and historical developments up to the current status. The relative merits of the approaches are discussed.
Rational design
Protein Engineering
Saturated mutagenesis
Directed Molecular Evolution
Protein design
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This account provides a personal view on the development of biocatalysis over the last two decades. Examples include the use of commercial enzymes, such as lipases, (recombinant) esterases, transaminases, and Baeyer–Villiger monooxygenases for the synthesis of optically pure compounds. The opportunity provided by modern protein engineering methods to tailor design an enzyme for a given scientific problem (substrate scope, selectivity, stability) is emphasized together with concepts to boost this technology in terms of timelines and success. 1 Introduction 2 Unexpected Discoveries 2.1 To Protect and Serve 2.2 ‘Abnormal’ Access to β-Amino Acids 3 Defined Enzyme Is Better Than Crude Extract 4 New Horizons Opened by Protein Engineering 4.1 Random Mutagenesis Can Give Random Results 5 Exploring Sequence and Structure Databases 5.1 Massive Alignment Identifies Evolutionary Variations 5.2 Fixing Wrong Annotations Can Yield a Toolbox of Novel Enzymes 6 Conclusion
Protein Engineering
Biocatalysis
Scope (computer science)
Directed Molecular Evolution
Toolbox
Protein design
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Protein Engineering
Glycobiology
Directed Molecular Evolution
High-Throughput Screening
Site-directed mutagenesis
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