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Intelligent host engineering for metabolic flux optimisation in biotechnology.
Munro, LJ, Kell, DB
The Biochemical journal. 2021;(20):3685-3721
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Abstract
Optimising the function of a protein of length N amino acids by directed evolution involves navigating a 'search space' of possible sequences of some 20N. Optimising the expression levels of P proteins that materially affect host performance, each of which might also take 20 (logarithmically spaced) values, implies a similar search space of 20P. In this combinatorial sense, then, the problems of directed protein evolution and of host engineering are broadly equivalent. In practice, however, they have different means for avoiding the inevitable difficulties of implementation. The spare capacity exhibited in metabolic networks implies that host engineering may admit substantial increases in flux to targets of interest. Thus, we rehearse the relevant issues for those wishing to understand and exploit those modern genome-wide host engineering tools and thinking that have been designed and developed to optimise fluxes towards desirable products in biotechnological processes, with a focus on microbial systems. The aim throughput is 'making such biology predictable'. Strategies have been aimed at both transcription and translation, especially for regulatory processes that can affect multiple targets. However, because there is a limit on how much protein a cell can produce, increasing kcat in selected targets may be a better strategy than increasing protein expression levels for optimal host engineering.
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Antisense Peptide Technology for Diagnostic Tests and Bioengineering Research.
Štambuk, N, Konjevoda, P, Pavan, J
International journal of molecular sciences. 2021;(17)
Abstract
Antisense peptide technology (APT) is based on a useful heuristic algorithm for rational peptide design. It was deduced from empirical observations that peptides consisting of complementary (sense and antisense) amino acids interact with higher probability and affinity than the randomly selected ones. This phenomenon is closely related to the structure of the standard genetic code table, and at the same time, is unrelated to the direction of its codon sequence translation. The concept of complementary peptide interaction is discussed, and its possible applications to diagnostic tests and bioengineering research are summarized. Problems and difficulties that may arise using APT are discussed, and possible solutions are proposed. The methodology was tested on the example of SARS-CoV-2. It is shown that the CABS-dock server accurately predicts the binding of antisense peptides to the SARS-CoV-2 receptor binding domain without requiring predefinition of the binding site. It is concluded that the benefits of APT outweigh the costs of random peptide screening and could lead to considerable savings in time and resources, especially if combined with other computational and immunochemical methods.
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Gene Targeting Facilitated by Engineered Sequence-Specific Nucleases: Potential Applications for Crop Improvement.
Miki, D, Wang, R, Li, J, Kong, D, Zhang, L, Zhu, JK
Plant & cell physiology. 2021;(5):752-765
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Abstract
Humans are currently facing the problem of how to ensure that there is enough food to feed all of the world's population. Ensuring that the food supply is sufficient will likely require the modification of crop genomes to improve their agronomic traits. The development of engineered sequence-specific nucleases (SSNs) paved the way for targeted gene editing in organisms, including plants. SSNs generate a double-strand break (DSB) at the target DNA site in a sequence-specific manner. These DSBs are predominantly repaired via error-prone non-homologous end joining and are only rarely repaired via error-free homology-directed repair if an appropriate donor template is provided. Gene targeting (GT), i.e. the integration or replacement of a particular sequence, can be achieved with combinations of SSNs and repair donor templates. Although its efficiency is extremely low, GT has been achieved in some higher plants. Here, we provide an overview of SSN-facilitated GT in higher plants and discuss the potential of GT as a powerful tool for generating crop plants with desirable features.
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Escherichia coli Extract-Based Cell-Free Expression System as an Alternative for Difficult-to-Obtain Protein Biosynthesis.
Smolskaya, S, Logashina, YA, Andreev, YA
International journal of molecular sciences. 2020;(3)
Abstract
Before utilization in biomedical diagnosis, therapeutic treatment, and biotechnology, the diverse variety of peptides and proteins must be preliminarily purified and thoroughly characterized. The recombinant DNA technology and heterologous protein expression have helped simplify the isolation of targeted polypeptides at high purity and their structure-function examinations. Recombinant protein expression in Escherichia coli, the most-established heterologous host organism, has been widely used to produce proteins of commercial and fundamental research interests. Nonetheless, many peptides/proteins are still difficult to express due to their ability to slow down cell growth or disrupt cellular metabolism. Besides, special modifications are often required for proper folding and activity of targeted proteins. The cell-free (CF) or in vitro recombinant protein synthesis system enables the production of such difficult-to-obtain molecules since it is possible to adjust reaction medium and there is no need to support cellular metabolism and viability. Here, we describe E. coli-based CF systems, the optimization steps done toward the development of highly productive and cost-effective CF methodology, and the modification of an in vitro approach required for difficult-to-obtain protein production.
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Practically useful protein-design methods combining phylogenetic and atomistic calculations.
Weinstein, J, Khersonsky, O, Fleishman, SJ
Current opinion in structural biology. 2020;:58-64
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Abstract
Our ability to design new or improved biomolecular activities depends on understanding the sequence-function relationships in proteins. The large size and fold complexity of most proteins, however, obscure these relationships, and protein-optimization methods continue to rely on laborious experimental iterations. Recently, a deeper understanding of the roles of stability-threshold effects and biomolecular epistasis in proteins has led to the development of hybrid methods that combine phylogenetic analysis with atomistic design calculations. These methods enable reliable and even single-step optimization of protein stability, expressibility, and activity in proteins that were considered outside the scope of computational design. Furthermore, ancestral-sequence reconstruction produces insights on missing links in the evolution of enzymes and binders that may be used in protein design. Through the combination of phylogenetic and atomistic calculations, the long-standing goal of general computational methods that can be universally applied to study and optimize proteins finally seems within reach.
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Engineering Plant Cytochrome P450s for Enhanced Synthesis of Natural Products: Past Achievements and Future Perspectives.
Shang, Y, Huang, S
Plant communications. 2020;(1):100012
Abstract
Cytochrome P450s (P450s) are the most versatile catalysts and are widely used by plants to synthesize a vast array of structurally diverse specialized metabolites that not only play essential ecological roles but also constitute a valuable resource for the development of new drugs. To accelerate the metabolic engineering of these high-value metabolites, it is imperative to identify and characterize pathway P450s, and to further improve their activities through protein engineering. In this review, we focus on P450 engineering and summarize the major strategies for enhancing the stability and activity of P450s and successful cases of P450 engineering. Studies in which the functions of P450s were altered to create de novo metabolic pathways or novel compounds are discussed as well. We also overview emerging tools, specifically DNA synthesis, machine learning, and de novo protein design, as well as the evolutionary patterns of P450s unveiled from a massive number of DNA sequences that could be integrated to accelerate the engineering of these enzymes. These approaches would greatly aid in the exploitation of plant-specialized metabolites or derivatives for various uses including medical applications.
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Catching a SPY: Using the SpyCatcher-SpyTag and Related Systems for Labeling and Localizing Bacterial Proteins.
Hatlem, D, Trunk, T, Linke, D, Leo, JC
International journal of molecular sciences. 2019;(9)
Abstract
The SpyCatcher-SpyTag system was developed seven years ago as a method for protein ligation. It is based on a modified domain from a Streptococcus pyogenes surface protein (SpyCatcher), which recognizes a cognate 13-amino-acid peptide (SpyTag). Upon recognition, the two form a covalent isopeptide bond between the side chains of a lysine in SpyCatcher and an aspartate in SpyTag. This technology has been used, among other applications, to create covalently stabilized multi-protein complexes, for modular vaccine production, and to label proteins (e.g., for microscopy). The SpyTag system is versatile as the tag is a short, unfolded peptide that can be genetically fused to exposed positions in target proteins; similarly, SpyCatcher can be fused to reporter proteins such as GFP, and to epitope or purification tags. Additionally, an orthogonal system called SnoopTag-SnoopCatcher has been developed from an S. pneumoniae pilin that can be combined with SpyCatcher-SpyTag to produce protein fusions with multiple components. Furthermore, tripartite applications have been produced from both systems allowing the fusion of two peptides by a separate, catalytically active protein unit, SpyLigase or SnoopLigase. Here, we review the current state of the SpyCatcher-SpyTag and related technologies, with a particular emphasis on their use in vaccine development and in determining outer membrane protein localization and topology of surface proteins in bacteria.
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Synthesis of Human Milk Oligosaccharides: Protein Engineering Strategies for Improved Enzymatic Transglycosylation.
Zeuner, B, Teze, D, Muschiol, J, Meyer, AS
Molecules (Basel, Switzerland). 2019;(11)
Abstract
Human milk oligosaccharides (HMOs) signify a unique group of oligosaccharides in breast milk, which is of major importance for infant health and development. The functional benefits of HMOs create an enormous impetus for biosynthetic production of HMOs for use as additives in infant formula and other products. HMO molecules can be synthesized chemically, via fermentation, and by enzymatic synthesis. This treatise discusses these different techniques, with particular focus on harnessing enzymes for controlled enzymatic synthesis of HMO molecules. In order to foster precise and high-yield enzymatic synthesis, several novel protein engineering approaches have been reported, mainly concerning changing glycoside hydrolases to catalyze relevant transglycosylations. The protein engineering strategies for these enzymes range from rationally modifying specific catalytic residues, over targeted subsite -1 mutations, to unique and novel transplantations of designed peptide sequences near the active site, so-called loop engineering. These strategies have proven useful to foster enhanced transglycosylation to promote different types of HMO synthesis reactions. The rationale of subsite -1 modification, acceptor binding site matching, and loop engineering, including changes that may alter the spatial arrangement of water in the enzyme active site region, may prove useful for novel enzyme-catalyzed carbohydrate design in general.
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Recent Progress in Chemical Modification of Proteins.
Sakamoto, S, Hamachi, I
Analytical sciences : the international journal of the Japan Society for Analytical Chemistry. 2019;(1):5-27
Abstract
Chemical modification of proteins is important for creating a myriad of engineered proteins and for elucidating the function and dynamics of proteins in live cells. A wide variety of chemical protein modification methods have been developed and can be categorized into three classes: (i) modification of proteins using the reactivity of naturally occurring amino acids; (ii) modification by bioorthogonal reactions using unnatural amino acids, most of which can be site-selectively incorporated into proteins-of-interest using genetic codon expansion techniques; and (iii) recognition driven chemical modification, which is the only approach that allows modification of endogenous proteins without any genetic manipulation even under heavily crowded and multi-molecular conditions, as in live cells and organisms. All of these approaches have merits and limitations. In this review, we summarize these approaches and discuss their characteristics with respect to specificity, reaction rate and versatility.
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Production of protein-based polymers in Pichia pastoris.
Werten, MWT, Eggink, G, Cohen Stuart, MA, de Wolf, FA
Biotechnology advances. 2019;(5):642-666
Abstract
Materials science and genetic engineering have joined forces over the last three decades in the development of so-called protein-based polymers. These are proteins, typically with repetitive amino acid sequences, that have such physical properties that they can be used as functional materials. Well-known natural examples are collagen, silk, and elastin, but also artificial sequences have been devised. These proteins can be produced in a suitable host via recombinant DNA technology, and it is this inherent control over monomer sequence and molecular size that renders this class of polymers of particular interest to the fields of nanomaterials and biomedical research. Traditionally, Escherichia coli has been the main workhorse for the production of these polymers, but the methylotrophic yeast Pichia pastoris is finding increased use in view of the often high yields and potential bioprocessing benefits. We here provide an overview of protein-based polymers produced in P. pastoris. We summarize their physicochemical properties, briefly note possible applications, and detail their biosynthesis. Some challenges that may be faced when using P. pastoris for polymer production are identified: (i) low yields and poor process control in shake flask cultures; i.e., the need for bioreactors, (ii) proteolytic degradation, and (iii) self-assembly in vivo. Strategies to overcome these challenges are discussed, which we anticipate will be of interest also to readers involved in protein expression in P. pastoris in general.