Table of Content

30 June 2022, Volume 3 Issue 3

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Invited Review



Artificial intelligence-assisted protein engineering

Jiahao BIAN, Guangyu YANG

2022, 3(3):  429-444.  doi:10.12211/2096-8280.2021-032

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Protein engineering is one of the important research fields of synthetic biology. However, de novo design of protein functions based on rational design is still challenging, because of the limited understanding on biological fundamentals such as protein folding and the natural evolution mechanism of enzymes. Directed evolution is capable of optimizing protein functions effectively by mimicking the principle of natural evolution in the laboratory without relying on structure and mechanism information. However, directed evolution is highly dependent on high-throughput screening methods, which also limits its applications on proteins which lack high-throughput screening methods. In recent years, artificial intelligence has been developed very rapidly for integrating into multidisciplinary fields. In synthetic biology, artificial intelligence-assisted protein engineering has become an efficient strategy for protein engineering besides rational design and directed evolution, which has shown unique advantages in predicting the structure, function, solubility of proteins and enzymes. Artificial intelligence models can learn the internal properties and relationships from given sequence-function data sets to make predictions on properties for virtual sequences. In this article, we review the application of artificial intelligence-assisted protein engineering. With the basic and process of the strategy introduced, three key points that affect the performance of the predictive model are analyzed: data, molecular descriptors and artificial intelligence algorithms. In order to provide useful tools for researchers who want to take advantage of this strategy, we summarize the main public database, diverse toolkits and web servers of the common molecular descriptors and artificial intelligence algorithms. We also comment on the functions, applications and websites of several artificial intelligence-assisted protein engineering platforms, through which a complete prediction task including protein sequences representation, feature analysis, model construction and output can be completed easily. Finally, we analyze some challenges that need to be solved in the artificial intelligence-assisted protein engineering, such as the lack of high-quality data, deviation in data sets and lacking of the universal models. However, with the development of automated gene annotations, ultra-high-throughput screening technologies and artificial intelligence algorithms, sufficient high-quality data and appropriate algorithms will be developed, which can enhance the performance of artificial intelligence-assisted protein engineering and thus facilitate the development of synthetic biology techniques.





Molecular chaperones promote protein stability and evolution

Yuqi TANG, Songtao YE, Jia LIU, Xin ZHANG

2022, 3(3):  445-464.  doi:10.12211/2096-8280.2022-013

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Native proteins are only marginally stable. Therefore, a few mutations or slight perturbation in the environment could easily destroy their functional structures, causing them to misfold or even aggregate. The proteome is also believed to be marginally stable as the malfunction of a handful of proteins could rapidly overload the ubiquitin-protease network, threatening the integrity of the entire proteome. The disruption of proteostasis would render tremendous side effects including tumors and diseases. Extensive molecular machineries, such as heat shock proteins, are employed by cells to assist certain protein folding, salvage misfolded proteins, and break down protein aggregates. Owing to this fact, many natively occurred molecular chaperones have the potency to be engineered as stabilizers for the expression of aggregation-prone proteins both in vitro and in vivo, or into specialized disaggregates towards disease-related proteins. Remarkably, these modifications could be achieved with minor changes in the primary sequence of typical molecular chaperones, which are often proved to be single-site mutations. Instead of focusing on particular molecular chaperones, an up-regulation of the entire proteostasis network components is proved to be a viable strategy in maintaining protein homeostasis. Mutations could also render proteins to evolve new or improved functions in given environments, even though most mutations are detrimental. Both theoretical and experimental studies have found that extra thermodynamic stability could promote evolvability by allowing a protein's native structure and function to tolerate random mutations more robustly. Increased mutational tolerance allows proteins to evolve faster to adapt to new environments. Molecular chaperones are also found to serve as a buffering system, alleviating stability constraints, and rescuing deleterious mutations that could mediate new or improved functions. Hopefully, with the advancement in biotechnology and computational analysis, more studies that reveal influences of molecular chaperones on protein stability and evolvability can provide better insights into deciphering the relationship between protein structures and functions, as well as fundamental theories exploring the pathogenesis of protein-related diseases.





Cell-free protein synthesis: from basic research to engineering applications

Jiaqi HOU, Nan JIANG, Lianju MA, Yuan LU

2022, 3(3):  465-486.  doi:10.12211/2096-8280.2021-064

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Cell-free protein synthesis (CFPS), also known as in vitro gene expression, is a multifunctional technique used to complement cell-based protein expression, which is at the core of cell-free synthetic biology. Since the CFPS system does not require a living cell, it can simulate the entire cellular transcription and translation process in vitro in a controlled environment, and allows for an in-depth study of individual components and biological networks. Therefore, as a platform technology, it is expected to overcome the loopholes caused by the limitations of cell membranes in the current in vivo manufacturing systems, which has a broad research prospect in fundamental and applied scientific research. The cell-free operation is simple and easy to control, and its advantages over in vivo protein expression include its nature with open systems, eliminating the dependence on living cells and using all system energy for the production of the target proteins. This article reviews the composition of CFPS systems and their development based on different component types, including different biological extracts or purified transcription and translation components. Furthermore, different CFPS reaction patterns are introduced, including batch and continuous exchange modes, and the research progress of CFPS systems in genetic circuits, protein engineering, and the construction of artificial life is described. Among them, the genetic circuit research progress mainly summarizes the latest applications and contributions of cell-free technology in the prototype design, biosensors, and in vitro metabolic engineering. The protein engineering research progress lists the advantages and advances of the CFPS systems for producing membrane proteins, virus-like particles, post-translational modifications, unnatural amino acid incorporation and protein evolution. In the construction of artificial "living systems", the synthesis of bacteriophages and the construction of artificial cells have opened up a novel frontier field. Finally, the opportunities and challenges of the CFPS platforms for future scientific research and industrial applications are highlighted.





Progress in the application of protein engineering in the developing of feed enzymes

Tao TU, Huiying LUO, Bin YAO

2022, 3(3):  487-499.  doi:10.12211/2096-8280.2022-027

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Feed enzymes are one of the hotspots for research in the field of feed additives. Because of their superior qualities of no residue, no pollution, and no drug resistance, feed enzymes have been widely promoted and applied in the feed industry. As functional additives, feed enzymes have tremendously fostered feed development. Since the catalytic performance of feed enzyme is the core factor in determining their application effectiveness, one of the primary challenges faced by scientists engaged in the R&D of feed enzymes is how to improve their overall performance. Based on the application requirement for feed enzymes, this review focuses on their thermal stability, pH dependence, protease resistance, and catalytic activities. We summarize the application of computer-aided protein rational design technology in the R&D of feed enzymes, and introduce effective molecular design strategies for improving their catalytic performance. In addition, we present the progress in improving the performance of key feed enzymes by protein engineering technology, demonstrating the application prospect of enzyme molecular design technology based on the knowledge of protein structures. We also prospect the future development of protein engineering technology in feed enzymes, including (1) application-oriented designs for feed enzymes, (2) molecular design of multifunctional enzymes for efficient utilization of feed materials, (3) improvement of the thermostability as well as the catalytic performance of feed enzymes to meet the requirement of feed pelleting and under digestive and intestinal conditions, and (4) innovation and development of enzyme molecular design technology. Improving the catalytic performance of feed enzymes comprehensively from the perspective of enzyme proteins using synthetic biology tools will promote the development of the environmental adaptive molecular design of feed enzymes to a new stage.





Application of enzyme catalysis in the preparation of vitamins and their derivatives

Panpan WANG, Hongwei YU

2022, 3(3):  500-515.  doi:10.12211/2096-8280.2021-070

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Enzymes, as a kind of natural catalysts, often have unique and excellent catalytic performance compared with chemical catalysts. The mining, modification and application of enzymes have always been a key research field of bioengineering. With the development of enzymes mining and modification technology, enzymatic catalysis has been widely used in industry. In the production of vitamins, vitamin C and vitamin B₁₂ have been produced by fermentation with a long history, and vitamin B₂ has also been produced by fermentation instead of chemical synthesis since the beginning of this century. In addition to the above vitamins, other vitamins are mainly synthesized by chemical routes. In the chemical process of vitamin synthesis, more and more cases of enzymatic catalysis to replace chemical catalysis have been explored for semi-chemo-based production. For examples: Biological enzymatic resolution instead of chemical chiral resolution in vitamin B₅ synthesis; Nitrile hydration catalyzed by nitrile hydratase instead of chemical catalyst in the synthesis of vitamin B₃; The synthesis of active vitamin D₃ [such as 25(OH) vitamin D₃] by P450 enzyme. Furthermore, many vitamin esters or glycoside derivatives are synthesized by lipases or glycosyltransferases. In this article, industrial applications of enzymes in this regard are reviewed, including screening, directed evolution and rational modification of the enzymes. Moreover, the applications of enzymatic catalysis in the synthesis of vitamins are summarized, including vitamin B₃, vitamin B₅ and vitamin D₃. The production of vitamin C is also highlighted because its fermentation process is similar to enzymatic catalysis. We also summarize the synthesis of several vitamin derivatives, mainly including ester derivatives of vitamin A, vitamin C and vitamin E, which are synthesized by lipases, and glycoside derivatives of vitamin C, which are synthesized by glycosyltransferases. Finally, we compare the advantages and disadvantages of chemical synthesis, fermentation and enzymatic catalysis in the production of vitamins. The characteristics and application potential of enzymatic catalysis are summarized. With the in-depth understanding of enzymatic catalysis mechanism, enzymes will play their unique advantages in the synthesis of vitamins and other natural products through integration of chemical engineering, computer aided design and other interdisciplinary knowledges.





Application of imine reductase in the synthesis of chiral amines

Lu YANG, Xudong QU

2022, 3(3):  516-529.  doi:10.12211/2096-8280.2021-054

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Chiral amines with bioactivities are important chiral auxiliaries, and also key intermediates for the synthesis of many natural products and chiral drugs. Among the top 200 drugs for market revenues in 2019, more than 30% contain chiral amine structures. Therefore, the development of efficient and effective methods to synthesize chiral amine compounds is of interest for research. Due to its high efficiency, environmental friendliness and economic competitiveness, more attention has been paid on the enzyme-catalyzed production of chiral amines by academia and industry. Imine reductases (IREDs) reviewed in this article are a class of NAD(P) H-dependent oxidoreductases that catalyze asymmetric reduction of imines to chiral amines. The reduction of C\underset{\text{??????}}{?}N bonds constitutes a physiological reaction present in a number of biosynthetic pathways, leading to a variety of metabolites. The imine reductases have excellent characteristics such as high catalytic efficiency, strong regioselectivity and stereoselectivity, etc., which stand out among many other methods for the synthesis of chiral amines, and attract attention and enthusiasm of many researchers. In the past decade, with the rapid development of bioinformatics, structural biology, high-throughput screening approaches and the continuous expansion of the gene databases, many imine reductases with different functions have been identified. Significant achievements have been made in the discovery of IREDs, protein engineering and multi-enzyme cascade applications, among which some successful modification cases have industrial application potentials. This review summarizes the structural characteristics, catalytic mechanisms and applications of IREDs, with emphasis on their protein engineering and applications in multi-enzyme cascade reactions, as well as the bottleneck, breakthrough and progress in asymmetric catalytic chiral amine biosynthesis. In addition, the challenges and potentials of the enzymatic synthesis of chiral amine compounds for industrial production, and the importance of novel artificial biosynthesis pathway design to overcome these challenges are highlighted.





Progress of biocatalytic deuteration of inert carbon-hydrogen bonds

Yujiao LOU, Jian XU, Qi WU

2022, 3(3):  530-544.  doi:10.12211/2096-8280.2021-067

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Deuterated compounds display unique properties, and thus have important applications in chemistry, biology, and related areas. For example, deuterated compounds are widely used in elucidating reaction mechanism in organic synthesis because deuterium atom has similar chemical reactivities to hydrogen atom but different magnetic property and mass. In pharmaceutical industry, deuterium-labeled compounds are used for improving the metabolism and pharmacokinetic properties of newly developed pharmaceuticals due to the higher chemical inertness of C—D bonds compared with the C—H bonds. The first deuterated drug, Austedo^TM, was approved in 2017 by the US Food and Drug Administration, which has greatly stimulated the research of deuterium incorporation in pharmaceuticals.Traditional chemical methods for introducing deuterium into compounds rely heavily on transition metal catalysis performed under harsh reaction conditions using complicated ligands with low selectivity, which limit their broad applications. Thus, developing an effective, highly selective, low-cost, and environmental-friendly process for deuteration is highly desirable. Comparing with chemical catalysts, enzymes offer a powerful route for producing chemicals due to their green nature and high selectivity. Thus, biocatalysis has been regarded as an attractive strategy for deuteration. So far, to the best of our knowledge, reported biocatalytic deuteration methods mainly include three types: hydrogen deuterium exchange (HDE), reduction deuteration and decarboxylation deuteration. The biocatalytic production of HDE is the most efficient synthesis process with good atom-economy, which mostly effectively introduces the isotope into the α-position of the carboxyl group. Bioreductive deuteration usually uses a NADH-dependent reductase with D₂O to construct deuterated chiral alcohols or amines. The decarboxylation deuteration offers good selectivity to introduce deuterium into the carboxyl group using carboxylic acids as low-cost starting materials. In this review, we describe recent advances in the three types of biocatalytic deuteration processes respectively. In these cases, D₂O is used as the main source for deuterium atoms. Although studies on the preparation of deuterated compounds by biocatalysis are still in the initial stage, it is expected to be flourish rapidly due to the importance of deuterated compounds.