Molecular engineering of industrial enzymes: recent advances and future prospects

Many enzymes are efficiently produced by microbes. However, the use of natural enzymes as biocatalysts has limitations such as low catalytic efficiency, low activity, and low stability, especially under industrial conditions. Many protein engineering technologies have been developed to modify natural enzymes and eliminate these limitations. Commonly used protein engineering strategies include directed evolution, site-directed mutagenesis, truncation, and terminal fusion. This review summarizes recent advances in the molecular engineering of industrial enzymes and discusses future prospects in this field. We expect this review to increase interest in and advance the molecular engineering of industrial enzymes.     

蛋白质定向进化的研究进展

在工业生物催化过程和生物细胞工厂构建方面,蛋白质定向进化被广泛地应用于酶的分子改造.蛋白质定向进化不仅可以针对某一目的蛋白进行改造,还可以改善代谢途径、优化代谢网络、获得期望表型细胞.为了获得更高效的突变效率,快捷、高通量的筛选方法,提高蛋白质定向进化的效果,研究者不断开发蛋白质体内、体外进化方法,取得了新的进展和应用.本文介绍了最近发展的蛋白质定向进化技术的原理、方法及特点,总结了突变文库的筛选方法和蛋白质定向进化的最新应用,最后讨论了蛋白质定向进化存在的挑战和未来发展方向.     

Recent progress in industrial biocatalysis

In recent years, several procedures have been reported for the development of biocatalytic processes. This review focuses on selected examples integrating biocatalysts into a variety of industrially interesting processes ranging from the manufacture of smaller, chiral speciality chemicals to the synthesis of more complex pharmaceutical intermediates. The use of rational protein design, multistep processes and de novo design of enzyme catalysts for the stereocontrolled preparation of important target structures is discussed.     

Integrating enzyme immobilization and protein engineering: An alternative path for the development of novel and improved industrial biocatalysts

Enzyme immobilization often achieves reusable biocatalysts with improved operational stability and solvent resistance. However, these modifications are generally associated with a decrease in activity or detrimental modifications in catalytic properties. On the other hand, protein engineering aims to generate enzymes with increased performance at specific conditions by means of genetic manipulation, directed evolution and rational design. However, the achieved biocatalysts are generally generated as soluble enzymes, ?thus not reusable- and their performance under real operational conditions is uncertain.Combined protein engineering and enzyme immobilization approaches have been employed as parallel or consecutive strategies for improving an enzyme of interest. Recent reports show efforts on simultaneously improving both enzymatic and immobilization components through genetic modification of enzymes and optimizing binding chemistry for site-specific and oriented immobilization. Nonetheless, enzyme engineering and immobilization are usually performed as separate workflows to achieve improved biocatalysts.In this review, we summarize and discuss recent research aiming to integrate enzyme immobilization and protein engineering and propose strategies to further converge protein engineering and enzyme immobilization efforts into a novel “immobilized biocatalyst engineering” research field. We believe that through the integration of both enzyme engineering and enzyme immobilization strategies, novel biocatalysts can be obtained, not only as the sum of independently improved intrinsic and operational properties of enzymes, but ultimately tailored specifically for increased performance as immobilized biocatalysts, potentially paving the way for a qualitative jump in the development of efficient, stable biocatalysts with greater real-world potential in challenging bioprocess applications.     

Directed evolution of enzymes and pathways for industrial biocatalysis

Directed evolution has become a powerful tool for developing enzyme and whole cell based biocatalysts. Significant recent advances include the creation of novel enzyme functions and the development of several new efficient directed evolution methods. The combination of directed evolution and rational design promises to accelerate the development of biocatalysts for applications in the pharmaceutical, chemical and food industries.     

Process implementation aspects for biocatalytic hydrocarbon oxyfunctionalization

Oxidoreductases catalyze a large variety of regio-, stereo-, and chemoselective hydrocarbon oxyfunctionalizations, reactions, which are important in industrial organic synthesis but difficult to achieve by chemical means. This review summarizes process implementation aspects for the in vivo application of the especially versatile enzyme class of oxygenases, capable of specifically introducing oxygen from molecular oxygen into a large range of organic molecules. Critical issues such as reaching high enzyme activity and specificity, product degradation, cofactor recycling, reactant toxicity, and substrate and oxygen mass transfer can be overcome by biochemical process engineering and biocatalyst engineering. Both strategies provide a growing toolset to facilitate process implementation, optimization, and scale-up. Major advances were achieved via heterologous overexpression of oxygenase genes, directed evolution, metabolic engineering, and in situ product removal. Process examples from industry and academia show that the combined use of different concepts enables efficient oxygenase-based whole-cell catalysis of various commercially interesting reactions such as the biosynthesis of chiral compounds, the specific oxyfunctionalization of complex molecules, and also the synthesis of medium-priced chemicals. Better understanding of the cell metabolism and future developments in both biocatalyst and bioprocess engineering are expected to promote the implementation of many and various industrial biooxidation processes.     

Biocatalytic ketone reduction: A green and efficient access to enantiopure alcohols

Chiral secondary alcohols play an important role in pharmaceutical, agrochemical, and chemical industries. In recent years, impressive steps forward have been achieved towards biocatalytic ketone reduction as a green and useful access to enantiopure alcohols. An increasing number of novel and robust enzymes are now accessible as a result of the ongoing progress in genomics, screening and evolution technologies, while process engineering provides further success in areas of biocatalytic reduction in meeting synthetic challenges. The versatile platform of these techniques and strategies offers the possibility to apply high substrate loading and thus to overcome the limitation of low volumetric productivity of usual enzymatic processes which is the bottleneck for their practical application. In addition, the integration of bioreduction with other enzymatic or chemical steps allows the efficient synthesis of more complex chiral products.     

Directed evolution strategies for improved enzymatic performance

The engineering of enzymes with altered activity, specificity and stability, using directed evolution techniques that mimic evolution on a laboratory timescale, is now well established. However, the general acceptance of these methods as a route to new biocatalysts for organic synthesis requires further improvement of the methods for both ease-of-use and also for obtaining more significant changes in enzyme properties than is currently possible. Recent advances in library design, and methods of random mutagenesis, combined with new screening and selection tools, continue to push forward the potential of directed evolution. For example, protein engineers are now beginning to apply the vast body of knowledge and understanding of protein structure and function, to the design of focussed directed evolution libraries, with striking results compared to the previously favoured random mutagenesis and recombination of entire genes. Significant progress in computational design techniques which mimic the experimental process of library screening is also now enabling searches of much greater regions of sequence-space for those catalytic reactions that are broadly understood and, therefore, possible to model.     

Biocatalysis in ionic liquids for lignin valorization: Opportunities and recent developments

Lignin holds tremendous potential as a renewable feedstock for upgrading to a number of high-value chemicals and products that are derived from the petroleum industry at present. Since lignin makes up a significant fraction of lignocellulosic biomass, co-utilization of lignin in addition to cellulose and hemicelluloses is vital to the economic viability of cellulosic biorefineries. The recalcitrant nature of lignin, originated from the molecule's compositional and structural heterogeneity, however, poses great challenges toward effective and selective lignin depolymerization and valorization. Ionic liquid (IL) is a powerful solvent that has demonstrated high efficiency in fractionating lignocellulosic biomass into sugar streams and a lignin stream of reduced molecular weight. Compared to thermochemical methods, biological lignin deconstruction takes place at mild temperature and pressure while product selectivity can be potentially improved via the specificity of biocatalysts (lignin degrading enzymes, LDEs). This review focuses on a lignin valorization strategy by harnessing the biomass fractionating capabilities of ILs and the substrate and product selectivity of LDEs. Recent advances in elucidating enzyme-IL interactions as well as strategies for improving enzyme activity in IL are discussed, with specific emphases on biocompatible ILs, thermostable and IL-tolerant enzymes, enzyme immobilization, and surface charge engineering. Also reviewed is the protein engineering toolsets (directed evolution and rational design) to improve the biocatalysts' activity, stability and product selectivity in IL systems. The alliance between IL and LDEs offers a great opportunity for developing a biocatalytic route for lignin valorization.     

Recent advances in heme biocatalysis engineering

Heme enzymes have the potential to be widely used as biocatalysts due to their capability to perform a vast variety of oxidation reactions. In spite of their versatility, the application of heme enzymes was long time-limited for the industry due to their low activity and stability in large scale processes. The identification of novel natural biocatalysts and recent advances in protein engineering have led to new reactions with a high application potential. The latest creation of a serine-ligated mutant of BM3 showed an efficient transfer of reactive carbenes into C═C bonds of olefins reaching total turnover numbers of more than 60,000 and product titers of up to 27 g/L⁻¹. This prominent example shows that heme enzymes are becoming competitive to chemical syntheses while being already advantageous in terms of high yield, regioselectivity, stereoselectivity and environmentally friendly reaction conditions. Advances in reactor concepts and the influencing parameters on reaction performance are also under investigation resulting in improved productivities and increased stability of the heme biocatalytic systems. In this mini review, we briefly present the latest advancements in the field of heme enzymes towards increased reaction scope and applicability.     

Biocatalysis for the synthesis of pharmaceuticals and pharmaceutical intermediates

Biocatalysis has been increasingly used for pharmaceutical synthesis in an effort to make manufacturing processes greener and more sustainable. Biocatalysts that possess excellent activity, specificity, thermostability and solvent-tolerance are highly sought after to meet the requirements of practical applications. Generating biocatalysts with these specific properties can be achieved by either discovery of novel biocatalysts or protein engineering. Meanwhile, chemoenzymatic routes have also been designed and developed for pharmaceutical synthesis on an industrial scale. This review discusses the recent discoveries, engineering, and applications of biocatalysts for the synthesis of pharmaceuticals and pharmaceutical intermediates. Key classes of biocatalysts include reductases, oxidases, hydrolases, lyases, isomerases, and transaminases.     

Computational modeling of carbohydrate processing enzymes reactions

Carbohydrate processing enzymes are of biocatalytic interest. Glycoside hydrolases and the recently discovered lytic polysaccharide monooxygenase for their use in biomass degradation to obtain biofuels or valued chemical entities. Glycosyltransferases or engineered glycosidases and phosphorylases for the synthesis of carbohydrates and glycosylated products. Quantum mechanics-molecular mechanics (QM/MM) methods are highly contributing to establish their different chemical reaction mechanisms. Other computational methods are also used to study enzyme conformational changes, ligand pathways, and processivity, e.g. for processive glycosidases like cellobiohydrolases. There is still a long road to travel to fully understand the role of conformational dynamics in enzyme activity and also to disclose the variety of reaction mechanisms these enzymes employ. Additionally, computational tools for enzyme engineering are beginning to be applied to evaluate substrate specificity or aid in the design of new biocatalysts with increased thermostability or tailored activity, a growing field where molecular modeling is finding its way.     

Extending the capabilities of nature's most versatile catalysts: directed evolution of mammalian xenobiotic-metabolizing P450s

Cytochrome P450 enzymes are amongst the most versatile enzymatic catalysts known. The ability to introduce a single atom of oxygen into an organic substrate has led to the diversification and exploitation of these enzymes throughout nature. Nowhere is this versatility more apparent than in the mammalian liver, where P450 monooxygenases catalyze the metabolic clearance of innumerate drugs and other environmental chemicals. In addition to the aromatic and aliphatic hydroxylations, N- and O-dealkylations, and heteroatom oxidations that are common in drug metabolism, many more unusual reactions catalyzed by P450s have been discovered, including reductions, group transfers and other biotransformations not typically associated with monooxygenases. A research area that shows great potential for development over the next few decades is the directed evolution of P450s as biocatalysts. Mammalian xenobiotic-metabolizing P450s are especially well suited to such protein engineering due to their ability to interact with relatively wide ranges of substrates with marked differences in structure and physicochemical properties. Typical characteristics, such as the low turnover rates and poor coupling seen during the metabolism of xenobiotics, as well as the enzyme specificity towards particular substrates and reactions, can be improved by directed evolution. This mini-review will cover the fundamental enabling technologies required to successfully engineer P450s, examine the work done to date on the directed evolution of mammalian forms, and provide a perspective on what will be required for the successful implementation of engineered enzymes.     

生物催化剂发现与改造的研究进展

生物催化是指将酶或生物有机体用于有用的化学转化的过程,在人们对传统化学催化的环境影响抱有忧虑的情况下,生物催化提供了一种有吸引力的选择。在过去的几十年里,对生物催化剂的研究每出现一次大的进步,生物催化的发展就会出现一次高潮。因此,生物催化剂的发现与改造已成为当今研究的热点。宏基因组文库技术的出现克服了许多微生物不可培养的障碍,人们能够从自然资源中获得丰富的潜在的生物催化剂。而基于理性设计的分子改造技术的发展,可以使得人们对潜在的生物催化剂进行快速而有效的改造以满足工业化生产的需求。随着生物催化剂发现与改造的手段不断进步,更多的优良生物催化剂得到了广泛的应用,生物催化在工业生产中也得到了更深入的应用。结合作者的研究工作,总结了生物催化剂发现与改良的一些研究进展,以为获得更多优良的、能够实现工业应用的生物催化剂奠定理论基础。     

From molecular engineering to process engineering: development of high-throughput screening methods in enzyme directed evolution

With increasing concerns in sustainable development, biocatalysis has been recognized as a competitive alternative to traditional chemical routes in the past decades. As nature’s biocatalysts, enzymes are able to catalyze a broad range of chemical transformations, not only with mild reaction conditions but also with high activity and selectivity. However, the insufficient activity or enantioselectivity of natural enzymes toward non-natural substrates limits their industrial application, while directed evolution provides a potent solution to this problem, thanks to its independence on detailed knowledge about the relationship between sequence, structure, and mechanism/function of the enzymes. A proper high-throughput screening (HTS) method is the key to successful and efficient directed evolution. In recent years, huge varieties of HTS methods have been developed for rapid evaluation of mutant libraries, ranging from in vitro screening to in vivo selection, from indicator addition to multi-enzyme system construction, and from plate screening to computation- or machine-assisted screening. Recently, there is a tendency to integrate directed evolution with metabolic engineering in biosynthesis, using metabolites as HTS indicators, which implies that directed evolution has transformed from molecular engineering to process engineering. This paper aims to provide an overview of HTS methods categorized based on the reaction principles or types by summarizing related studies published in recent years including the work from our group, to discuss assay design strategies and typical examples of HTS methods, and to share our understanding on HTS method development for directed evolution of enzymes involved in specific catalytic reactions or metabolic pathways.

     

Engineered enzymes for chemical production

In order to enable competitive manufacturing routes, most biocatalysts must be tailor-made for their processes. Enzymes from nature rarely have the combined properties necessary for industrial chemical production such as high activity and selectivity on non-natural substrates and toleration of high concentrations of organic media over the wide range of conditions (decreasing substrate, increasing product concentrations, solvents, etc.,) that will be present over the course of a manufacturing process. With the advances in protein engineering technologies, a variety of enzyme properties can be altered simultaneously, if the appropriate screening parameters are employed. Here we discuss the process of directed evolution for the generation of commercially viable biocatalysts for the production of fine chemicals, and how novel approaches have helped to overcome some of the challenges.     

Selective immobilization of Bacillus subtilis lipase A from cell culture supernatant: Improving catalytic performance and thermal resistance

Bacillus subtilis lipase A (BSLA) has been extensively studied through protein engineering; however, its immobilization and behavior as an insoluble biocatalyst have not been extensively explored. In this work, for the first time, a direct immobilization of recombinant BSLA from microbial culture supernatant was reported, using chemically modified porous with different electrostatic, hydrophobic, hydrophilic, and hydrophilic−hydrophobic enzyme-support interactions. The resulting biocatalysts were evaluated based on their immobilization kinetics, activity expression (pH 7.4), thermal stability (50 °C), solvent resistance and substrate preference. Biocatalysts obtained using glyoxyl silica support resulted in the selective immobilization of BSLA, resulting in an activity recovery of 50 % and an outstanding aqueous stabilization factor of 436, and 9.5 in isopropyl alcohol, compared to the free enzyme. This selective immobilization methodology of BSLA allows to efficiently generate immobilized biocatalysts, thus avoiding laborious purification steps from cell culture supernatant, which is usually a limiting step when large amounts of enzyme variants or candidates are assessed as immobilized biocatalysts. Direct enzyme immobilization from cell supernatant provides an interesting tool which can be used to facilitate the development and assessment of immobilized biocatalysts from engineered enzyme variants and mutant libraries, especially in harsh conditions, such as high temperatures or non-aqueous solvents, or against non-water-soluble substrates. Furthermore, selective immobilization approaches from cell culture supernatant or clarified lysates could help bridging the gap between protein engineering and enzyme immobilization, allowing for the implementation of immobilization steps in high throughput enzyme screening platforms for their potential use in directed evolution campaigns.     

Tools and ingredients for the biocatalytic synthesis of carbohydrates and glycoconjugates

The presence of multiple functional groups and stereocentres in carbohydrates and glycoconjugates make them challenging targets for synthesis. Although progress in chemical synthesis and engineering is impressive, there is still a need to selectively introduce and remove protecting groups in the total synthesis of target molecules of increasing complexity. Multiple hydroxyl-groups with similar reactivities have to be differentiated in order to form the desired glycosidic bonds in a regio- and stereospecific way. To complement the existing chemical tools and ingredients, biocatalysts for selective carbon–carbon bond formation and glycosylation reactions have been developed. The availability of auxiliary ingredients like transfer reagents is a prerequisite for the development of viable biocatalytic process steps. In the case of dihydroxyacetone-phosphate-dependent aldolases, e.g. fructose-1,6-bisphosphate aldolase (EC 4.1.2.13), the large-scale availability of dihydroxyacetone-phosphate (DHAP) eliminates the need to synthesize the donor DHAP. For the pyruvate-dependent aldolases, e.g. the N-acetylneuraminic acid aldolase (EC 4.1.3.3) and acetaldehyde-dependent aldolases like the 2-deoxy-d-ribose-5-phosphate aldolase (4.2.1.4), the donors pyruvate and acetaldehyde are also available on a large scale. A broad range of natural and recombinant aldolases have been produced in stable lyophilized form. Recombinant transketolase together with a new synthesis of hydroxypyruvates has provided a platform technology for the preparation of monosaccharides, whereby the carbon backbone is extended by a two-carbon unit (C2-elongation). Natural and recombinant glycosyltransferases have been prepared on a large-scale to establish biocatalytic glycosylations in water as highly regio- and stereospecific reaction methodologies without the need for laborious protecting group manipulations, solubility adaptations and complex synthetic schemes. In order to simplify the synthetic manipulations for specific glycosylations, toolkits for β-1,4-galactosylations, α-1,3-galactosylations and α-1,3-fucosylations have been developed for rapid quantitative conversions. The introduction of matched pairs of biocatalysts and transfer reagents as ingredients together with the optimized reaction methodology as tool provide an important starting point for biocatalytic glycomics.     

Structure–function relationships of epoxide hydrolases and their potential use in biocatalysis

Background

Chiral epoxides and diols are important synthons for manufacturing fine chemicals and pharmaceuticals. The epoxide hydrolases (EC 3.3.2.-) catalyze the hydrolytic ring opening of epoxides producing the corresponding vicinal diol. Several isoenzymes display catalytic properties that position them as promising biocatalytic tools for the generation of enantiopure epoxides and diols.

Scope of review

This review focuses on the present data on enzyme structure and function in connection to biocatalytic applications. Available data on biocatalysis employed for purposes of stereospecific ring opening, to produce chiral vicinal diols, and kinetic resolution regimes, to achieve enantiopure epoxides, are discussed and related to results gained from structure–activity studies on the enzyme catalysts. More recent examples of the concept of directed evolution of enzyme function are also presented.

Major conclusions

The present understanding of structure–activity relationships in epoxide hydrolases regarding chemical catalysis is strong. With the ongoing research, a more detailed view of the factors that influence substrate specificities and stereospecificities is expected to arise. The already present use of epoxide hydrolases in synthetic applications is expected to expand as new enzymes are being isolated and characterized. Refined methodologies for directed evolution of desired catalytic and physicochemical properties may further boost the development of novel and useful biocatalysts.

General significance

The catalytic power of enzymes provides new possibilities for efficient, specific and sustainable technologies to be developed for production of useful chemicals.     

Incorporation of non-natural modules into proteins: structural features beyond the genetic code

The biotechnological application of enzymes necessitates a permanent quest for new biocatalysts. Among others, improvement of catalytic activity, modification of substrate specificity, or increase in stability of the enzymes are desirable goals. The exploration of homologous enzymes from various sources or DNA-based methods, like site-directed mutagenesis or directed evolution, yield an incredible variety of biocatalysts but they all rely on the restricted number of canonical amino acids. Chemistry offers an almost unlimited palette of additional modifications which can endow the proteins with improved or even completely new properties. Numerous techniques to furnish proteins with non-natural amino acids or non-proteinogenic modules have been introduced and are reviewed with special focus on expressed protein ligation, a method that combines the potential of protein biosynthesis and chemical synthesis. An erratum to this article can be found at