Biological enzyme catalysis technology and application
Enzymes are very powerful catalysts that use complex active sites to process chemical transformations. The tremendous rate acceleration and unparalleled selectivity that can be achieved by enzymes make them attractive catalysts that can be used in sustainable manufacturing processes. The field of biocatalysis has evolved to a stage where it is now considered a key enabling technology for the development of a greener and more efficient chemical industry. Several major methodological innovations have underpinned the rapid progress, including：
1) Providing fast, accurate and low-cost DNA synthesis and sequencing services.
2) the development of advanced bioinformatics tools and computational modeling methods.
3) and increasingly complex experimental workflows for high-throughput structural and biochemical enzyme characterization.
However, due to the inherent complexity of biological systems and the poor applicability of natural enzymes to non-natural products, biocatalytic processes are often developed with long lead times and huge investments, which severely limit the diffusion of biocatalytic technologies. Natural enzymes, usually require protein engineering to optimize their properties for practical applications. In recent years, high-throughput protein engineering strategies, most notably directed evolution, have emerged for the development of biocatalysts to efficiently and selectively process non-natural substrates and operate effectively under commercially viable process conditions.
Intra-organismal catalysis
In-organism catalysis takes place in living host cells through the organic combination of multiple enzymes that catalyze reactions step by step according to metabolic pathways. Metabolic engineering, on the other hand, is the design and construction of these metabolic pathways to obtain products more efficiently. Since bioconversion in living cells does not require the addition of exogenous cofactors or steps such as protein purification, in vivo catalysis becomes a cost-effective approach.
In vivo fermentation and metabolic engineering Metabolic engineering uses molecular biology techniques or genetic engineering such as DNA recombination to modify or construct cellular activities and metabolic pathways by manipulating intracellular enzymes, transport and regulatory functions to improve the yield of desired products or to obtain new products1. A successful metabolic engineering is based on the analysis of cellular functions to design improved strains, which are then constructed by genetic engineering. With the increasing research on metabolic engineering, many have classified metabolic engineering with their own understanding of it1,2, among which Nielsen J. subdivided biological metabolic engineering according to different methods and purposes3: 1. insertion of heterologous genes into the host to produce heterologous proteins; 2. expansion of microbial applications of substrates for more efficient use of raw materials; 3. Production of completely new metabolic pathways through strategies such as gene shuffling or protein directed evolution to increase the rate of production; 4. Engineering the metabolic pathways of specific organisms to have the ability to degrade multiple compounds simultaneously by inserting them into other organisms; 5. Engineering cell physiology to improve the physicochemical properties and morphology of cells; 6. Designing metabolic pathways to avoid negative effects of byproducts on cells, thus Eliminate or reduce the generation of by-products; 7. Increase yield and productivity by increasing the activity of biosynthetic pathways.
Biological in vitro catalysis
Biological in vitro (in vitro) catalysis, on the other hand, can simplify the process by engineering the performance of multiple enzymes under actual production conditions, with multiple enzymes participating in the cascade reaction simultaneously, reducing the production of some intermediates or by-products, avoiding the toxicity of by-products to cells, the separation of intermediates, and improving the yield and purity, while saving production costs4.
There are various types of enzyme-catalyzed cascade reaction systems, and the most common ones are currently classified into the following four categories5: (A) linear cascade reactions; (B) orthogonal cascade reactions; (C) parallel cascade reactions; and (D) cyclic cascade reactions. In the actual cascade reactions, usually not only one reaction type is applied, but multiple types are combined, and oxidoreductases and transaminases are the most frequently applied enzymes among them.
A. The conversion of a single substrate into a single end product after enzyme catalysis into one or more intermediates is called a linear cascade reaction and it is the simplest and most intuitive of the four cascade systems;
B. The orthogonal cascade reaction is characterized by the enzyme converting the substrate to a product and then participating in another enzyme reaction in the presence of a co-substrate in order to regenerate the cofactor or co-substrate, or to remove the generated by-products so that the reaction proceeds in a positive direction. The most classical orthogonal cascade is the coupling of a carbonyl reductase to a dehydrogenase enzyme resulting in regeneration of the coenzyme.
C. A process in which two or more substrates are converted into two or more products by different biocatalytic reaction systems, but they share the same set of coenzymes or co-substrate regeneration systems is called parallel cascade;
D. Cyclic cascade reactions in which multiple substrates can be selectively converted into an intermediate, which is subsequently converted into the desired compound or the initial raw material for repeated cyclic reactions in which the desired compound is continuously accumulated.
In vivo vs. in vitro comparison and analysis of advantages and disadvantages
In vivo biotransformation
Advantages：
1、Coenzyme cycle (such as ATP, NADPH, SAM, etc.) can be realized in vivo without additional supplementation.
2, the internal environment is stable, the activity and stability of related enzymes are not easily affected.
Disadvantages：
1、Synthesis of compounds with active metabolic pathways in the cell will limit their accumulation concentration (e.g. pyruvate), i.e. they are easily converted into other substances, resulting in a low final yield.
2. not easy to synthesize compounds that are toxic to cells.
3. the kinetics of metabolic pathways are not easily controlled and modified
4. the product concentration is usually low and the isolation and purification is complicated.
Need to balance the competition between product production pathway and cell survival pathway for substances.
In vitro enzyme cascade
Advantages：
1、Reducing reaction steps and reducing costs.
2、The reaction system can be flexibly selected according to the physicochemical characteristics of the product and the requirements of the separation process, which makes it easier to carry out systematic process optimization and can achieve high spatial and temporal yields and extraction yields.
3. Suitable bulk products can be selected as starting materials, and the highest atomic economy (100%) can be achieved.
Disadvantages：
1、When expensive coenzymes such as ATP and NADPH need to be used, the construction of the coenzyme cycle system needs to be considered at the same time.
2、The stability of some enzymes in vitro is poor, and there are difficulties in large-scale stable production.
The collaboration of multiple enzymes in vitro is often less orderly and efficient than in vivo.
Application of bioenzyme catalysis in drug synthesis
A variety of biocatalysts are available for the synthesis of drugs and drug intermediates in solving environmental problems in the pharmaceutical industry, including oxidases, reductases, hydrolases, cleavage enzymes, isomerases, and transaminases. In this paper, the most common oxidation and reduction reactions are used as examples to briefly discuss the application of bioenzyme catalysis in drug synthesis.
Oxidation reaction
Biooxidation reactions are now widely used in the pharmaceutical industry, and the use of oxidases for biocatalysis is increasingly being explored. For example, the hepatitis C virus protease inhibitors telaprevir and boceprevir and the proton pump inhibitor esomeprazole use oxidases in their industrial production. As another example, the phytoestrogen pinoresinol has been much studied by the pharmaceutical community because of its protective effect on the organism in a variety of disease processes. A two-step, one-pot synthesis of turpentine alcohol using vanillyl alcohol oxidase from Penicillium simplicissimum and bacterial laccase has been reported using inexpensive eugenol as a starting material. Under optimized conditions, the synthesis of pinoresinol could reach a semi-preparative scale of 1.6 g/L (Figure 2).
Figure 2. Synthesis of pinoresinol by tandem reaction using vanillyl alcohol oxidase and bacterial laccase
Reduction reaction
The use of bioenzyme catalysts is highly effective for regioselectivity and stereoselectivity of carbonyl functional groups, especially important in the reductive synthesis of chiral drug intermediates. For example new microbial strains have been isolated from the environment with good carbonyl reduction activity as well as desired biochemical properties including thermal stability and tolerance to organic solvents.
Optically pure tert-butyl 6-cyano-(3R,5R)-dihydroxyhexanoate is a key chiral precursor of Lipitor (atorvastatin calcium) and is produced by the enzymatic reduction of tert-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate. It has been reported that a new carbonyl reductase, KlAKR, was identified in the yeast Kluyveromyces lactis, capable of asymmetrically reducing tert-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate (Figure 3). The enzyme activity was also improved using a semi-rational design strategy, and the mutant Y295W/W296L, obtained after two rounds of site-saturation mutagenesis screening based on homology modeling and molecular docking, was 11.25-fold more efficient in catalysis than wild-type KlAKR.
Figure 3. Reduction reaction catalyzed by reductase KlAKR
New enzyme catalysts are continuously developed by screening new microorganisms, or identifying new genes through genome mining, and then optimizing and screening these biological enzymes through powerful enzyme engineering tools, including rational design, irrational design, and high-throughput screening platforms. Not only developing new enzymes but also finding the environment to exert their activity and applying them to suitable reactions.
Research Prospects of Biological Enzymes
The tremendous advances made in the last decade in enzyme design and engineering, bottom-up or ab initio enzyme design, where entirely new catalytic centers are created within the protein host, could provide a universal solution for the speed and scope of future biocatalyst delivery. If design is to meet or even exceed the level of practical utility achieved by more established top-down approaches to biocatalyst development, the field of research must now address two core challenges：
First, it is important to design highly active enzymes with efficiencies that more closely resemble natural systems. Currently, even for relatively simple transformations, many designs must be generated and experimentally tested to identify some that exhibit the desired activity, and extensive evolutionary optimization is required to bridge the efficiency gap with natural enzymes. The design challenge is amplified when targeting multi-step reactions, where carefully planned conformational adjustments are required to accurately identify multiple chemical states. Striking a balance between active site preorganization and conformational kinetics is critical to the future success of enzyme design.
Second, it is important to expand the range of chemistry achievable with ab initio enzymes and to develop catalysts for valuable chemical processes that can be implemented on a large scale. Here, a broader collaboration between organic chemists and protein designers is particularly valuable in identifying suitable active site arrangements for new target translations. The range of accessible chemicals can be greatly expanded by engineering cellular translations to introduce new functional amino acids into proteins that can be used to modulate the catalysis of metal ion cofactors or as genetically encoded substitutes for small molecule organocatalysts. In the future, enzyme designers and engineers will continue to advance the field by developing catalysts for increasingly complex transformations.
References
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2.Cameron, D. C., & Tong, I.-T. (1993). Cellular and metabolic engineering. Applied Biochemistry and Biotechnology, 38(1-2), 105–140. doi:10.1007/bf02916416
3.Nielsen, J. (2001). Metabolic engineering. Applied Microbiology and Biotechnology, 55(3), 263–283. doi:10.1007/s002530000511
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5.Ricca, E., Brucher, B., & Schrittwieser, J. H. (2011). Multi-Enzymatic Cascade Reactions: Overview and Perspectives. Advanced Synthesis & Catalysis, 353(13), 2239–2262. doi:10.1002/adsc.201100256