Biocatalytic synthesis of hydroxylated natural products using aldolases and related enzymes

Synthetic building blocks bearing hydroxylated chiral centers are important targets for biocatalysis. Many C-C bond forming enzymes have recently been investigated for new applications and new strategies towards the synthesis of natural products and related oxygenated compounds. Several old catalysts have been studied to increase our functional knowledge of natural aldolase-type enzymes, and new mutated catalysts or catalytic antibodies have been tested for their synthetic utility.     

Towards novel processes for the fine-chemical and pharmaceutical industries

In response to the need in the pharmaceutical industry for more complex, chiral molecules, fine-chemical companies are embracing new manufacturing technologies to produce compounds of these specifications. In particular, recent developments in biocatalysis combined with novel process engineering are providing improved methods for the production of valuable chemical intermediates.     

内消旋-二氨基庚二酸脱氢酶不对称合成非天然手性D-氨基酸的研究进展

内消旋-二氨基庚二酸脱氢酶不对称合成非天然的手性D-氨基酸是目前生物催化领域的研究热点。内消旋-二氨基庚二酸脱氢酶具有优良的立体选择性,利用其进行酶催化不对称合成光学纯的手性D-氨基酸,被广泛用于医药、食品、化妆品、精细化学品等领域。为了促进生物催化法在合成手性D-氨基酸方向的进一步发展,本文对内消旋-二氨基庚二酸脱氢酶催化合成D-氨基酸的现状进行了综述。重点介绍了Corynebacterium glutamicum、Ureibacillus thermosphaericus、Symbiobacterium thermophilum来源的内消旋-二氨基庚二酸脱氢酶在新酶的挖掘、催化性能、晶体结构解析、分子改造、功能与催化机制、合成D-氨基酸新途径等方面的研究进展,并对内消旋-二氨基庚二酸脱氢酶的未来研究方向及策略进行了展望。本综述将进一步加深人们对内消旋-二氨基庚二酸脱氢酶的认识,也为具有挑战性的生物合成任务提供信息借鉴。     

Biocatalysis for pharmaceuticals--status and prospects for a key technology

In reviewing how biocatalysis can be applied to improve chiral synthesis for pharmaceuticals it becomes clear that there will be many opportunities using a simple enzyme system but that many of the more useful applications will require the whole cell because of the requirement for cofactors. An assessment is made of the opportunities to apply metabolic engineering to construct de novo metabolic pathways for the biosynthesis of useful advanced intermediates and a conceptual example is provided for the biosynthesis of cis-aminoindanol. We predict that in the future novel pathways will be assembled for a one-step biosynthesis of many semisynthetic natural products.     

工业生物催化前线动态及名家观点

随着现代生物技术的进步,尤其是酶的快速筛选和活力优化技术的发展,使酶的获取更加容易、酶的操作更加简单,进而促使生物催化成为手性合成的便利工具。综述了一些著名的国际化工或制药公司最近在生物催化技术研发和应用方面的动态信息以及相关技术的一些评论,以便我国从事工业生物催化工作的相关人士能从中获得有益启示。     

手性技术与生物催化

简要介绍了手性,手性技术与生物催化的基本概念。手性,是指一个有机分子具有不对称性,形成两种空间排布方式不同的对映异构体。手性技术即生产手性化合物的技术,手性化合物的制备方法主要有手性源、外消旋体拆分、不对称合成等几种。生物催化,即利用酶或微生物等生物材料催化进行某种化学反应,被认为是手性化合物生产取得突破的关健技术。文章还介绍了生物催化外消旋体拆分、生物催化不对称合成等几种生产手性化合物的应用实例。     

迅速发展中的不对称生物催化技术

本文概述了生物催化技术近年来迅速发展的背景、现状和前景,特别谈到酶在手性合成领域的广泛应用;结合国内外实例,分别介绍了生物催化的不对称氧化还原反应和水解酶催化的对映选择性合成两个主要方面的研究与开发动态.     

The production of fine chemicals by biotransformations

Today, biocatalysis is a standard technology for the production of chemicals. An analysis of 134 industrial biotransformations reveals that hydrolases (44%) and redox biocatalysts (30%) are the most prominent categories. Most products are chiral (89%) and are used as fine chemicals. In the chemical industry, successful product developments involve on average a yield of 78%, a volumetric productivity of 15.5 g/(L.h) and a final product concentration of 108 g/L. By contrast, the pharmaceutical industry focuses on time-to-market. The implications of this for future research and development on biocatalysis are discussed.     

Biocatalysis in drug discovery and development

Enzyme catalysis, enabled by advances in protein engineering and directed evolution, is beginning to transform chemical synthesis in the pharmaceutical industry. This review presents recent examples of the creative use of biocatalysis to enable drug discovery and development. We illustrate how increased access to novel biotransformations and the rise of cascade biocatalysis allowed fundamentally new syntheses of novel medicines, representing progress toward more sustainable pharmaceutical manufacturing. Finally, we describe the opportunities and challenges the industry must address to ensure the reduction to practice of biotechnological innovations to develop new therapies in a faster, more economical, and environmentally benign way.     

Strategies for discovery and improvement of enzyme function: state of the art and opportunities

Developments in biocatalysis have been largely fuelled by consumer demands for new products, industrial attempts to improving existing process and minimizing waste, coupled with governmental measures to regulate consumer safety along with scientific advancements. One of the major hurdles to application of biocatalysis to chemical synthesis is unavailability of the desired enzyme to catalyse the reaction to allow for a viable process development. Even when the desired enzyme is available it often forces the process engineers to alter process parameters due to inadequacies of the enzyme, such as instability, inhibition, low yield or selectivity, etc. Developments in the field of enzyme or reaction engineering have allowed access to means to achieve the ends, such as directed evolution, de novo protein design, use of non‐conventional media, using new substrates for old enzymes, active‐site imprinting, altering temperature, etc. Utilization of enzyme discovery and improvement tools therefore provides a feasible means to overcome this problem. Judicious employment of these tools has resulted in significant advancements that have leveraged the research from laboratory to market thus impacting economic growth; however, there are further opportunities that have not yet been explored. The present review attempts to highlight some of these achievements and potential opportunities.     

Directed evolution of an industrial biocatalyst: 2-deoxy-D-ribose 5-phosphate aldolase

Aldolases are emerging as powerful and cost efficient tools for the industrial synthesis of chiral molecules. They catalyze enantioselective carbon-carbon bond formations, generating up to two chiral centers under mild reaction conditions. Despite their versatility, narrow substrate ranges and enzyme inactivation under synthesis conditions represented major obstacles for large-scale applications of aldolases. In this study we applied directed evolution to optimize Escherichia coli 2-deoxy-D-ribose 5-phosphate aldolase (DERA) as biocatalyst for the industrial synthesis of (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranoside. This versatile chiral precursor for vastatin drugs like Lipitor (atorvastatin) is synthesized by DERA in a tandem-aldol reaction from chloroacetaldehyde and two acetaldehyde equivalents. However, E. coli DERA shows low affinity to chloroacetaldehyde and is rapidly inactivated at aldehyde concentrations useful for biocatalysis. Using high-throughput screenings for chloroacetaldehyde resistance and for higher productivity, several improved variants have been identified. By combination of the most beneficial mutations we obtained a tenfold improved variant compared to wild-type DERA with regard to (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranoside synthesis, under industrially relevant conditions.     

Biocatalytic synthesis of optically active tertiary alcohols

The enzymatic preparation of optically pure tertiary alcohols under sustainable conditions has received much attention. The conventional chemical synthesis of these valuable building blocks is still hampered by the use of harmful reagents such as heavy metal catalysts. Successful examples in biocatalysis used esterases, lipases, epoxide hydrolases, halohydrin dehalogenases, thiamine diphosphate-dependent enzymes, terpene cyclases, -acetylases, and -dehydratases. This mini-review provides an overview on recent developments in the discovery of new enzymes, their functional improvement by protein engineering, the design of chemoenzymatic routes leading to tertiary alcohols, and the discovery of entirely new biotransformations.     

Biocatalysis for pharmaceutical intermediates: the future is now

Biocatalysis is continuing to gain momentum and is now becoming a key component in the toolbox of the process chemist, with a place alongside chemocatalysis and chromatographic separations. The pharmaceutical industry demands a speed of development that must be on a parallel with conventional chemistry and high optical purity for complex compounds with multiple chiral centres. This review describes how these demands are being addressed to make biocatalysis successful, particularly by the use of micro-scale technology for high-speed catalyst screening and process development alongside discipline integration of biology and engineering with chemistry. Developments in recombinant technology will further expand the repertoire of biocatalysis in the coming years to new chemistries and enable catalyst design to fit the process. Further development of biocatalysis for green chemistry and high productivity processes can also be expected.     

Nitrile-converting enzymes as a tool to improve biocatalysis in organic synthesis: recent insights and promises

Nitrile-converting enzymes, including nitrilase and nitrile hydratase (NHase), have received increasing attention from researchers of industrial biocatalysis because of their critical role as a tool in organic synthesis of carboxylic acids and amides from nitriles. To date, these bioconversion approaches are considered as one of the most potential industrial processes using resting cells or purified enzymes as catalysts for production of food additives, pharmaceutical, and agrochemical precursors. This review focuses on the distribution and catalytic mechanism research of nitrile-converting enzymes in recent years. Molecular biology aspects to improve the biocatalytic performance of microbial nitrilase and NHase are demonstrated. The process developments of microbial nitrilase and NHase for organic synthesis are also discussed.     

Trends and innovations in industrial biocatalysis for the production of fine chemicals

Biocatalysis has become an established technology for the industrial manufacture of fine chemicals. In recent years, a multitude of chemical companies have embraced biocatalysis for the manufacture of desired stereoisomers, and new or improved methods for the synthesis of enantiomerically pure alpha- and beta-amino acids, amines, amides, peptides, nitriles, alcohols, organic acids and epoxides have emerged. Furthermore, the selectivity and mild operational conditions of biocatalysts are increasingly applied in industry to modify complex target molecules. These recent innovations in the manufacture of industrial fine chemicals using biocatalysis are discussed from an industrial perspective.     

Extremozymes

Extremozymes offer new opportunities for biocatalysis and biotransformations as a result of their extreme stability. From recent work, major approaches to extending the range of applications of extremozymes have emerged. Both the discovery of new extremophilic species and the determination of genome sequences provide a route to new enzymes, with the possibility that these will lead to novel applications. Of equal importance, protein engineering and directed evolution provide approaches to improve enzyme stability and modify specificity in ways that may not exist in the natural world.     

The importance of sourcing enzymes from non-conventional fungi for metabolic engineering and biomass breakdown

A wealth of fungal enzymes has been identified from nature, which continue to drive strain engineering and bioprocessing for a range of industries. However, while a number of clades have been investigated, the vast majority of the fungal kingdom remains unexplored for industrial applications. Here, we discuss selected classes of fungal enzymes that are currently in biotechnological use, and explore more basal, non-conventional fungi and their underexploited biomass-degrading mechanisms as promising agents in the transition towards a bio-based society. Of special interest are anaerobic fungi like the Neocallimastigomycota, which were recently found to harbor the largest diversity of biomass-degrading enzymes among the fungal kingdom. Enzymes sourced from these basal fungi have been used to metabolically engineer substrate utilization in yeast, and may offer new paths to lignin breakdown and tunneled biocatalysis. We also contrast classic enzymology approaches with emerging ‘omics’-based tools to decipher function within novel fungal isolates and identify new promising enzymes. Recent developments in genome editing are expected to accelerate discovery and metabolic engineering within these systems, yet are still limited by a lack of high-resolution genomes, gene regulatory regions, and even appropriate culture conditions. Finally, we present new opportunities to harness the biomass-degrading potential of undercharacterized fungi via heterologous expression and engineered microbial consortia.     

Bacterial mandelic acid degradation pathway and its application in biotechnology

Mandelic acid and its derivatives are an important class of chemical synthetic blocks, which is widely used in drug synthesis and stereochemistry research. In nature, mandelic acid degradation pathway has been widely identified and analysed as a representative pathway of aromatic compounds degradation. The most studied mandelic acid degradation pathway from Pseudomonas putida consists of mandelate racemase, S-mandelate dehydrogenase, benzoylformate decarboxylase, benzaldehyde dehydrogenase and downstream benzoic acid degradation pathways. Because of the ability to catalyse various reactions of aromatic substrates, pathway enzymes have been widely used in biocatalysis, kinetic resolution, chiral compounds synthesis or construction of new metabolic pathways. In this paper, the physiological significance and the existing range of the mandelic acid degradation pathway were introduced first. Then each of the enzymes in the pathway is reviewed one by one, including the researches on enzymatic properties and the applications in biotechnology as well as efforts that have been made to modify the substrate specificity or improving catalytic activity by enzyme engineering to adapt different applications. The composition of the important metabolic pathway of bacterial mandelic acid degradation pathway as well as the researches and applications of pathway enzymes is summarized in this review for the first time.     

Highlights in Biocatalysis – Historical Landmarks and Current Trends

Biocatalysis has ancient roots, yet it is developing into a key tool for synthesis in a wide range of applications. Important events in the history of enzyme technology from the 19^th century onwards are highlighted. Considering the most relevant progress steps, the production of penicillanic acid and the impact of genetic engineering are traced in more detail. Applied biocatalysis has been defined as the application of a biocatalyst to achieve a desired conversion selectively, under controlled, mild conditions in a bioreactor. Biocatalysts are currently used to produce a wide range of products in the fields of food manufacture (such as bread, cheese, beer), fine chemicals (e.g., amino acids, vitamins), and pharmaceuticals (e.g., derivatives of antibiotics). They not only provide access to innovative products and processes, but also meet criteria of sustainability. In organic synthesis, recombinant technologies and biocatalysts have greatly widened the scope of application. Examples of current applications and processes are given. Recent developments and trends are presented as a survey, covering new methods for accessing biodiversity with new enzymes, directed evolution for improving enzymes, designed cells, and integrated downstream processing.     

Are Lipases Still Important Biocatalysts? A Study of Scientific Publications and Patents for Technological Forecasting

The great potential of lipases is known since 1930 when the work of J. B. S. Haldane was published. After eighty-five years of studies and developments, are lipases still important biocatalysts? For answering this question the present work investigated the technological development of four important industrial sectors where lipases are applied: production of detergent formulations; organic synthesis, focusing on kinetic resolution, production of biodiesel, and production of food and feed products. The analysis was made based on research publications and patent applications, working as scientific and technological indicators, respectively. Their evolution, interaction, the major players of each sector and the main subject matters disclosed in patent documents were discussed. Applying the concept of technology life cycle, S-curves were built by plotting cumulative patent data over time to monitor the attractiveness of each technology for investment. The results lead to a conclusion that the use of lipases as biocatalysts is still a relevant topic for the industrial sector, but developments are still needed for lipase biocatalysis to reach its full potential, which are expected to be achieved within the third, and present, wave of biocatalysis.