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Industry perspective

When the scientific community started to investigate the applications of enzymes in the organic synthesis the expectations were very high. A completely new

synthetic chemistry without organic solvents, without heavy metals and eliminating environmental pollution seemed to be within reach. Since then a noteworthy number of bio-catalytic processes have found their way into industrial application (1-4). However, the main benefit of bio-catalysis chemists appreciated and still appreciate is its treasure chest of synthetic alternatives. Where chemical methods fail, enzymes can be found that exhibit the needed regio- and stereoselectivity or the high tolerance of multiple functionalities. Today the challenges we are facing due to the increasing global industrialisation and the increasing global population make the old visions even more desirable (5). The conceivable or already existing ecological problems and the shortage of resources made scientist all over the world work hard to identify solutions which allow us but also next generations to manage with those funds earth is providing to us. These efforts pushed the biotechnology to a next level in many different fields of industry. In this article we will focus on the application of bio-catalysis in the field of fine and speciality chemistry.

The first example of a bio-catalyst application as a tool in organic synthesis was the production of L-Phenylacetylcarbinol (L-PAC) – the key intermediate in the synthesis of ephedrine (6). In a whole cell transformation the conversion of benzaldehyde and pyruvate to L-PAC is catalysed by an enzyme from the class of lyases; more precisely a pyruvate decarboxylase. Though this process was already established in the 1920th additional examples for industrial applications of bio-catalytic organic synthesis were rare in the following decades. The reason for this was that enzymes were evolved by nature to work under physiologic conditions. This means that enzymes are produced by their host organisms only in small amounts. In these organisms they catalyse very low concentrations of substrates to very low concentrations of product and they are often very specific for a certain substrate. Mainly starting in the 1970th tremendous improvements made in the fields of microbiology and molecular biology which helped to overcome these hurdles boosted the number of innovative studies, inventions and applied processes. Two decades ago scientist were happy to isolate minimal quantities of a wild-type enzyme from its natural host and to convert milligrams of substrate in a complex aqueous medium. Today the production of a tailor made enzyme variant optimised regarding a broad variety of reaction parameters in a complex evolution process in a highly efficient expression host on multi-cubic metre scale is state of the art. And also the application of such enzymes under completely non-physiological conditions (e.g. in organic solvent, under extreme pH values or in the presence of high substrate concentrations) is nothing extraordinary for chemists and process engineers anymore. The first enzyme class that attracted the interest of the organic chemists and were applied on industrial scale were hydrolases, enzymes that hydrolyse bonds under water consumption. In some of the processes established it is made use of the capability of many enzymes to catalyse the forward- as well as the back-reaction. As far as the hydrolases are concerned in the chemical synthesis the enzymes are mainly used for the resolution of racemic mixtures.For example various enantiomerically pure L-amino acids are produced applying this method (Scheme 1a) (7). Within the last decades a broad range of enzyme classes have been investigated and processes for industrial application have been developed.Among the most intensively studied enzymes are Transaminases which belong to the class of transferases. These convert ketones into chiral amines. Due to the high synthetic potential they are currently investigated intensively by various companies.However, there is only one example for an industrial application until now; the synthesis of a key intermediate of the antihyperglycemic Sitagliptin (Scheme 1b) (8).

Biocatalysis; enzymes; sustainability; bio-based chemicals; designer microbes.

The tremendously fast advancements in the fields of molecular biology and biotechnology which made customised bio-catalysts available on industrial scale lead to the successful implementation of many biotechnological process in the chemical and pharmaceutical industry. A broad variety of enzyme classes are used to catalyse multiple kinds of conversions which were formerly carried out by classical chemical means. Initially, chemists were mainly interested in using bio-catalysis as a new tool in the kit of synthetic strategies developed during the last two centuries. Reduced production costs and reduced emissions were appreciated side-effects. Today the mandatory need for completely new synthetic approaches caused by the increasing scarcity of resources and the growing challenges due to environmental pollution is boosting bio-catalysis to a new level. Technologies like metabolic engineering, systems biology and bioinformatics are used to identify sustainable sources for crucial compounds and materials which are no longer based on fossil resources.

ABSTRACT

KEYWORDS

CHRISTIAN LEGGEWIE, PASCAL DUENKELMANNevocatal GmbH, Merowingerplatz 1a, Düsseldorf, 40225, Germany

BiocatalysisThe way out of resource dissipation andenvironmental pollution

Pascal Dünkelmann

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Christian Leggewie

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In many cases the pharmaceutical or speciality chemicals industry uses enzyme as isolated catalysts or in form of whole-cell transformations for the synthesis of expensive chiral compounds. But there are also many examples for applications of enzymes in the synthesis of commodities in large volumes. For example acrylamide is produced starting from acrylonitrile on a multi-thousand-ton scale per year (Scheme 1c) applying a nitrile hydratase (9). Alcohol dehydrogenases belonging to the class of oxidoreductases have to be mentioned here as well. These have been studied extremely well in the last years and today there are multiple examples for industrial application of these enzymes which convert aldehydes or ketones into alcohols (10). Among other applications alcohol dehydrogenases have found broad usage in the synthesis of pharmaceutical intermediates. In order to give an example of how powerful molecular biologic tools can be used to generate highly efficient bio-catalyst we wish to report here the successful evolution of an enzyme that is now used to produce (R)-3-quinuclidinol, a key intermediate in the synthesis of Solifenacin a pharmaceutical ingredient against overactive bladder (Scheme 2).

The last production step of the initial chemical process to produce (R)-quinuclidinol was a resolution of the racemic compound using chiral auxiliaries. So the maximum yield was only 50 percent. This process was no longer economically feasible and a more efficient process was needed.Enzyme catalysis suggested itself as the mild reaction conditions and the high selectivity of a bio-catalytic conversion seemed promising for obtaining a highly pure product without losing too much material in the downstream processing. The first step made in this approach was a screening of a library of wild-type enzymes. In this screening various proteins showing activity in the desired reaction were identified.

However, the best enzyme showed 20 percent conversion in 24 h and an enantiomeric excess of only 87 percent. This was far from being sufficient for an economically feasible process. In order to improve the performance of the bio-catalyst the protein structure of the enzyme was studied. In this context the active side of the enzyme – formed by the amino acids – could be elucidated in detail. Based on these results a new library of enzyme variants was generated. These mutants contained exchanges of specific amino acids which are involved in the formation of the active side but are not involved in the catalytic process. The library was then again screened for improved performance. The best enzyme identified in this screening now showed complete conversion of the substrate in 24 h and an enantiomeric excess of 99 percent. The active sides of the wild-type enzyme as well as of the enzyme variant are depicted in Scheme 3.

Obviously, the exchange of a small amino acid against a more bulky one had a strong impact on the conversion.The reduced space in the cavity is leading to a fixation of the substrate during the catalytic process resulting in an improved enantiomeric excess.Further developments on the process concerning co-factor regeneration, substrate loading, etc. provided an efficient procedure from a technical as well as from an economic perspective. Validation batches of this process are currently running. The above given examples for applications of enzymes all have in common that one special synthetic step previously carried out by classical chemical means is replaced by a bio-catalytic approach. Even though the number of reaction steps is not reduced effects can be achieved that are beneficial economically as well as ecologically.A comparison of the chemical asymmetric reduction of ketones with the described application of an alcohol dehydrogenase shows this quite clearly. While the efficiency of the bio-processes regarding space-time-yield is comparable or even superior the use of complex and expensive heavy metal-based hydrogenation catalysts can be circumvented. The amount of organic solvents needed can be reduced and the amount of energy consumed for heating and cooling is less. Such effects are of course even more considerable when the bio-catalytic approach enables a completely new synthetic approach reducing the number of synthetic steps that are necessary to obtain the target molecule. An impressive example for such an approach is the formation of Ursodeoxycholic acid (Scheme 4). The chemical synthesis of this API used to treat gallstones starting from cholic acid would include 7 chemical steps comprising several steps for attaching and removing protection groups (11). An alternative biotechnological approach consists of three steps (12). The first synthetic step is a selective enzymatic oxidation of the hydroxy-functions in 7- and 12-position catalysed by hydroxysteroid dehydrogenases. This is followed by a selective reduction of the keto-function in 7-position by an enzyme of the same class.

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Scheme 1. a) Synthesis of L-Methionine applying an amino acylase; b) Synthesis of Sitagliptine applying a transaminase; c) Synthesis of acrylamide applying a nitrile hydratase.

Scheme 2. Synthesis of (R)-3-Quinuclidinol starting from 3-Quinuclidinone applying an alcohol dehydrogenase optimised by evolution of a wild-type enzyme.

Scheme 3. In-silico models of the active sides of the wild-type alcohol dehydrogenase (ADH) and the alcohol dehydrogenase optimised by enzyme evolution.

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The final product can be obtained after chemical removal of the keto-function in 12-position by a Wolff-Kishner-reaction.A hurdle for alternative bio-catalytic syntheses containing intermediates that are different from those used in chemical synthesis is that they are often not considered by the chemists. To consider an enzyme turning a ketone into an enantiomerically pure alcohol when the chemical method using a metal-catalyst is already known is obvious.But to contemplate intermediates that hardly occur in organic syntheses and maybe even to start with a different substrate is not always self-evident.An enzyme class with very promising synthetic potential that has to be mentioned in this context are the thiamine-diphosphate-dependent lyases (13). These are capable of forming enantiomerically pure 2-hydroxy-ketones which can easily be transferred into various other compound; enzymatically or chemically. However, due to the fact that the named compounds are difficult to obtain from chemical reactions in an enantiomerically pure form they are not part of the usual “construction kit”.But there are many potential possibilities to use such enzymes – e.g. as already realised in the synthesis of ephedrine mentioned above – not only for speciality chemicals but also for commodities. We have successfully been working on a process to generate an aliphatic 2-hydroxy-ketone (Scheme 5) that can be used to produce a compound that is used in the cosmetics industry on multi-ton scale.

In this case the targets for the enzyme evolution process were the stability in the presence of the substrates and the selectivity (to avoid other cross coupling products).The performed enzyme screening and evolution yielded in an efficiency increase of the intended process by two orders of magnitude compared to the initial method using the wild-type enzyme.To date, almost all (fine-) chemicals are manufactured on the basis of fossil resources.

However, many experts agree that now at least half of the crude oil reserves were consumed and the so called peak oil has been reached already.Although the chemical industry has a share of only 7 percent of total oil consumption, there is an urgent demand to produce bulk chemicals from renewable resources due to the shortage of oil supply in the future. In recent years the predicted oil limitation led to an increased effort to produce various kinds of fuel, e.g. gasoline, diesel and jet fuel, based on renewable raw materials. In most cases these fuels are produced by engineered microorganisms through fermentation. In contrast to the first generation of biofuels which used sugar from food crops as a carbon and energy source and thus created a competition of fuel production and food supply, the second or third generation of biofuels will process non-food materials like lignocellulose or even CO/CO2 that amongst others could origin from the waste stream of steel mills or the thermal processing of lignocellulosic material. For more information about technologies, challenges and opportunities to produce biofuels by designer microbes please see the recently published review by Keasling and colleagues (14).For the production of bio-based chemicals, however, similar hurdles and challenges have to be considered as in the microbial production of biofuels. But there are promising examples for the fermentative production of some platform molecules and chemicals (15). Classically, amino acids were produced by microbial fermentation and huge markets are served by biotechnological production plants.A potent microbial producer of L-amino acids is the gram-positive bacterium Corynebacterium glutamicum, which is known for decades as a reliable production host.One of the main commercial products is L-Lysine with an annual market size of 1.5 million tons, which is produced by traditionally optimized C. glutamicum strains (16). Recently, however, it was reported that an engineered C. glutamicum strain exceeded the efficiency of previous known production strains significantly (17). That strain was made by modern biotechnological methods which included systems biology, metabolic engineering and in silico modelling. Certainly L-Lysine is a natural molecule from the C. glutamicum metabolism.

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Scheme 5. Selective coupling of two different aldehydes to one 2-hydroxy-ketone applying a thiamine-diphosphate-dependent lyase. The formation of the other three theoretically possible cross-coupling products is suppressed by enzyme evolution.

Scheme 4. Synthetic approach to Ursodeoxycholic acid using enzymatic as well as chemicals steps; HSDH: Hydroxysteroid dehydrogenase.

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Existing metabolic pathways and regulatory elements can be modified for an efficient L-Lysine production. Although the successful development of such an engineered production strain is quite ambitious, it is even more challenging to produce heterologous molecules or even non-natural products with designed microorganisms (18).Today many scientists from academia as well as from industry investigate the opportunities to produce natural as well as non-natural chemicals based on renewable raw materials. Industrial biotechnology companies are already working on the design and improvement of engineered microbes. Particularly C2 to C6 platform chemicals like alcohols, diols, diamines or dicarboxylic acids are of interest for so called “drop in” solutions for various product categories including food additives, pharmaceuticals, solvents and monomers for the production of diverse kinds of polymers (15).In addition, more complex molecules are of extensive industrial interest and we are working on the development of bio-based C6 to C8 platform chemicals.Today only a few bio-based chemicals like lactic acid and 1,3-propanediol are commercialized. But there are many more product opportunities.For example we are currently working on the development of sustainable biotechnological methods for the synthesis of precursors for the rubber production.This project is focused on the use of local renewable materials in the manufacture of rubber upstream products and aims at identifying new pathways for the development and for the manufacture of efficient biocatalysts.So far, rubber production was exclusively based on fossil materials. Now evocatal and partners are aiming at the implementation of biological methods that help to omit this dependence and to preserve resources as well as to reduce emissions.To expand the list of such “green chemicals” several hurdles have to be taken and focused research has to be done to

a) better connect several biological disciplines like metabolic engineering, systems biology and bioinformatics with chemistry and process engineering, b) identify alternative microbes as suitable production strains which are tolerant against products and intermediates of desired pathways, c) enable the utilisation of various carbon sources which do not compete with food supply and therefore permitting the production of “true green chemicals” and d) develop and design pathways for the desired bio-based products. The implementation of these technologies is certainly a challenge, but both the ecologic and economic benefits will be tremendous. Therefore, the development of bio-based chemicals will be in the focus of industrial biotechnology and biocatalysis in the next years and decades.

REFERENCES AND NOTES

1. B.M. Nestl, B.A. Nebel et al., Curr Opin Chem Biol., 15, p. 187 (2011).2. R. Wohlgemuth, Curr Opin Biotech., 21, p. 713 (2010).3. A. Schmid, J.S. Dordick et al., Nature, 409, p. 258 (2001).4. C. Wandrey, A. Liese et al., Org Process Res Dev., 4, p. 286 (2000).5. H.-P. Meyer, Org Process Res Dev., 15, p. 180 (2011).6. C. Neuberg, L. Liebermann, Biochem. Z., 121, p. 311 (1921).7. T. Tosa, T. Mori et al., Enzymologia, 31, p. 214 (1966).8. C.K. Savile, J.M. Janey et al., Science, 329, p. 305 (2010).9. S. Shimizu, J Ogawa et al., New Enzymes for Organic Synthesis,

Springer: New York, p. 45 (1997).10. M. Hall, A.S. Bommarius, Chem. Rev., 111, p. 4088 (2011).11. H. Sawada, H. Taguchi, US Patent 4579819 (1986).12. R. Bovara, G. Carrera et al., Biotech. Letters, 18, p. 305 (1996).13. P. Dünkelmann, M. Müller, Speciality Chemicals Magazine, 31, p. 16

(2011).14. P.P. Peralty-Yahya, F. Zhang et al., Nature, 488, p. 320 (2012).15. Y.-S. Jang, B. Kim et al., Biotechnol. Bioeng., 109, p. 2437 (2012).16. J. Becker, C. Wittmann, Curr. Opin. Biotechnol., 23, p. 631 (2012).17. J. Becker, O. Zelder et al., Metab. Eng., 13, p. 159 (2011).18. J.W. Lee, D. Na et al., Nat. Chem. Biol., 8, p. 536 (2012).

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