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Dispelling the Myths--Biocatalysis in Industrial Synthesis

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Science  14 Mar 2003:
Vol. 299, Issue 5613, pp. 1694-1697
DOI: 10.1126/science.1079237

Abstract

Biocatalysis has emerged as an important tool in the industrial synthesis of bulk chemicals, pharmaceutical and agrochemical intermediates, active pharmaceuticals, and food ingredients. However, the number and diversity of the applications are modest, perhaps in part because of perceived or real limitations of biocatalysts, such as limited enzyme availability, substrate scope, and operational stability. Recent scientific breakthroughs in genomics, directed enzyme evolution, and the exploitation of biodiversity should help to overcome these limitations. As a result, we expect many new industrial applications of biocatalysis to be realized, from single-step enzymatic conversions to customized multistep microbial synthesis by means of metabolic pathway engineering.

Microbial cells and the enzymes therein have been used for millennia in the production of valuable food products. Several decades ago, the first applications of biocatalysis in the chemical industry were implemented. Examples include the use of acylases, hydantoinases, and aminopeptidases in the production of optically pure amino acids, and the use of nitrile hydratase in the enzymatic production of the bulk chemical acrylamide from acrylonitrile (1–3). The recognition that lipase and other enzymes can be used in organic media (4, 5) and even in the solid phase (6) has further increased the potential of enzymes as catalysts in organic synthesis.

Today, both the academic and the industrial community see biocatalysis as a highly promising area of research, especially for the development of sustainable technologies for the production of chemicals (green chemistry) (7) and more selective and complex active ingredients in pharmaceuticals and agrochemicals. High stereoselectivities and hence enantiomerically pure products are a particularly attractive feature of biocatalysis. Large-scale industrial applications of enzyme catalysis include the thermolysin-catalyzed synthesis of the low-calorie sweetener aspartame (8) and the synthesis of semi-synthetic β-lactam antibiotics with the use of acylases (9). Further, acrylamide (10) and nicotinamide (3) are produced with the help of a nitrile hydratase (11), and the organic solvent 1,5-dimethyl-2-piperidone (1,5-DMPD) is manufactured using a nitrilase (12). There is also a multitude of smaller scale applications, particularly for manufacturing pharmaceutical ingredients (Fig. 1) (1–3, 13).

Figure 1

Examples of molecules that are manufactured using biocatalysis, ranging from nonchiral compounds to products with multiple chiral centers.

Yet, despite widespread research efforts in academia and industry, the number and diversity of biocatalyst applications remain rather modest (14). This situation may be attributed to several perceived limitations of biocatalysis, including the availability of the biocatalysts, their substrate scope, and their operational stability. It may in part also be due to the reluctance of the chemical community to explore the potential of biocatalysts in more depth.

Several recent scientific breakthroughs should help to overcome some of these limitations and expand the applications of biocatalysts. Advances in genomics, directed evolution, gene and genome shuffling (15, 16), and the exploration of Earth's biodiversity aided by bioinformatics and high-throughput screening (17, 18) facilitate the discovery and optimization of enzymes customized to fit to required process conditions. These techniques may, for example, lead to enhanced solvent resistance, increased process stability, change of pH and temperature optima, and enhanced and even reversed enantioselectivity. These exciting developments set the stage for a number of paradigm shifts that will change the perception of chemists with regard to the scope and limitations of biocatalysis for large-scale industrial synthesis.

Availability

Traditionally, active biocatalysts have been obtained by screening a broad variety of microorganisms, ranging from archaea to fungal systems, frequently isolated from extreme environments. These biocatalysts were used either as isolated enzymes or in the form of whole cell preparations. Later, recombinant systems were developed in which the genes encoding the desired enzyme were overexpressed in a more limited set of industrial host microorganisms. The resulting “designer bugs” (19) have an elevated level of the desired enzyme, as well as zero or reduced levels of enzymes catalyzing unwanted side reactions, because the genes coding for the latter enzymes are not transferred from the source microorganism.

Enzyme activity has successfully been modified and/or enhanced with site-directed mutagenesis, in which one or a few amino acid residues are rationally and directionally replaced. Improvements by such directed protein engineering have not always led to the desired result; furthermore, the method is rather time-consuming. As a consequence, the application of such biocatalysts in industrial synthesis has been hampered.

High-throughput screening and modern molecular biology techniques such as directed evolution (15), in combination with tremendous progress in genomics and bioinformatics, have led to a substantial increase in the availability of enzymes. Dedicated efforts to explore the worldwide biodiversity for novel enzymes at a genetic and functional level will further expand the arsenal of biocatalysts available for industrial application (18). Examples include the discovery of novel aldolases and nitrilases (see below). Novel strategies for accessing the genetic and functional diversity of nonculturable microorganisms by cloning the soil metagenome can also increase the availability of biocatalysts (20).

Scope of Reactions

Despite popular belief, the use of enzymes is not limited to the production of compounds that are similar to the natural substrate that the enzyme was made to convert. Many enzymes have a scope much broader than the natural starting materials and products (1–3,13, 14, 21). Moreover, directed evolution has opened the path to biocatalysts with broader substrate ranges, as well as to enzymes dedicated to a single specific transformation. However promising, the time required and limitations associated with patent and licensing issues related to some directed-evolution techniques may hamper broad industrial application.

Stability

The operational stability of biocatalysts applied in industrial processes has been improved steadily over the years through the use of genetic engineering, improvements in their formulation (such as immobilization), or process alterations (1, 2, 10, 12,14). Because biocatalytic transformations in industrial synthesis often involve organic molecules not soluble in water, the enzymes must operate efficiently in nonaqueous media. In recent years, various simple, scalable, and often low-cost techniques have been developed to generate highly active biocatalyst preparations for use in organic solvents. In addition, the enantioselectivity and/or regioselectivity of the enzyme can be enhanced. Enzymes may be activated by using a variety of excipients that will enhance or modulate enzyme activity through several different mechanisms (22). The excipients include nonbuffer salts, solid-state buffers, crown ethers, and cyclodextrins. Also, nonionic liquids may be used as solvents (22).

Some industrial enzymes are remarkably stable as the result of a careful development and selection process. For example, the xylose isomerase enzyme, used in the production of d-fructose fromd-glucose, is stable and active at temperatures up to 70°C for prolonged reaction times of several months. Recently, an interesting application of xylose isomerase in the form of a cross-linked enzyme crystal (CLEC) was described. In this form, the enzyme can be used as a biocatalyst in a packed bed reactor while simultaneously acting as a (chiral) separation matrix. Several new substrates for xylose isomerase CLECs were recently discovered, including both the d- and the l-form of various tetroses, pentoses, and hexoses. With the use of this new reactor and separation concept, l-ribose has been produced froml-arabinose (21).

Accessibility of Either Enantiomer

Kinetic and dynamic kinetic resolution processes. It is still a widespread and often erroneous perception that enzymes can only be used for the production of one of the two possible enantiomers. A racemic mixture of enantiomers (mirror images) can be biocatalytically converted to a new mixture of compounds with different chemical and physical properties (2, 3). The two compounds can then easily be separated. This so-called kinetic resolution is based on a difference in the rates of reaction of the two enantiomers. For example, the aminopeptidase from Pseudomonas putida ATCC 12633 converts racemic amino acid amides into a mixture of l–amino acid and unchanged d–amino acid amide (Fig. 2A, R2= H). The reaction is highly stereoselective. In a single run, both thel-acid and the d-amide can be obtained in enantiomerically pure form, albeit at a maximal theoretical yield of 50% (23). Evidently, to avoid the production of undesired waste material, recycling procedures should be applied. However, such procedures are commercially feasible only when the volume is large enough or when the starting material is too expensive to discard. Therefore, much research effort is being devoted to the development of biocatalytic schemes with 100% theoretical yield and 100% enantiomeric excess (e.e.) (24).

Figure 2

(A) Kinetic resolution. (B) Spontaneous and (C) chemically catalyzed dynamic kinetic resolutions.

Dynamic kinetic resolution is the resolution of a racemic mixture in combination with in situ racemization of the unwanted stereoisomer or enantiomer. Well-known examples include the production ofd-para-hydroxyphenylglycine (Fig. 2B) using ad-hydantoinase in combination with ad-carbamoylase while the unconvertedl-hydantoin spontaneously racemizes under the reaction conditions (2). Alternatively, hydantoin racemases have been described that actively catalyze the racemization of the hydantoin compounds but leave the d-N-carbamoyl–amino acid intermediate and the resulting d–amino acid untouched (1). Kinetic resolution processes have also been developed for chiral alcohols (Fig. 2C). They use a lipase under conditions that favor the racemization of the undesired enantiomer via a redox mechanism catalyzed by a transition metal catalyst in a one-pot procedure (25).

Chiral synthesis. Another 100% yield–100% e.e. approach to obtain enantiopure products is by stereoselective conversions of prochiral substrates, such as activated olefins (for example, by ammonia lyases), aldehydes (for example, using hydroxynitrile lyases), ketones (through reduction or by using aminotransferases), and symmetric substrates such as dinitriles (using nitrilases and/or nitrile hydratases) (1–3, 13, 14, 19,24). For instance, nitrilases are known to stereoselectively convert the prochiral substrate 3hydroxyglutaronitrile, albeit with moderate selectivity (20% e.e.). Genomic libraries consisting of 200 genetically diverse nitrilase genes were screened for this conversion with the use of ultrahigh-throughput methods. Some of the nitrilases found could be used to exclusively synthesize (R)-4-cyano-3-hydroxybutyric acid, whereas others could be used to synthesize the corresponding (S)-enantiomer (17). This particular case shows that effective use of high-throughput screening techniques can provide access to either enantiomer (Fig. 3).

Figure 3

Access to mirror-image molecules through biocatalysis. From a common starting point, enantiomerically complementary compounds and epimers are produced by different enzymes.

To obtain enzymes that can address both enantiomers, one can also use directed-evolution techniques to evolve a known enzyme with nonoptimal characteristics into an enzyme fit for industrial synthesis. In directed evolution, a collection of random gene mutations is subjected to a selection regime that discriminates between fit and nonfit enzymes (15, 16). A prerequisite for obtaining enzymes with opposite stereoselectivity is a reliable high-throughput e.e. screening method (26). Using this methodology, a hydantoinase that was (R)-selective for methionine hydantoin has been evolved into an (S)-selective enzyme (27). It also proved possible to change an (R)-selective lipase into an (S)-selective lipase after several rounds of directed evolution (28).

Redox conversions. Statistical analysis of industrial biotransformations (14) shows that a large percentage of known transformations should be classified as redox conversions. An excellent example of a biomediated aromatic hydroxylation is the formation of 6-hydroxynicotinic acid from niacin (3). Enzymatic oxidations of methyl groups on aromatic rings have also been described (3). Although large-scale commercial applications are still limited, we expect an increasing number of such applications to emerge. Interesting conversions include the reduction of prochiral ketones, the oxidation of alcohols, the epoxidation of alkenes, and the hydroxylation of nonactivated carbon atoms (1–3, 14,24). Redox conversions may be catalyzed by isolated enzymes or by cosubstrate-dependent enzymes in whole-cell biocatalytic processes. Often these whole-cell conversions involve apolar substrates and products that are generally insoluble in water, requiring dedicated technical solutions (3, 12). Also, biocatalytic redox conversions may face fierce competition from alternative synthetic routes to the target molecules.

Reactions That Challenge Organic Chemistry

Stereoselective carbon-carbon bond formation.Carbon-carbon bond formations are pivotal processes in organic chemistry, but on an industrial scale relatively few enzymatic processes are known. Recently, the formation of enantiopure (S)-meta-phenoxybenzaldehyde cyanohydrin using the recombinant (S)-hydroxynitrile lyase (HNL) from the rubber tree Hevea brasiliensis has been implemented on an industrial scale (29). The compound is used in the synthesis of pyrethroid insecticides. One of the oldest examples of enzyme-catalyzed C-C bond formation is the synthesis of enantiopure hydroxynitriles using (R)-HNL from almonds (Prunus amygdalus). Large-scale application of this versatile enzyme had been hampered by its limited availability. Recently, the gene has been cloned and overexpressed in an industrial host organism. With both the (R)- and (S)-HNL enzymes readily available, the number of industrial applications will undoubtedly grow (29, 30) (Fig. 3).

A major challenge in stereoselective C-C bond formation is the aldol condensation. Selectivity (in terms of cross-condensation versus self-condensation) and enantioselectivity are difficult to achieve in this group of reactions. Many chemical methods have been developed, but they involve complex reagents, auxiliaries, or catalysts to prevent the formation of unwanted side products. In addition, protecting groups are required for the product or for additional functionality present in the reactants. In general, forming C-C bonds without having to protect any groups remains a major challenge for synthetic organic chemists.

Large steps toward this goal have been accomplished with C-C bond–forming enzymes that use unmasked aldehydes as reagents. These methods cannot easily be scaled up because they require phosphorylated aldehydes or the presence of a keto acid functionality. A selective aldol condensation of two different aldehydes can be accomplished using 2-deoxyribose-5-phosphate aldolase (DERA) (31). Several successful enzyme-catalyzed cross-couplings have been realized in fair to very good yields, as exemplified by the total synthesis of epothilones (32). Also, the synthetic scheme could be extended to the use of threonine aldolases that couple glycine as the donor to a variety of aldehydes to obtain the corresponding amino acid alcohols (33).

Great advances in the customization of C-C bond–forming catalysts have also been reported. Through directed evolution, the phosphate dependence and enantioselectivity ofd-2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase could be modified (33, 34). This again shows the possibility of accessing both (R)- and (S)-enantiomers with the use of biocatalysis (Fig. 3).

Combining bio- and chemocatalysis. As discussed above, biocatalytic procedures are very useful for the production of enantiopure compounds. Nonetheless, advanced organic synthesis is likely to remain an essential tool in the production of pharmaceuticals and agrochemicals. The best industrial synthesis routes of the future are probably based on a combination of bio- and chemocatalysis.

An illustration of this principle can be found in the synthesis of conformationally restricted cyclic amino acid derivatives. The synthesis of such compounds has been accomplished starting from enantiopure linear unsaturated amino acid derivatives obtained by an aminopeptidase-catalyzed process (Fig. 2) (23). These versatile building blocks can subsequently be converted into enantiopure, highly functionalized cyclic amino acid derivatives by means of transition metal catalysts (35). The resulting compounds are used either as pharmaceutical intermediates or as scaffolds for the construction of combinatorial libraries, used in drug discovery and development. Combinatorial biocatalysis uses the natural diversity of enzymatic reactions to expand the array of combinatorial methods for the generation and optimization of lead compounds (36).

Metabolic pathway engineering. By channeling metabolic pathways in microorganisms toward a desired metabolite through rational introduction and removal of genes—metabolic engineering—a wide range of natural compounds, such as amino acids, organic acids, and nucleotides, can be produced. In recent years, metabolic engineering has also been used to synthesize compounds that do not naturally occur. Examples include the engineering of pathways to the blue dye indigo (37), the antibiotic nucleus 7-ADCA (38), and 1,3-propanediol, a polymer raw material (39).

Particularly interesting are the developments in metabolic engineering that are directed toward the synthesis of specialty and commodity products, such as vanillin, shikimic acid, and adipic acid, using renewable resources (40, 41). At the pharmaceutical end, promising libraries of new, biologically active polyketide compounds have been synthesized by combining genes and pathways from a variety of actinomycetes in heterologous hosts (42). Similarly, access to nonnatural peptides has been demonstrated using customized peptide synthetases that catalyze nonribosomal peptide synthesis (43). These developments show that engineering metabolic pathways, by integration of genes that are accessible from the numerous genomics programs, holds great promise for commercial manufacturing of both naturally occurring and nonnatural chemical compounds.

Conclusions

In the past decades, biocatalysis has emerged as a powerful technology for the production of several important industrial products, including organic solvents, polymer raw materials, enantiomerically pure building blocks for pharmaceuticals and agrochemicals, active pharmaceutical ingredients, antibiotics, and food ingredients such as sweeteners and vitamins. With the help of directed-evolution techniques, ever-increasing genetic information, and powerful enzyme discovery tools, numerous novel applications of biocatalysis are likely to emerge in industrial synthesis in the coming years. Progress in chemocatalysis and in biocatalysis is synergistic, and integration of the respective best technologies available is required to reach more efficient and sustainable production processes.

  • * To whom correspondence should be addressed. E-mail: hans.schoemaker{at}dsm.com

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