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Protein engineering of microbial enzymes
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Protein engineering of microbial enzymes Dominique Bo¨ttcher and Uwe T Bornscheuer Protein engineering has emerged as an important tool to overcome the limitations of natural enzymes as biocatalysts. Recent advances have mainly focused on applying directed evolution to enzymes, especially important for organic synthesis, such as monooxygenases, ketoreductases, lipases or aldolases in order to improve their activity, enantioselectivity, and stability. The combination of directed evolution and rational protein design using computational tools is becoming increasingly important in order to explore enzyme sequencespace and to create improved or novel enzymes. These developments should allow to further expand the application of microbial enzymes in industry. Address Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, University of Greifswald, Felix-Hausdorff-Str. 4, 17487 Greifswald, Germany Corresponding author: Bornscheuer, Uwe T ([email protected])
Current Opinion in Microbiology 2010, 13:274–282 This review comes from a themed issue on Ecology and Industrial Microbiology Edited by Erick Vandamme Available online 17th February 2010 1369-5274/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2010.01.010
instance for pig liver esterase [8,9] and horse liver alcohol dehydrogenase (Evocatal GmbH, Du¨sseldorf, Germany), it is often not trivial to get correctly folded and active protein in high amounts. Another important source of biocatalysts are plant enzymes, where for instance hydroxynitrile lyases from Manihot esculenta [10] and Hevea brasiliensis [11] could be expressed in E. coli and the yeast Pichia pastoris, respectively. Independent of the reaction system and enzyme investigated for a given biocatalytic process, very often the enzyme does not meet the requirements for a large-scale application and its properties have to be optimized. This usually includes not only the chemoselectivity, regioselectivity and especially stereoselectivity of the biocatalyst, but also process-related aspects such as long-term stability at certain temperatures or pH-values and activity in the presence of high substrate concentrations to achieve highest productivity. Beside rather classical strategies such as immobilization, additives or process engineering, molecular biology techniques nowadays represent probably the most important methodology to tailor-design the enzyme for a given process. Two different strategies are used: rational protein design and directed (molecular) evolution, which are increasingly applied in a synergistic manner (Figure 1). In the following sections, selected examples for protein engineering of biocatalysts of microbial origin, which are particularly useful for organic synthesis, are given. Readers are also referred to consult recent general literature on protein engineering of biocatalysts [12–14].
Introduction Biocatalysts are extensively used in the industrial production of bulk chemicals and pharmaceuticals and over 300 processes have been already implemented [1–4]. In the vast majority of processes, enzymes of microbial origin are used as the microbial kingdom represents a huge – and still only partially explored – reservoir for biocatalysts with desired properties. Furthermore, the metagenome approach [5–7] substantially facilitates the discovery of novel enzymes from microbial sources and hence the number of potentially useful biocatalysts increased exponentially in the past decade. In contrast, enzymes from animal tissues are less preferred as these often occur as mixture of isoenzymes differing in substrate specificity and product safety (i.e. viral infections), which often restricts their industrial use. Although this can be overcome by expression of the corresponding genes in microbial hosts as shown for Current Opinion in Microbiology 2010, 13:274–282
Examples for protein engineering of microbial enzymes Dehydrogenases
The application of enantioselective alcohol dehydrogenases plays an important role for the asymmetric synthesis of enantiomerically and diastereomerically pure alcohols, which are important building blocks for pharmaceuticals, agro, and fine chemicals. To meet process conditions, highly active and also solvent stable enzymes are required. For example an alcohol dehydrogenase from Pyrococcus furiosus was recently engineered for improved activity at low temperature for the production of enantiopure (2S,5S)hexanediol. A mutant obtained by error-prone polymerase chain reaction (epPCR) showed a 10-fold higher maximum specific activity compared to the wild-type enzyme [15]. In a rational design approach the enantiopreference of a secondary alcohol dehydrogenase from Thermoanaerobacter www.sciencedirect.com
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Figure 1
The appropriate method for protein engineering of enzymes should be decided case-by-case on the basis of the availability and quality of the structural and mechanistic information and the feasibility of a high-throughput screening (HTS) system for screening or selection.
ethanolicus was reversed by just a single point mutation (I86A). This mutant not only shows anti-Prelog stereospecificity, but also accepts more sterically demanding substrates [16]. One recent example for altered coenzyme specificity was shown for the glucose dehydrogenase from Haloferax mediterranei, where a single mutation G206D was sufficient to change the enzyme preference from NADP+ to NAD+ [17]. Oxygenases
Oxygenases are enzymes, which introduce one (monooxygenases) or two (dioxygenases) oxygen atoms into their substrates. Typically NADH or NADPH serve as reduction equivalents via electron-transfer proteins such as reductases. The major interest in these enzymes for organic synthesis is due to their high chemoselectivity, regioselectivity and stereoselectivity. P450 monooxygenases catalyze a broad range of interesting reactions, but their application is hampered by their www.sciencedirect.com
low specific activity, narrow substrate range, usually low stability and the need for a complex electron-transfer system. In addition, most P450 enzymes are only applicable in whole cell systems. Nevertheless, the recent progress made in the protein engineering of several P450 enzymes might allow for their application in biocatalysis in the near future [18]. Most recently published P450 engineering approaches summarized below aimed for broader substrate specificities (Figure 2). The directed evolution of self-sufficient P450 BM-3 (CYP102A1) from Bacillus megaterium yielded among others a variant that shows improved conversion and selectivity on astemizole, a potent histamine receptor antagonist used for the treatment of allergy symptoms [19]. Li et al. further improved a previously isolated cytochrome P450 BM-3 variant via site-directed and random mutagenesis to produce indigo and indirubin. Three resulting mutants exhibited higher hydroxylation activity towards indole in comparison to the parent enzyme, two P450 BM-3 variants (D168H/3X, E435T/3X) showed an up to sixfold increase in catalytic efficiency, and the mutant Current Opinion in Microbiology 2010, 13:274–282
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Figure 2
Substrates for protein engineering of P450 monooxygenases. Top row: [19], second row: [20–24], third row: [25], and fourth row: [26].
D168H/3X exhibited higher regioselectivity [20]. Another example for engineering regioselectivity of this enzyme was based on rational design using site-directed mutagenesis. The mutants V78A/F87A/I263G and S72Y/V78A/ F87A were able to hydroxylate lauric acid at the d-position, g-position and b-position [21]. A thermophilic cytochrome P450 (CYP175A1) was engineered by site-directed mutagenesis to catalyze the hydroxylation of the non-natural substrate testosterone [22]. Applying DNA family shuffling methodology on different cytochrome P450 enzymes resulted in an expansion of the enzyme substrate range (e.g. activity towards different luciferin derivatives) [23,24]. Other recent evolution advances on P450 enzymes yielded variants with improved activity and regioselectivity or stereoselectivity in the oxidation of terpenes [25] or with improved 11-deoxycortisol and progesterone hydroxylation activity [26] or they have been improved for activity towards biomimetic NADH cofactors [27]. A very elegant approach using in vivo-directed evolution was applied to enhance the activity of two mediumCurrent Opinion in Microbiology 2010, 13:274–282
chain-length terminal alkane hydroxylases (AlkB and CYP153A6) on small alkanes. An evolution system based on enhanced growth on butane of a Pseudomonas putida strain complemented with evolved AlkB and CYP153A6 variants served for the identification of best mutants. The resulting variants exhibited higher rates of 1-butanol production from butane and maintained their preference for terminal hydroxylation [28]. Another important group of enzymes are Baeyer–Villiger Monooxygenases (BVMO). Although several BVMOs have already been recombinantly expressed and characterized, so far directed evolution and rational protein design have been applied to mainly three of them to alter their substrate specificity and enantioselectivity: cyclohexanone monooxygenase (CHMO) from Acinetobacter calcoaceticus NCIMB 9871 [29], cyclopentanone monooxygenase (CPMO) from Comamonas testosteroni [30] NCIMB 9872 and phenylacetone monooxygenase (PAMO) from Thermobifida fusca. Because of its thermostability and the stability in organic solvents, especially the PAMO is a very interesting candidate for application in organic synthesis. In order to broaden the limited substrate scope and to improve the selectivity, the enzyme was recently engineered by two research groups. www.sciencedirect.com
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First, based on structure comparison of the PAMO crystal structure and a structure model of the sequence-related CPMO, several active-site residues were selected for sitedirected mutagenesis. One mutant (M446G) was able to convert a number of aromatic ketones, amines and sulfides, whereas the wild-type enzyme showed no activity. In addition the mutant represents the first BVMO, which is able to convert indole into indigo [31]. The other rational approach was recently published by Reetz and Wu. After an alignment of eight different BVMOs and induced-fit docking experiments, five different libraries were designed and randomized by saturation mutagenesis (CASTing). Mutants were found that show high activity and enantioselectivity towards different 2aryl and 2-alkylcyclohexanones and a bicyclic ketone. The best mutant also maintained thermostability of the wild-type enzyme [32]. Recently, the activity and enantioselectivity of a BVMO from Pseudomonas fluorescens with substrate preference for aliphatic ketones could be further enhanced in our group using directed evolution [33]. An extensive coverage of the state-of-the-art research regarding BVMOs can be found in recent reviews [34,35]. Transferases
Within the enzyme class of transferases, transaminases are highly versatile biocatalysts for the synthesis of optically active amines or a-amino acids by transfer of an amino group from a donor substrate to an acceptor compound utilizing the cofactor pyridoxal-5-phosphate. aTransaminases require the presence of a carboxylic acid function in a-position to the keto or amine functionality and hence only allow the formation of a-amino acids, vtransaminases are much more useful as they in principle accept any ketone (or amine). In the last decade, especially v-transaminases have been extensively studied and also overexpressed in microbial hosts such as E. coli with the enzyme from Vibrio fluvialis as probably the most intensively studied v-transaminase. The current status for the use of transaminases is covered in a recent review [36]. Recently, the substrate specificity of the v-aminotransferase from Vibrio fluvialis was rationally designed for the kinetic resolution of aliphatic chiral amines. Two mutant enzymes showed significant changes in their substrate specificity. They catalyzed transamination of a broad range of aliphatic amines without losing the original activities toward aromatic amines and enantioselectivity [37]. A mesophilic v-aminotransferase from Athrobacter citreus was evolved to a thermostable aminotransferase via epPCR. Through five rounds of mutagenesis, the specific activity was improved 260-fold from 5.9 to 1583 U/g and www.sciencedirect.com
the optimum temperature could be elevated from 30 to 558C [38]. Hydrolases
The majority of industrially used enzymes are lipases and thus many examples of engineered lipases can be found in literature and recent reviews [39,40]. Candida antarctica lipase B (CAL-B) is probably the most useful lipase for organic synthesis and numerous examples for its application [41] and engineering can be found in literature. The enzyme has been recently improved concerning thermostability [42], activity [43,44], enantioselectivity [45] and also solubility [46]. In one of these examples a very interesting approach, the so-called circular permutation (CP) was used [43,44,47]. Lutz et al. linked the native N-termini and C-termini of the gene encoding CAL-B and subsequently linearized it by random digestion to yield variants bearing alternative N-termini and C-termini. Surprisingly, this not only lead to active lipase, some variants also showed higher catalytic efficiency in comparison to the wild type (up to 11-fold against p-nitrophenol butyrate and 175-fold against 6,8-difluoro-4-methylumbelliferyl octanoate, while KM values were nearly the same). For the most active variant (cp283), kinetic experiments demonstrated that CP of this enzyme did not compromise the enantioselectivity in the resolution of some chiral secondary alcohols [44]. Among the field of hydrolases only a few esterases found their application in organic chemistry. Nevertheless, some examples for their successful improvement by directed evolution, rational protein design and combinations of these methods have been described. The synthetically useful esterase BS2 – which differs only by 11 amino acids from a para-nitrobenzyl esterase from Bacillus subtilis (BsubpNBE), being the first example for directed evolution of an esterase – was evolved by rational design in our group and the enantioselectivity towards an ester of the tertiary alcohol 2-phenyl-3-butyn2-yl acetate could be increased sixfold to E = 19, and towards linalyl acetate inverted from (R)-preference to (S)-preference with E = 6 [48]. In a later study, this mutant (G105A) showed a good enantioselectivity towards 2-phenyl-3-butyn-2-yl acetate (E = 54) in 20%, v/v, DMSO, and an E-value of >100 towards the trifluoromethyl analog [49]. Another point mutation (E188D) gave similar high enantioselectivity towards both substrates as well as a series of other tertiary alcohol acetates [50]. Using a focused directed evolution approach, we recently created a library covering three residues by saturation mutagenesis and identified a double mutant (E188W/M193C) with an (S)-preference and an E-value of 70 towards a tertiary alcohol thus leading to a variant with completely inverted enantioselectivity [51]. Interestingly, the two single mutations showed substantially Current Opinion in Microbiology 2010, 13:274–282
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lower and even undesired selectivity: E188W shows only E = 26 for the (S)-enantiomer and M193C was still (R)selective (E = 16). Thus, only the simultaneous saturation mutagenesis at several amino acid positions resulted in the significant inversion of enantiopreference and an increase of enantioselectivity. An esterase from Pseudomonas fluorescens DSM50106 (PFEI) was recently improved in our group by the OSCARR method yielding two mutants with a 10-fold higher catalytic efficiency towards long chain p-nitrophenyl esters (octanoate and dodecanoate) [52] and both mutants (F126I and G120S/F126I) showed also a slightly decreased catalytic efficiency towards the typical esterase substrates p-nitrophenyl acetate and p-nitrophenyl butanoate. Thus, the substrate specificity of this esterase could be shifted to lipase-like properties. Very recently the same enzyme was further rationally designed along the active-site entrance to widen the substrate tunnel and to study the influence on substrate specificity and enantioselectivity [53]. Optically pure epoxides and diols are important building blocks for the production of pharmaceuticals. One way to prepare enantiopure epoxides is by hydrolytic kinetic resolution of racemic epoxides using enantioselective epoxide hydrolases. Epoxide hydrolases can also be used to produce optically enriched vicinal (R,R)-diols or (S,S)diols in 100% theoretical yield through regioselective hydrolysis of meso-epoxides [54]. The epoxide hydrolase from Aspergillus niger M200 was recently engineered via saturation mutagenesis and it could be shown that amino acid exchanges in the substrate tunnel region can lead to significant improvements in enantioselectivity and activity [55]. Another commonly applied epoxide hydrolase from Agrobacterium radiobacter (EchA) was evolved using sitedirected mutagenesis. For this, position F108, which flanks the nucleophilic aspartate and forms part of the substrate-binding pocket, was saturated using degenerate primers and resulted in mutants with higher activity toward cis-disubstituted meso-epoxides. Mutant F108C converted cis-2,3-epoxybutane to enantiopure (2R,3R)2,3-butanediol with a sevenfold improved activity, and mutant F108A hydrolyzed cyclohexene oxide to (1R,2R)1,2-cyclohexanediol (99%ee) with 150-fold higher activity than the wild-type enzyme [56]. A range of haloalkane dehalogenases of microbial origin have been discovered and characterized. These enzymes mainly catalyze the hydrolysis of chloroalkanes, or bromoalkanes to the corresponding alcohol and halide ion, and also iodoalkanes are accepted as substrates. They generally exhibit a broad substrate tolerance, with preference to primary carbon–halogen bonds [57]. Pavlova et al. identified key residues in entrance tunnels to the Current Opinion in Microbiology 2010, 13:274–282
active site by rational design of Rhodococcus rhodochrous haloalkane dehalogenase and randomized this region by directed evolution methods. The most active variant bearing large aromatic residues at two out of three randomized positions and two positions modified by site-directed mutagenesis, showed up to 32-fold higher activity than the wild type towards 1,2,3-trichloropropane [58]. Hydantoinases are valuable enzymes for the production of optically pure D-amino and L-amino acids. They catalyze the reversible hydrolytic cleavage of hydantoins and 50 -monosubstituted hydantoins. In combination with carbamoylases (N-carbamoyl-D-amidohydrolases), the reaction yields L-amino or D-amino acids depending on the stereoselectivity of the enzymes. Nowadays, hydantoinases from, for example, Arthrobacter sp., Nocardia sp., Bacillus sp. and Pseudomonas sp. have been described and a range of D-amino acids are accessible by using them. A recent example for hydantoinase engineering is the rational design of Bacillus stearothermophilus D-hydantoinase for altered substrate specificity towards hydroxyphenylhydantoin and resulted in the mutant M63I/ F159S exhibiting about 200-fold higher specificity for hydroxyphenylhydantoin than the wild-type enzyme [59]. In another approach the solubility of Burkholderia pickettii was improved by protein engineering. A combination of random mutagenesis using epPCR and DNA shuffling techniques gave several mutants with reduced aggregation. It was found that mainly three amino acid residues (A18, Y30, and K34) are involved in protein solubility. A triple mutant A18T/Y30N/K34E could be overexpressed in highly soluble form in E. coli [60]. Later, the thermostability of the same highly soluble D-carbamoylase variant was improved using directed evolution. The most thermostable mutant, Q12L, showed a 7 8C increase in thermostability [61]. D-carbamoylase
Lyases
A broad range of aldolases has been described in literature and these biocatalysts are very attractive for organic synthesis as they allow to create two stereocenters in a single reaction step [62,63]. However, narrow substrate ranges and enzyme inactivation under synthesis conditions represented major obstacles for the large-scale application of aldolases. Again, these limitations could be overcome by directed evolution. The Wong group described an in vivo selection assay for the directed evolution of an L-rhamnulose aldolase starting from an L-rhamnulose-1-phosphate aldolase (RhaD) [64]. This assay enabled the identification of the desired RhaD variant in an epPCR-derived library, which in contrast to the wild type accepts dihydroxyacetone (DHA) as donor instead of the very expensive and unstable phosphorylated analog DHAP. A related enzyme utilizing www.sciencedirect.com
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galactose derivatives (KDPGal) was subjected to a combination of epPCR, DNA shuffling and site-directed mutagenesis yielding a variant with 60-fold improved catalytic efficiency in the synthesis of a shikimate pathway product [65]. Recently a library of randomly generated mutants of thermophilic Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase was screened for improved synthesis of 2keto-3-deoxygluconate at lower temperatures yielding a single mutant (V193A) that displayed a threefold increase in activity compared to the wild-type enzyme. The increased specific activity of this mutant at suboptimal temperatures between 40 and 608C could be observed, not only for the aldol condensation of pyruvate with glyceraldehyde, but also for several non-natural acceptor aldehydes [66]. A broad range of further examples of aldolases improved by protein engineering methods can be found in a number of recent reviews [67–70]. Halohydrin dehalogenases (also known as haloalcohol dehalogenases or halohydrin hydrogen-halide lyases) catalyze the cofactor-independent dehalogenation of vicinal haloalcohols by an intramolecular substitution reaction, producing the corresponding epoxide. These enzymes are involved in the bacterial degradation of several important environmental pollutants, such as epichlorohydrin or 1,3-dichloro-2-propanol [57]. The catalytic function of a halohydrin dehalogenase from Agrobacterium radiobacter was improved by ProSAR-driven evolution (protein sequence activity relationship), introduced by Fox et al., to obtain ethyl (R)-4cyano-3-hydroxybutyrate, which is involved in the synthesis of Atorvastatin (Lipitor) [71]. The new enzyme had a 4000-fold volumetric productivity in the cyanation process compared to the wild type. This example also demonstrates the efficiency of combining theoretical consideration with laboratory methods for the improvement of biocatalyst function.
Promiscuous and novel enzyme activities Some enzymes already catalyze reactions on alternative functional groups, but at a very slow rate, compared to their main catalytic function. This ‘catalytic promiscuity’ is now a major research theme and the current status has been covered in recent reviews [72–74]. For example the promiscuous enzyme xylose reductase from Neurospora crassa, which prefers D-xylose over Larabinose, has been engineered via semi-rational design approaches by site-saturation mutagenesis, combinatorial active site-saturation testing (CASTing) and finally one mutant (L109Q) with ninefold higher preference for Dxylose could be identified [75]. www.sciencedirect.com
De Groeve et al. modified substrate specificity of Cellulomonas uda cellobiose phosphorylase from cellobiose to lactose by directed evolution. This enzyme already exhibits a low rate of lactose phosphorylase activity, which is unusual since this enzyme class is known to have a strict sugar phosphate donor specificity. After a single round of random mutagenesis, clones with improved lactose phosphorylase activity could be enriched by an in vivo selection on minimal medium containing lactose as sole carbon source. Subsequent site-directed and site-saturation mutagenesis resulted in the mutant T508I/N667A with 7.5 times higher specific activity on lactose than the wild-type enzyme [76]. The bromoperoxidase A2 from Streptomyces aureofaciens was converted into a lipase by structure comparison with lipase A from Bacillus subtilis, which shows a similar structure but only very low sequence identity. Using epPCR and site-directed mutagenesis mutants with improved hydrolytic activities and substrate preferences toward long chain substrates could be obtained [77]. In a recent example we could show that it is possible to interconvert enzymes within the class of a/b-hydrolase fold enzymes. Starting from an esterase from Pseudomonas fluorescence, the introduction of distinct point mutations together with the exchange of a loop sequence – containing a mechanistically important tyrosine residue and possibly enabling the substrate access to the active site – derived from an epoxide hydrolase, activity could be generated in the esterase scaffold. The resulting chimera not only showed reasonable activity only 800-fold lower than a true epoxide hydrolase, but also exhibited high enantioselectivity (E > 100) in the kinetic resolution of pnitrostyrene oxide [78].
Conclusions Protein engineering via directed evolution or rational design has emerged as a very powerful tool to design and alter the properties of enzymes. This technology quickly found its application for a broad range of proteins, with the vast majority being of interest for biocatalysis. Consequently, a diverse set of molecular biology tools to create well-balanced mutant libraries as well as suitable high-throughput screening methods have been developed to make the application of directed evolution more easy and feasible. Within just a decade, directed evolution has emerged standard methodology in protein engineering and therefore can be used complementary or in combination with rational protein design to meet the demands for industrially applicable biocatalysts exhibiting desired chemoselectivity, regioselectivity, and stereoselectivity as well as withstands process conditions (i.e. high substrate concentrations, solvents, temperatures, long-term stability). Current Opinion in Microbiology 2010, 13:274–282
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Acknowledgements We thank the German Research Foundation (DFG, Grant Bo1862/4-1) and the Deutsche Bundesstiftung Umwelt (DBU, Grant AZ13198-32) for financial support.
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37. Cho BK, Park HY, Seo JH, Kim J, Kang TJ, Lee BS, Kim BG: Redesigning the substrate specificity of v-aminotransferase for the kinetic resolution of aliphatic chiral amines. Biotechnol Bioeng 2008, 99:275-284.
53. Schliessmann A, Hidalgo A, Berenguer J, Bornscheuer UT: Increased enantioselectivity by engineering bottleneck mutants in an esterase from Pseudomonas fluorescens. ChemBioChem 2009, 10:2920-2923.
38. Martin AR, DiSanto R, Plotnikov I, Kamat S, Shonnard D, Pannuri S: Improved activity and thermostability of (S)aminotransferase by error-prone polymerase chain reaction for the production of a chiral amine. Biochem Eng J 2007, 37:246-255.
54. Drauz K, Waldmann H (Eds): Enzyme Catalysis in Organic Synthesis, vol II, edn 2. Weinheim: VCH; 2002.
39. Kourist R, Brundiek H, Bornscheuer UT: Protein engineering and discovery of lipases. Eur J Lipid Sci Technol 2010, 112:64-74. 40. Shu Z-Y, Jiang H, Lin R-F, Jiang Y-M, Lin L, Huang J-Z: Technical methods to improve yield, activity and stability in the development of microbial lipases. J Mol Catal B Enzym 2010, 62:1-8. 41. Anderson EM, Larsson KM, Kirk O: One biocatalyst – many applications: the use of Candida antarctica B-lipase in organic synthesis. Biocatal Biotransform 1998, 16:181-204. 42. Zhang N, Suen WC, Windsor W, Xiao L, Madison V, Zaks A: Improving tolerance of Candida antarctica lipase B towards irreversible thermal inactivation through directed evolution. Protein Eng 2003, 16:599-605. 43. Yu Y, Lutz S: Improved triglyceride transesterification by circular permuted Candida antarctica lipase B. Biotechnol Bioeng 2009, 105:44-50. 44. Qian Z, Fields CJ, Lutz S: Investigating the structural and functional consequences of circular permutation on lipase B from Candida antarctica. ChemBioChem 2007, 8:1989-1996. The natural termini of lipase B from C. antarctica were linked by a six amino acid peptide. The resulting library of CAL-B variants with shifted N-termini and C-termini yielded over 60 active hydrolase variants, and several show significantly improved catalytic performance and enantioselectivity. 45. Skjot M, De Maria L, Chatterjee R, Svendsen A, Patkar S, Ostergaard P, Brask J: Understanding the plasticity of the a/bhydrolase fold: lid swapping on the Candida antarctica lipase B results in chimeras with interesting biocatalytic properties. ChemBioChem 2009, 10:520-527. The authors replaced the lid of Candida antarctica lipase B with corresponding lid regions from homologous enzymes and yielded chimeras with increased activity towards esters of branched carboxylic acids and an increased enantioselectivity towards profens. 46. Jung S, Park S: Improving the expression yield of Candida antarctica lipase B in Escherichia coli by mutagenesis. Biotechnol Lett 2008, 30:717-722. 47. Qian Z, Lutz S: Improving the catalytic activity of Candida antarctica lipase B by circular permutation. J Am Chem Soc 2005, 127:13466-13467. 48. Henke E, Bornscheuer UT, Schmid RD, Pleiss J: A molecular mechanism of enantiorecognition of tertiary alcohols by carboxylesterases. ChemBioChem 2003, 4:485-493. 49. Heinze B, Kourist R, Fransson L, Hult K, Bornscheuer UT: Highly enantioselective kinetic resolution of two tertiary alcohols using mutants of an esterase from Bacillus subtilis. Protein Eng Des Sel 2007, 20:125-131. 50. Kourist R, Bartsch S, Bornscheuer UT: Highly enantioselective synthesis of arylaliphatic tertiary alcohols using mutants of an esterase from Bacillus subtilis. Adv Synth Catal 2007, 349:1393-1398. 51. Bartsch S, Kourist R, Bornscheuer UT: Complete inversion of enantioselectivity towards tertiary alcohols by a double mutant of a Bacillus subtilis esterase. Angew Chem Int Ed 2008, 47:1508-1511. Using a focused directed evolution approach, an esterase library was created covering three residues by saturation mutagenesis and one www.sciencedirect.com
55. Kotik M, Stepanek V, Kyslik P, Maresova H: Cloning of an epoxide hydrolase-encoding gene from Aspergillus niger M200, overexpression in E. coli, and modification of activity and enantioselectivity of the enzyme by protein engineering. J Biotechnol 2007, 132:8-15. 56. van Loo B, Kingma J, Heyman G, Wittenaar A, Spelberg JHL, Sonke T, Janssen DB: Improved enantioselective conversion of styrene epoxides and meso-epoxides through epoxide hydrolases with a mutated nucleophile-flanking residue. Enzyme Microb Technol 2009, 44:145-153. The influence of mutations near the substrate-binding pocket on activity and enantioselectivity of the epoxide hydrolase was studied by sitesaturation mutagenesis. The mutant F108C showed sevenfold improved activity and excellent enantioselectivity in the conversion of cis-2,3epoxybutane. Mutant F108A showed 150-fold higher activity and excellent enantioselectivity on cyclohexene oxide. 57. Janssen DB: Biocatalysis by dehalogenating enzymes. Adv Appl Microbiol 2007, 61:233-252. 58. Pavlova M, Klvana M, Prokop Z, Chaloupkova R, Banas P, Otyepka M, Wade R, Tsuda M, Nagata Y, Damborsky J: Redesigning dehalogenase access tunnels as a strategy for degrading an anthropogenic substrate. Nat Chem Biol 2009, 5:727-733. The authors identified key residues in entrance tunnels to the active site by rational design of Rhodococcus rhodochrous haloalkane dehalogenase and randomized this region by directed evolution. 59. Lee S, Chang Y, Shin D, Han J, Seo M, Fazelinia H, Maranas CD, Kim H: Designing the substrate specificity of D-hydantoinase using a rational approach. Enzyme Microb Technol 2009, 44:170-175. This article describes the rational design of a Bacillus stearothermophilus D-hydantoinase resulting in a mutant with 200-fold higher specificity for hydroxyphenylhydantoin than the wild-type enzyme. 60. Jiang S, Li C, Zhang W, Cai Y, Yang Y, Yang S, Jiang W: Directed evolution and structural analysis of N-carbamoyl-D-amino acid amidohydrolase provide insights into recombinant protein solubility in Escherichia coli. Biochem J 2007, 402:429-437. 61. Yu H, Li J, Zhang D, Yang Y, Jiang W, Yang S: Improving the thermostability of N-carbamyl-D-amino acid amidohydrolase by error-prone PCR. Appl Microbiol Biotechnol 2009, 82:279-285. 62. Breuer M, Hauer B: Carbon–carbon coupling in biotransformation. Curr Opin Biotechnol 2003, 14:570-576. 63. Fessner W-D: Enzymatic synthesis using aldolases. In Stereoselective Biocatalysis. Edited by Patel RN. Marcel Dekker; 2000:239-265. 64. Sugiyama M, Hong Z, Greenberg WA, Burk MJ: In vivo selection for the directed evolution of L-rhamnulose aldolase from Lrhamnulose-1-phosphate aldolase (Rha D). Bioorg Med Chem 2007, 15:5905-5911. 65. Ran N, Frost JW: Directed evolution of 2-keto-3-deoxy-6phosphogalactonate aldolase to replace 3-deoxy-D-arabinoheptulosonic acid 7-phosphate synthase. J Am Chem Soc 2007, 129:6130-6139. 66. Wolterink-van Loo S, Siemerink MA, Perrakis G, Kaper T, Kengen SW, van der Oost J: Improving low-temperature activity of Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase. Archaea 2009, 2:233-239. Current Opinion in Microbiology 2010, 13:274–282
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67. Dean SM, Greenberg WA, Burk MJ: Recent advances in aldolase-catalyzed asymmetric synthesis. Adv Synth Catal 2007, 349:1308-1320.
73. Aharoni A, Gaidukov L, Khersonsky O, McQ. Gould S, Roodveldt C, Tawfik DS: The ‘evolvability’ of promiscuous protein functions. Nat Genet 2004, 37:73-76.
68. Samland A, Sprenger GA: Microbial aldolases as C–C bonding enzymes – unknown treasures and new developments. Appl Microbiol Biotechnol 2006, 71:253-264.
74. Hult K, Berglund P: Enzyme promiscuity: mechanism and applications. Trends Biotechnol 2007, 25:231-238.
69. Franke D, Hsu CC, Wong CH: Directed evolution of aldolases. Methods Enzymol 2004, 388:224-238.
75. Nair N, Zhao H: Evolution in reverse: engineering a D-xylosespecific xylose reductase. ChemBioChem 2008, 9:1213-1215.
70. Bolt A, Berry A, Nelson A: Directed evolution of aldolases for exploitation in synthetic organic chemistry. Arch Biochem Biophys 2008, 474:318-330.
76. de Groeve MRM, de Baere M, Hoflack L, Desmet T, Vandamme EJ, Soetaert W: Creating lactose phosphorylase enzymes by directed evolution of cellobiose phosphorylase. Protein Eng Des Sel 2009, 22:393-399.
71. Fox RJ, Davis SC, Mundorff E, Newman LM, Gavrilovic V, Ma SK, Chung LM, Ching C, Tam S, Muley S et al.: Improving catalytic function by ProSAR-driven enzyme evolution. Nat Biotechnol 2007, 25:338-344.
77. Chen B, Cai Z, Wu W, Huang Y, Pleiss J, Lin Z: Morphing activity between structurally similar enzymes: from heme-free bromoperoxidase to lipase. Biochemistry 2009, 48:1149611504.
72. Bornscheuer UT, Kazlauskas RJ: Catalytic promiscuity in biocatalysis: using old enzymes to form new bonds and follow new pathways. Angew Chem Int Ed 2004, 43:6032-6040.
78. Jochens H, Stiba K, Savile C, Fujii R, Yu J, Gerassenkov T, Kazlauskas RJ, Bornscheuer UT: Converting an esterase into an epoxide hydrolase. Angew Chem Int Ed 2009, 48:3532-3535.
Current Opinion in Microbiology 2010, 13:274–282
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Protein engineering of microbial enzymes Dominique Bo¨ttcher and Uwe T Bornscheuer Protein engineering has emerged as an important tool to overcome the limitations of natural enzymes as biocatalysts. Recent advances have mainly focused on applying directed evolution to enzymes, especially important for organic synthesis, such as monooxygenases, ketoreductases, lipases or aldolases in order to improve their activity, enantioselectivity, and stability. The combination of directed evolution and rational protein design using computational tools is becoming increasingly important in order to explore enzyme sequencespace and to create improved or novel enzymes. These developments should allow to further expand the application of microbial enzymes in industry. Address Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, University of Greifswald, Felix-Hausdorff-Str. 4, 17487 Greifswald, Germany Corresponding author: Bornscheuer, Uwe T ([email protected])
Current Opinion in Microbiology 2010, 13:274–282 This review comes from a themed issue on Ecology and Industrial Microbiology Edited by Erick Vandamme Available online 17th February 2010 1369-5274/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2010.01.010
instance for pig liver esterase [8,9] and horse liver alcohol dehydrogenase (Evocatal GmbH, Du¨sseldorf, Germany), it is often not trivial to get correctly folded and active protein in high amounts. Another important source of biocatalysts are plant enzymes, where for instance hydroxynitrile lyases from Manihot esculenta [10] and Hevea brasiliensis [11] could be expressed in E. coli and the yeast Pichia pastoris, respectively. Independent of the reaction system and enzyme investigated for a given biocatalytic process, very often the enzyme does not meet the requirements for a large-scale application and its properties have to be optimized. This usually includes not only the chemoselectivity, regioselectivity and especially stereoselectivity of the biocatalyst, but also process-related aspects such as long-term stability at certain temperatures or pH-values and activity in the presence of high substrate concentrations to achieve highest productivity. Beside rather classical strategies such as immobilization, additives or process engineering, molecular biology techniques nowadays represent probably the most important methodology to tailor-design the enzyme for a given process. Two different strategies are used: rational protein design and directed (molecular) evolution, which are increasingly applied in a synergistic manner (Figure 1). In the following sections, selected examples for protein engineering of biocatalysts of microbial origin, which are particularly useful for organic synthesis, are given. Readers are also referred to consult recent general literature on protein engineering of biocatalysts [12–14].
Introduction Biocatalysts are extensively used in the industrial production of bulk chemicals and pharmaceuticals and over 300 processes have been already implemented [1–4]. In the vast majority of processes, enzymes of microbial origin are used as the microbial kingdom represents a huge – and still only partially explored – reservoir for biocatalysts with desired properties. Furthermore, the metagenome approach [5–7] substantially facilitates the discovery of novel enzymes from microbial sources and hence the number of potentially useful biocatalysts increased exponentially in the past decade. In contrast, enzymes from animal tissues are less preferred as these often occur as mixture of isoenzymes differing in substrate specificity and product safety (i.e. viral infections), which often restricts their industrial use. Although this can be overcome by expression of the corresponding genes in microbial hosts as shown for Current Opinion in Microbiology 2010, 13:274–282
Examples for protein engineering of microbial enzymes Dehydrogenases
The application of enantioselective alcohol dehydrogenases plays an important role for the asymmetric synthesis of enantiomerically and diastereomerically pure alcohols, which are important building blocks for pharmaceuticals, agro, and fine chemicals. To meet process conditions, highly active and also solvent stable enzymes are required. For example an alcohol dehydrogenase from Pyrococcus furiosus was recently engineered for improved activity at low temperature for the production of enantiopure (2S,5S)hexanediol. A mutant obtained by error-prone polymerase chain reaction (epPCR) showed a 10-fold higher maximum specific activity compared to the wild-type enzyme [15]. In a rational design approach the enantiopreference of a secondary alcohol dehydrogenase from Thermoanaerobacter www.sciencedirect.com
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Figure 1
The appropriate method for protein engineering of enzymes should be decided case-by-case on the basis of the availability and quality of the structural and mechanistic information and the feasibility of a high-throughput screening (HTS) system for screening or selection.
ethanolicus was reversed by just a single point mutation (I86A). This mutant not only shows anti-Prelog stereospecificity, but also accepts more sterically demanding substrates [16]. One recent example for altered coenzyme specificity was shown for the glucose dehydrogenase from Haloferax mediterranei, where a single mutation G206D was sufficient to change the enzyme preference from NADP+ to NAD+ [17]. Oxygenases
Oxygenases are enzymes, which introduce one (monooxygenases) or two (dioxygenases) oxygen atoms into their substrates. Typically NADH or NADPH serve as reduction equivalents via electron-transfer proteins such as reductases. The major interest in these enzymes for organic synthesis is due to their high chemoselectivity, regioselectivity and stereoselectivity. P450 monooxygenases catalyze a broad range of interesting reactions, but their application is hampered by their www.sciencedirect.com
low specific activity, narrow substrate range, usually low stability and the need for a complex electron-transfer system. In addition, most P450 enzymes are only applicable in whole cell systems. Nevertheless, the recent progress made in the protein engineering of several P450 enzymes might allow for their application in biocatalysis in the near future [18]. Most recently published P450 engineering approaches summarized below aimed for broader substrate specificities (Figure 2). The directed evolution of self-sufficient P450 BM-3 (CYP102A1) from Bacillus megaterium yielded among others a variant that shows improved conversion and selectivity on astemizole, a potent histamine receptor antagonist used for the treatment of allergy symptoms [19]. Li et al. further improved a previously isolated cytochrome P450 BM-3 variant via site-directed and random mutagenesis to produce indigo and indirubin. Three resulting mutants exhibited higher hydroxylation activity towards indole in comparison to the parent enzyme, two P450 BM-3 variants (D168H/3X, E435T/3X) showed an up to sixfold increase in catalytic efficiency, and the mutant Current Opinion in Microbiology 2010, 13:274–282
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Figure 2
Substrates for protein engineering of P450 monooxygenases. Top row: [19], second row: [20–24], third row: [25], and fourth row: [26].
D168H/3X exhibited higher regioselectivity [20]. Another example for engineering regioselectivity of this enzyme was based on rational design using site-directed mutagenesis. The mutants V78A/F87A/I263G and S72Y/V78A/ F87A were able to hydroxylate lauric acid at the d-position, g-position and b-position [21]. A thermophilic cytochrome P450 (CYP175A1) was engineered by site-directed mutagenesis to catalyze the hydroxylation of the non-natural substrate testosterone [22]. Applying DNA family shuffling methodology on different cytochrome P450 enzymes resulted in an expansion of the enzyme substrate range (e.g. activity towards different luciferin derivatives) [23,24]. Other recent evolution advances on P450 enzymes yielded variants with improved activity and regioselectivity or stereoselectivity in the oxidation of terpenes [25] or with improved 11-deoxycortisol and progesterone hydroxylation activity [26] or they have been improved for activity towards biomimetic NADH cofactors [27]. A very elegant approach using in vivo-directed evolution was applied to enhance the activity of two mediumCurrent Opinion in Microbiology 2010, 13:274–282
chain-length terminal alkane hydroxylases (AlkB and CYP153A6) on small alkanes. An evolution system based on enhanced growth on butane of a Pseudomonas putida strain complemented with evolved AlkB and CYP153A6 variants served for the identification of best mutants. The resulting variants exhibited higher rates of 1-butanol production from butane and maintained their preference for terminal hydroxylation [28]. Another important group of enzymes are Baeyer–Villiger Monooxygenases (BVMO). Although several BVMOs have already been recombinantly expressed and characterized, so far directed evolution and rational protein design have been applied to mainly three of them to alter their substrate specificity and enantioselectivity: cyclohexanone monooxygenase (CHMO) from Acinetobacter calcoaceticus NCIMB 9871 [29], cyclopentanone monooxygenase (CPMO) from Comamonas testosteroni [30] NCIMB 9872 and phenylacetone monooxygenase (PAMO) from Thermobifida fusca. Because of its thermostability and the stability in organic solvents, especially the PAMO is a very interesting candidate for application in organic synthesis. In order to broaden the limited substrate scope and to improve the selectivity, the enzyme was recently engineered by two research groups. www.sciencedirect.com
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First, based on structure comparison of the PAMO crystal structure and a structure model of the sequence-related CPMO, several active-site residues were selected for sitedirected mutagenesis. One mutant (M446G) was able to convert a number of aromatic ketones, amines and sulfides, whereas the wild-type enzyme showed no activity. In addition the mutant represents the first BVMO, which is able to convert indole into indigo [31]. The other rational approach was recently published by Reetz and Wu. After an alignment of eight different BVMOs and induced-fit docking experiments, five different libraries were designed and randomized by saturation mutagenesis (CASTing). Mutants were found that show high activity and enantioselectivity towards different 2aryl and 2-alkylcyclohexanones and a bicyclic ketone. The best mutant also maintained thermostability of the wild-type enzyme [32]. Recently, the activity and enantioselectivity of a BVMO from Pseudomonas fluorescens with substrate preference for aliphatic ketones could be further enhanced in our group using directed evolution [33]. An extensive coverage of the state-of-the-art research regarding BVMOs can be found in recent reviews [34,35]. Transferases
Within the enzyme class of transferases, transaminases are highly versatile biocatalysts for the synthesis of optically active amines or a-amino acids by transfer of an amino group from a donor substrate to an acceptor compound utilizing the cofactor pyridoxal-5-phosphate. aTransaminases require the presence of a carboxylic acid function in a-position to the keto or amine functionality and hence only allow the formation of a-amino acids, vtransaminases are much more useful as they in principle accept any ketone (or amine). In the last decade, especially v-transaminases have been extensively studied and also overexpressed in microbial hosts such as E. coli with the enzyme from Vibrio fluvialis as probably the most intensively studied v-transaminase. The current status for the use of transaminases is covered in a recent review [36]. Recently, the substrate specificity of the v-aminotransferase from Vibrio fluvialis was rationally designed for the kinetic resolution of aliphatic chiral amines. Two mutant enzymes showed significant changes in their substrate specificity. They catalyzed transamination of a broad range of aliphatic amines without losing the original activities toward aromatic amines and enantioselectivity [37]. A mesophilic v-aminotransferase from Athrobacter citreus was evolved to a thermostable aminotransferase via epPCR. Through five rounds of mutagenesis, the specific activity was improved 260-fold from 5.9 to 1583 U/g and www.sciencedirect.com
the optimum temperature could be elevated from 30 to 558C [38]. Hydrolases
The majority of industrially used enzymes are lipases and thus many examples of engineered lipases can be found in literature and recent reviews [39,40]. Candida antarctica lipase B (CAL-B) is probably the most useful lipase for organic synthesis and numerous examples for its application [41] and engineering can be found in literature. The enzyme has been recently improved concerning thermostability [42], activity [43,44], enantioselectivity [45] and also solubility [46]. In one of these examples a very interesting approach, the so-called circular permutation (CP) was used [43,44,47]. Lutz et al. linked the native N-termini and C-termini of the gene encoding CAL-B and subsequently linearized it by random digestion to yield variants bearing alternative N-termini and C-termini. Surprisingly, this not only lead to active lipase, some variants also showed higher catalytic efficiency in comparison to the wild type (up to 11-fold against p-nitrophenol butyrate and 175-fold against 6,8-difluoro-4-methylumbelliferyl octanoate, while KM values were nearly the same). For the most active variant (cp283), kinetic experiments demonstrated that CP of this enzyme did not compromise the enantioselectivity in the resolution of some chiral secondary alcohols [44]. Among the field of hydrolases only a few esterases found their application in organic chemistry. Nevertheless, some examples for their successful improvement by directed evolution, rational protein design and combinations of these methods have been described. The synthetically useful esterase BS2 – which differs only by 11 amino acids from a para-nitrobenzyl esterase from Bacillus subtilis (BsubpNBE), being the first example for directed evolution of an esterase – was evolved by rational design in our group and the enantioselectivity towards an ester of the tertiary alcohol 2-phenyl-3-butyn2-yl acetate could be increased sixfold to E = 19, and towards linalyl acetate inverted from (R)-preference to (S)-preference with E = 6 [48]. In a later study, this mutant (G105A) showed a good enantioselectivity towards 2-phenyl-3-butyn-2-yl acetate (E = 54) in 20%, v/v, DMSO, and an E-value of >100 towards the trifluoromethyl analog [49]. Another point mutation (E188D) gave similar high enantioselectivity towards both substrates as well as a series of other tertiary alcohol acetates [50]. Using a focused directed evolution approach, we recently created a library covering three residues by saturation mutagenesis and identified a double mutant (E188W/M193C) with an (S)-preference and an E-value of 70 towards a tertiary alcohol thus leading to a variant with completely inverted enantioselectivity [51]. Interestingly, the two single mutations showed substantially Current Opinion in Microbiology 2010, 13:274–282
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lower and even undesired selectivity: E188W shows only E = 26 for the (S)-enantiomer and M193C was still (R)selective (E = 16). Thus, only the simultaneous saturation mutagenesis at several amino acid positions resulted in the significant inversion of enantiopreference and an increase of enantioselectivity. An esterase from Pseudomonas fluorescens DSM50106 (PFEI) was recently improved in our group by the OSCARR method yielding two mutants with a 10-fold higher catalytic efficiency towards long chain p-nitrophenyl esters (octanoate and dodecanoate) [52] and both mutants (F126I and G120S/F126I) showed also a slightly decreased catalytic efficiency towards the typical esterase substrates p-nitrophenyl acetate and p-nitrophenyl butanoate. Thus, the substrate specificity of this esterase could be shifted to lipase-like properties. Very recently the same enzyme was further rationally designed along the active-site entrance to widen the substrate tunnel and to study the influence on substrate specificity and enantioselectivity [53]. Optically pure epoxides and diols are important building blocks for the production of pharmaceuticals. One way to prepare enantiopure epoxides is by hydrolytic kinetic resolution of racemic epoxides using enantioselective epoxide hydrolases. Epoxide hydrolases can also be used to produce optically enriched vicinal (R,R)-diols or (S,S)diols in 100% theoretical yield through regioselective hydrolysis of meso-epoxides [54]. The epoxide hydrolase from Aspergillus niger M200 was recently engineered via saturation mutagenesis and it could be shown that amino acid exchanges in the substrate tunnel region can lead to significant improvements in enantioselectivity and activity [55]. Another commonly applied epoxide hydrolase from Agrobacterium radiobacter (EchA) was evolved using sitedirected mutagenesis. For this, position F108, which flanks the nucleophilic aspartate and forms part of the substrate-binding pocket, was saturated using degenerate primers and resulted in mutants with higher activity toward cis-disubstituted meso-epoxides. Mutant F108C converted cis-2,3-epoxybutane to enantiopure (2R,3R)2,3-butanediol with a sevenfold improved activity, and mutant F108A hydrolyzed cyclohexene oxide to (1R,2R)1,2-cyclohexanediol (99%ee) with 150-fold higher activity than the wild-type enzyme [56]. A range of haloalkane dehalogenases of microbial origin have been discovered and characterized. These enzymes mainly catalyze the hydrolysis of chloroalkanes, or bromoalkanes to the corresponding alcohol and halide ion, and also iodoalkanes are accepted as substrates. They generally exhibit a broad substrate tolerance, with preference to primary carbon–halogen bonds [57]. Pavlova et al. identified key residues in entrance tunnels to the Current Opinion in Microbiology 2010, 13:274–282
active site by rational design of Rhodococcus rhodochrous haloalkane dehalogenase and randomized this region by directed evolution methods. The most active variant bearing large aromatic residues at two out of three randomized positions and two positions modified by site-directed mutagenesis, showed up to 32-fold higher activity than the wild type towards 1,2,3-trichloropropane [58]. Hydantoinases are valuable enzymes for the production of optically pure D-amino and L-amino acids. They catalyze the reversible hydrolytic cleavage of hydantoins and 50 -monosubstituted hydantoins. In combination with carbamoylases (N-carbamoyl-D-amidohydrolases), the reaction yields L-amino or D-amino acids depending on the stereoselectivity of the enzymes. Nowadays, hydantoinases from, for example, Arthrobacter sp., Nocardia sp., Bacillus sp. and Pseudomonas sp. have been described and a range of D-amino acids are accessible by using them. A recent example for hydantoinase engineering is the rational design of Bacillus stearothermophilus D-hydantoinase for altered substrate specificity towards hydroxyphenylhydantoin and resulted in the mutant M63I/ F159S exhibiting about 200-fold higher specificity for hydroxyphenylhydantoin than the wild-type enzyme [59]. In another approach the solubility of Burkholderia pickettii was improved by protein engineering. A combination of random mutagenesis using epPCR and DNA shuffling techniques gave several mutants with reduced aggregation. It was found that mainly three amino acid residues (A18, Y30, and K34) are involved in protein solubility. A triple mutant A18T/Y30N/K34E could be overexpressed in highly soluble form in E. coli [60]. Later, the thermostability of the same highly soluble D-carbamoylase variant was improved using directed evolution. The most thermostable mutant, Q12L, showed a 7 8C increase in thermostability [61]. D-carbamoylase
Lyases
A broad range of aldolases has been described in literature and these biocatalysts are very attractive for organic synthesis as they allow to create two stereocenters in a single reaction step [62,63]. However, narrow substrate ranges and enzyme inactivation under synthesis conditions represented major obstacles for the large-scale application of aldolases. Again, these limitations could be overcome by directed evolution. The Wong group described an in vivo selection assay for the directed evolution of an L-rhamnulose aldolase starting from an L-rhamnulose-1-phosphate aldolase (RhaD) [64]. This assay enabled the identification of the desired RhaD variant in an epPCR-derived library, which in contrast to the wild type accepts dihydroxyacetone (DHA) as donor instead of the very expensive and unstable phosphorylated analog DHAP. A related enzyme utilizing www.sciencedirect.com
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galactose derivatives (KDPGal) was subjected to a combination of epPCR, DNA shuffling and site-directed mutagenesis yielding a variant with 60-fold improved catalytic efficiency in the synthesis of a shikimate pathway product [65]. Recently a library of randomly generated mutants of thermophilic Sulfolobus acidocaldarius 2-keto-3-deoxygluconate aldolase was screened for improved synthesis of 2keto-3-deoxygluconate at lower temperatures yielding a single mutant (V193A) that displayed a threefold increase in activity compared to the wild-type enzyme. The increased specific activity of this mutant at suboptimal temperatures between 40 and 608C could be observed, not only for the aldol condensation of pyruvate with glyceraldehyde, but also for several non-natural acceptor aldehydes [66]. A broad range of further examples of aldolases improved by protein engineering methods can be found in a number of recent reviews [67–70]. Halohydrin dehalogenases (also known as haloalcohol dehalogenases or halohydrin hydrogen-halide lyases) catalyze the cofactor-independent dehalogenation of vicinal haloalcohols by an intramolecular substitution reaction, producing the corresponding epoxide. These enzymes are involved in the bacterial degradation of several important environmental pollutants, such as epichlorohydrin or 1,3-dichloro-2-propanol [57]. The catalytic function of a halohydrin dehalogenase from Agrobacterium radiobacter was improved by ProSAR-driven evolution (protein sequence activity relationship), introduced by Fox et al., to obtain ethyl (R)-4cyano-3-hydroxybutyrate, which is involved in the synthesis of Atorvastatin (Lipitor) [71]. The new enzyme had a 4000-fold volumetric productivity in the cyanation process compared to the wild type. This example also demonstrates the efficiency of combining theoretical consideration with laboratory methods for the improvement of biocatalyst function.
Promiscuous and novel enzyme activities Some enzymes already catalyze reactions on alternative functional groups, but at a very slow rate, compared to their main catalytic function. This ‘catalytic promiscuity’ is now a major research theme and the current status has been covered in recent reviews [72–74]. For example the promiscuous enzyme xylose reductase from Neurospora crassa, which prefers D-xylose over Larabinose, has been engineered via semi-rational design approaches by site-saturation mutagenesis, combinatorial active site-saturation testing (CASTing) and finally one mutant (L109Q) with ninefold higher preference for Dxylose could be identified [75]. www.sciencedirect.com
De Groeve et al. modified substrate specificity of Cellulomonas uda cellobiose phosphorylase from cellobiose to lactose by directed evolution. This enzyme already exhibits a low rate of lactose phosphorylase activity, which is unusual since this enzyme class is known to have a strict sugar phosphate donor specificity. After a single round of random mutagenesis, clones with improved lactose phosphorylase activity could be enriched by an in vivo selection on minimal medium containing lactose as sole carbon source. Subsequent site-directed and site-saturation mutagenesis resulted in the mutant T508I/N667A with 7.5 times higher specific activity on lactose than the wild-type enzyme [76]. The bromoperoxidase A2 from Streptomyces aureofaciens was converted into a lipase by structure comparison with lipase A from Bacillus subtilis, which shows a similar structure but only very low sequence identity. Using epPCR and site-directed mutagenesis mutants with improved hydrolytic activities and substrate preferences toward long chain substrates could be obtained [77]. In a recent example we could show that it is possible to interconvert enzymes within the class of a/b-hydrolase fold enzymes. Starting from an esterase from Pseudomonas fluorescence, the introduction of distinct point mutations together with the exchange of a loop sequence – containing a mechanistically important tyrosine residue and possibly enabling the substrate access to the active site – derived from an epoxide hydrolase, activity could be generated in the esterase scaffold. The resulting chimera not only showed reasonable activity only 800-fold lower than a true epoxide hydrolase, but also exhibited high enantioselectivity (E > 100) in the kinetic resolution of pnitrostyrene oxide [78].
Conclusions Protein engineering via directed evolution or rational design has emerged as a very powerful tool to design and alter the properties of enzymes. This technology quickly found its application for a broad range of proteins, with the vast majority being of interest for biocatalysis. Consequently, a diverse set of molecular biology tools to create well-balanced mutant libraries as well as suitable high-throughput screening methods have been developed to make the application of directed evolution more easy and feasible. Within just a decade, directed evolution has emerged standard methodology in protein engineering and therefore can be used complementary or in combination with rational protein design to meet the demands for industrially applicable biocatalysts exhibiting desired chemoselectivity, regioselectivity, and stereoselectivity as well as withstands process conditions (i.e. high substrate concentrations, solvents, temperatures, long-term stability). Current Opinion in Microbiology 2010, 13:274–282
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Acknowledgements We thank the German Research Foundation (DFG, Grant Bo1862/4-1) and the Deutsche Bundesstiftung Umwelt (DBU, Grant AZ13198-32) for financial support.
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