Engineering specialized metabolic pathways—is there a room for enzyme improvements?

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Engineering specialized metabolic pathways—is there a room for enzyme improvements? Arren Bar-Even1 and Dan Salah Tawfik2 Recent advances in enzyme engineering enable dramatic improvements in catalytic efficiency and/or selectivity, as well as de novo engineering of enzymes to catalyze reactions where natural enzymes are not available. Can these capabilities be utilized to transform biosynthesis pathways? Metabolic engineering is traditionally based on combining existing enzymes to give new, or modified, pathways, within a new context and/or organism. How efficient, however, are the individual enzyme components? Is there room to improve pathway performance by enzyme engineering? We discuss the differences between enzymes in central versus specialized, or secondary metabolism and highlight unique features of specialized metabolism enzymes participating in the synthesis of natural products. We argue that, for the purpose of metabolic engineering, the catalytic efficiency and selectivity of many enzymes can be improved with the aim of achieving higher rates, yields and product purities. We also note the relative abundance of spontaneous reactions in specialized metabolism, and the potential advantage of engineering enzymes that will catalyze these steps. Specialized metabolism therefore offers new opportunities to integrate enzyme and pathway engineering, thereby achieving higher metabolic efficiencies, enhanced production rates and improved product purities. Addresses 1 Department of Plant Sciences, Weizmann Institute of Science, Rehovot 76100, Israel 2 Department Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel Corresponding authors: Bar-Even, Arren ([email protected]) and Salah Tawfik, Dan ([email protected])

Current Opinion in Biotechnology 2013, 24:310–319 This review comes from a themed issue on Plant biotechnology Edited by Natalia Dudareva and Dean DellaPenna For a complete overview see the Issue and the Editorial Available online 26th October 2012 0958-1669/$ – see front matter, # 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.copbio.2012.10.006

Introduction Metabolic engineering is tackling emerging challenges in the pharmaceutical industry [1–3], energy market [4–7] and green chemistry [8–10]. Many engineered pathways make use of enzymatic apparatus derived from central metabolism. However, specialized (also known as secondary) metabolism offers a wider range of metabolic and Current Opinion in Biotechnology 2013, 24:310–319

biosynthetic routes, and a broader range of products, hence providing fertile engineering grounds. Here, we highlight some fundamental differences between central and specialized metabolism. We focus on inherent differences in their enzyme components, and discuss the implications for metabolic engineering. Metabolic engineering generally regards the reassembly of existing, natural enzymes within a new context. The tacit assumption is that individual enzyme components are optimal with respect to their native function. Engineering efforts therefore aim almost entirely at optimizing enzymes toward alternative reactions, or reaction conditions that differ from the natural ones, and not on increasing catalytic efficiency toward the native substrate and reaction (exceptions include attempts to improve Rubisco [11,12], although others argued that this enzyme is optimal [13,14]). The optimality assumption, however, is derived from the textbook examples of enzyme ‘superstars’, such as carbonic anhydrase and triosephosphate isomerase [15]. The latter, however, do not reflect the ‘average’ enzyme [16], and, as discussed below, they definitely do not reflect enzymes in specialized metabolism. To achieve an economic advantage, engineered pathways must carry high fluxes, sustain high yields, and have a minimal effect on growth rates and on other organismal traits. They should therefore be economical in terms of protein production (i.e. the amount of enzymes required) and other cellular resources consumed for product formation (e.g. energy or reducing equivalents) [17,18]. In nature, these demands shaped the properties of individual enzymes and of entire pathways. They resulted in remarkably high catalytic efficiencies, exquisite specificities and tight regulation of expression levels and enzyme action (e.g. allosteric regulation). We argue, however, that specialized metabolism enzymes and pathways have not been shaped to the same extent. We point out the discrepancies between certain features of specialized metabolism, and of natural product biosynthesis in particular, and the requirements for highly efficient engineered pathways. We suggest that, for purposes of metabolic engineering in heterologous bacterial and fungal systems, more efficient pathways could be engineered through the optimization of individual enzymes, that is, by increasing the turnover number (kcat) and/or catalytic efficiency (kcat/KM) of slow enzymes, by refining enzyme specificity, by engineering tighter regulation, and possibly by introducing enzymes for reactions that may occur spontaneously in nature. www.sciencedirect.com

Engineering specialized metabolic pathways – a room for enzyme improvements? Bar-Even and Salah Tawfik 311

Specialized metabolism – biochemistry’s ‘Panda’s Thumb’ Our view of metabolism comes primarily from central metabolism pathways. The genetics of these pathways portrayed a clear picture of well-organized, sequential and tightly regulated pathways. The individual enzymes seem highly efficient, exquisitely specific and tightly regulated. This picture is, however, incomplete. For example, over a third of Escherichia coli’s enzymes catalyze two or more transformations [19]. Further, underneath the well-organized facade lies ‘underground metabolism’ [20] suggesting that pathways could be redirected, and that enzymes can act on alternative substrates or yield other products [21,22]. Indeed, loose enzyme specificity, underground metabolism, and other manifestations of noise and ‘messiness’, comprise the origins of evolutionary innovations [23]. Messiness is highly pronounced in specialized metabolism. As compared to central metabolism, specialized metabolism superficially appears to be ‘low tech’ as apparent in the biosynthesis of natural products – these are specific to small species groups (clades), and have often diverged relatively recently. The production rates associated with specialized metabolism are typically low, and these pathways often have little effect on growth. In our view, specialized metabolism comprises the most notable example at the molecular level for the sluggish, tinkering nature of evolutionary processes – the ‘Panda’s Thumb’ and Darwin’s ‘old wheels, springs and pulleys, only slightly altered’ of biochemistry. The exact nature and the underlying forces behind these differences are not entirely understood but we try to speculate on them in the below sections. These features of specialized metabolism are demonstrated with several examples including betalain biosynthesis (Figure 1). Betalains are plant and fungi pigments of red-violet and yellow-orange colors [24]. They are of interest for the food industry, serving as acid-resistant food colorants and possessing anti-oxidant and radical scavenging activities [25,26], and are also being considered as sensitized-dyes for solar cells [27]. Although betalains have not been extensively studied, this pathway is illustrative of the points we wish to convey. Specialized metabolism enzymes are slow

A global analysis of published enzymatic parameters indicated that, compared to their central metabolism counterparts, enzymes employed in specialized metabolism tend to be significantly slower, in term of kcat and kcat/KM (Figure 2) [16]. Specifically, kcat values are, on average, 30-fold lower in specialized metabolism enzymes. The high efficiency of enzymes in central metabolism is expected as the transformations catalyzed by these pathways are directly linked to growth rate, are www.sciencedirect.com

shared by a large number of organisms, indicating their ancient origins. The selection pressures acting to optimize their catalytic parameters are strong, and have been persistently acting for a long time. Notably, since core metabolic enzymes mediate high fluxes, they comprise a large fraction of the cellular proteome (e.g. [28]) despite being catalytically efficient. Central metabolism enzymes also tend to be substrate and product specific and allosterically regulated [19]. Overall, it seems that little was left to chance in central metabolism. The catalytic inefficiency of specialized metabolism enzymes is also somewhat expected. These pathways usually carry relatively low and transient fluxes – in some cases to avoid competing with primary metabolism – that are rarely coupled to growth rates. The selection pressures acting to increase rate are therefore weaker [29]. In many cases, central metabolic enzymes were recruited to specialized metabolism but their catalytic respecialization seems partial [29]. In addition, as discussed below, spontaneous reactions sometimes constrain the rate of specialized metabolic pathways, thus masking the selective advantages associated with higher enzyme rates. It could also be is that some specialized metabolism enzymes have not been under selection for high catalytic efficiency with a single substrate, but have actually evolved as generalists, thus promoting chemical diversity rather than a single product (see also below). In betalains biosynthesis, for example, a key step is the glycosylation of hydroxyl groups (as in many other natural products). The glycosyltransferase that glycosylate betanidin is a slow enzyme (reaction ‘R7’ in Figure 1; vmax  10 mmol min1 mg1 [30], or even slower [31,32]). Glycosyltransferases from central metabolism, such as corn sucrose synthase (vmax  128 mmol min1 mg1 [30]), or E. coli glycogen synthase (vmax  500 mmol min1 [33]) are at least an order of magnitude faster. Specialized metabolism also makes use of certain reaction types that are inherently slow and are uncommon in central metabolism. For example, oxygenase reactions which are rare in central metabolism are prevalent in specialized metabolism. The median kcat for oxygenase enzymes of the EC class 1.14.X.X (3 s1) is considerably lower than that of enzymes operating in central metabolism (80 s1, see Figure 2). Specifically, tyrosinase, the oxygenase thought to initiate the betalain pathway in some organisms (reactions ‘R1’ and ‘R2’) [24], is a rather slow enzyme. Mushroom tryrosinases, for example, have kcat of 3–7 s1 [34]. In beet, a cytochrome P450 oxidase catalyzes the oxidation of dopa (reaction ‘R2’) [35]. However, P450s are also slow enzymes with kcat values that are usually <5 s1. Another example is the enzyme 4,5-DOPA dioxygenase that oxidizes dopa to 4,5-secodopa (reaction ‘R4’) [24]. The specific activity of the beet enzyme is also low, <1 mmol min1 mg1 [36]. Current Opinion in Biotechnology 2013, 24:310–319

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Figure 1

Figure 2 COOA

HO

1

Cumulative distribution

Tyrosine R1

O2

HO

COOA NH2

HO

Dopa

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O

Dopaquinone

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0.2

4,5-seco-Dopa

R3

2.5 5.2

R5

18

79

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O

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R

COOH N H

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H2N

cyclo-Dopa

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amino-acids R6

HOOC

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R8

(b)

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+

N H

COOH

Betanidin

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HOOC

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Betaxanthins

OH

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UDP-Glucose

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HO

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0.2

HO

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1

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The current view of the major routes for betalains synthesis. Other betalain pigments and metabolic routes for their synthesis are given in Ref. [24]. (R1) Tyrosinase or another oxidase; (R2) tyrosinase or cytochrome P450; (R3) spontaneous cyclization; (R4) Dopa dioxygenase; (R5) spontaneous cyclization; (R6) spontaneous aldimine condensation; (R7) glycosyltransferase and (R8) spontaneous aldimine condensation.

Specialized metabolism enzymes exhibit broad specificity

Many of the enzymes participating in specialized metabolism catalyze the same reaction with a relatively broad range of substrates or catalyze similar yet different reactions on the same substrate (so-called generalists). As all other facets of evolution, generalists could be the outcome of both chance and necessity. Chance, owing to the recruitment of promiscuous enzymes to a newly formed pathway with some optimization [37], but not necessarily Current Opinion in Biotechnology 2013, 24:310–319

Enzymes operating within different metabolic groups have different kcat and kcat/KM values (adopted from Ref. [16]). Enzyme-substrate pairs were assigned to the metabolic modules in which they participate based on the categorization in the KEGG database [16]. All modules were classified into four central groups: Central-CE (carbohydrate-energy) metabolism, involving the main carbon and energy flow; Central-AFN (amino-acids, fatty-acids and nucleotide) metabolism; Intermediate metabolism, including the biosynthesis and degradation of various common cellular components, such as co-factors and co-enzymes and Secondary metabolism related to metabolites which are produced in specific cells or tissues, under specific conditions and/or in relatively limited quantities. (A) The cumulative distribution of kcat values for enzyme-substrate pairs belonging to different metabolic contexts. The distributions are significantly different with p-value <0.0005, rank-sum test, except those of intermediate and specialized metabolisms ( p-value <0.05). (B) The cumulative distribution of kcat/KM values for enzymesubstrate pairs belonging to different metabolic contexts. Central-CE (carbohydrate and energy) metabolism exhibits significantly higher kcat/ KM values as compared to all other metabolic groups ( p-value <0.0005, rank-sum test).

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exquisite specificity. Necessity suggests that certain specialized metabolic enzymes actually evolved as generalists, with the explicit aim of acting on multiple substrates, thus generating more than one product that in turn could be further modified to an even broader range of products by the subsequent enzyme(s) in the pathway. Many terpene cyclases, for example, make several products starting from a single substrate [38,39]. Subsequent P450 enzymes are able to hydroxylate these multiple terpene cyclase products, often inserting an oxygen into the same relative positions on the different molecules, and thus yielding a spectrum of dozens of different compounds (e.g. [40]). These compounds are produced, for example, to combat pathogens, pests or herbivores, whereby the ensemble of products exhibit overlapping as well as distinct effects, thus serving to broaden the protection spectrum while maintaining efficacy toward abundant threats. Hence, broad specificity can sometimes represent a more efficient approach of achieving certain metabolic goals. While loose specificity can be tolerated, or be advantageous in the original organism, it will most surely be disadvantageous when specialized pathways are removed from original context in the wild-type plant or microorganism and recruited for the mass production of a single product. Under these conditions broad specificity may lead to an increased ‘underground metabolism’ [20], thus giving rise to waste or toxic products. Tyrosinases, for example, exhibit broad specificity, catalyzing the hydroxylation of various monophenols and the subsequent oxidation of o-diphenols to quinones [41]. P450s are also known to exhibit broad specificity [42–44]. Similarly, bacterial multicomponent monooxygenases (BMMs) catalyze a variety of oxidations including of aromatic compounds to give mono-phenols and diphenols. They exhibit broad substrate as well as product specificity (i.e. the same substrate yielding a mixture of ophenols, m-phenols or p-phenols) [45]. This broad specificity can result in the undesired modification of cellular metabolites and macromolecules. Tyrosinase, for instance, was shown to induce protein cross-linking [46]. Notably, tyrosine 3-monooxygenase, which also catalyzes the oxidation of tyrosine to DOPA, achieves a considerably higher substrate specificity (e.g. [47]), suggesting that the broad specificity of tyrosinase does not represent an inherent limitation of this chemistry. Loose specificity also relates to the same enzyme repeating the same reaction more than once, that is, within the same active-site, the product of one step comprises the substrate for the next one [48,49]. This often results in multiple products, intricate mechanisms of controlling the product’s chemistry and poor kinetics [48]. The outcome is inefficient flux control. Tyrosinases also illustrate www.sciencedirect.com

this point, catalyzing two sequential oxidation steps within the same active site (reactions ‘R1’ and ‘R2’) [41]. The production of betalamic acid starts with oxidation of tyrosine to give dopa. However, tyrosinase can oxidize tyrosine directly to dopaquinone at the same active site, thus limiting the flux toward betalamic acid (which requires reactions R4 and R5 to occur on the product of R1 rather than R2). This is likely to limit the overproduction of betalains, particularly in a foreign host. Using the betalains biosynthetic system from beet, in which the formation of DOPA is decoupled from its oxidation to cyclo-DOPA [35], might therefore be advantageous as it enables tighter control of the different pathway fluxes. The biosynthesis of phytosterols provides another example. The same SAM-dependent methyltransferase (SMT) catalyzes methyl additions to different sterol sidechains, and sometimes accept the product of its previous methylation cycle. For example, it can methylate cycloartenol to 24(28)-methylenecycloartanol, which can then undergo further methylation [50]. The specific activity of this enzyme, regardless of the exact substrate, is very low (103 mmol/min/mg) [50]. Enzymes supporting parallel routes

The broad specificity of specialized metabolism enzymes can give rise to parallel metabolic routes catalyzed by the same set of enzymes. Generally speaking, redundant pathways that start and end with the same metabolites can be found both in central and specialized metabolism. These redundant alternatives sometimes operate the same reaction set but differ in the order of the reactions. Several examples of such a redundancy are apparent in central metabolism. For example, the Entner–Doudoroff glycolytic pathway operating in thermophylic archaea is modified such that the phosphorylation step that normally occurs at the level of glucose, takes places at the level of 2-keto-3-deoxy-gluconate [51]. Hence, the usual reaction sequence phosphrylation ! oxidation ! dehydration is replaced by the sequence oxidation ! dehydration ! phosphrylation [51]. Another central metabolism example is the biosynthesis of cysteinyl-tRNAcys. In bacteria and plants the intermediate O-acetyl-serine is first substituted with hydrogen sulfide to produce cystein, which is then ligated with tRNAcys. In some species, however, the order is reversed: O-acetyl-serine is first attached to tRNAcys and then converted to cysteinyl-tRNAcys [52]. In central metabolism, however, the redundant orders are mutually exclusive and are catalyzed by unique enzymes. Glucokinase, for example, does not phosphorylate 2-keto-3deoxy-gluconate. Further, these redundant pathways do not operate in the same organism let alone at the same time. In specialized metabolism, redundancy is sometimes mediated by unique enzymes, just as in central Current Opinion in Biotechnology 2013, 24:310–319

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metabolism. For example, in the flowering plant Mirabilis Jalapa, cyclo-DOPA is glucosylated by a specific glucosyltransferase to form a cyclo-DOPA-5-O-glucoside that only then spontaneously condensates with betalamic acid [53]. However, the loose specificity of specialized metabolism enzymes often results in the absence of a strict reaction order. The enzymes may catalyze their reaction either before, or after, additional reactions on the same starting compound took place. For example, betalamic acid is condensed with cyclo-dopa that is produced via tyrosinase-dependent oxidation of tyrosine (reaction ‘R6’ in Figure 1). However, betalamic acid can also condense with tryrosine. Tyrosinase would then oxidize the tryrosine moiety of betalamic acid, thus yielding the same product, betanidin [54,55]. The synthesis of phenylpropanoids and monolignols, two lignin building blocks, comprises another example [56]. The O-methyltransferases, dehydrogenases, reductases and hydroxylases operating in these pathways may display loose specificity that results in multiple routes that ultimately lead to the same alcohol product [56]. In the production of brassinosteroids, oxygenases and reductases may operate at varying points along the pathway leading from campesterol to brassinosteroid, resulting in several alternative routes [57]. The synthesis of astaxanthin presents a similar redundancy. Crt oxygenases can operate in different sequential orders, providing several routes for the transformation of b-carotene to astaxanthin [58]. Similarly, the degradation of toluene and o-xylene to the corresponding catechols can proceed via one of two distinct monooxygenases, toluene o-xylene monooxygenase (ToMO) and phenol hydroxylase (PH), or by their sequential operation, where ToMO can operate before or after PH [59]. In chlorophyll biosynthesis, the trans-reduction of the double bond in ring D of divinylchlorophyllide may take place before or after the reduction of the vinyl group of ring B, and both alternatives are catalyzed by the same two enzymes [60]. Finally, the synthesis of a-carotene, a precursor of lutein, is mediated by b-lycopen and e-lycopen cyclases, whereby e-lycopen cyclase can either precede or proceed b-lycopen cyclase [61]. The existence of various alternative pathways operating in parallel limits the kinetic efficiency of the individual enzymes owing to substrate–product binding events that do not lead to high conversion rates [48]. It also complicates the regulation of pathway operation, and gives rise to a large variety of intermediates some of which might posses toxic or inhibitory effects [29]. Within the original organism and context, these intermediates might lead to potentially useful chemical diversities, and are typically alleviated by compartmentalization, regulation of expression and/or low metabolite concentrations. However, upon engineering for high production rates, yield and purity, the longer way might actually be better. Namely, Current Opinion in Biotechnology 2013, 24:310–319

having discrete enzymes, each catalyzing one defined step, might offer kinetic and product purity advantages over the use of enzymes that catalyzed two or more sequential reactions with no defined order. Spontaneous reactions are more common in specialized metabolism

In central metabolism, nearly all transformations are enzyme catalyzed (the cyclization of glutamate 5-semialdehyde to pyrroline 5-carboxylate in the biosynthesis of proline being one exception [62]). Spontaneous reactions seem to be far more common in specialized metabolism. A plausible argument is that enzymes are yet to be discovered for some of the reactions that are now deemed spontaneous – a scenario which is less likely in the highly researched central metabolism. While this argument might be true, it is important to note that many specialized metabolism reactions proceed at a considerable spontaneous rate – a phenomenon that is relatively rare in central metabolism. The biosynthesis of betalains presents several examples of spontaneously occurring reactions. Specifically, dopaquinone spontaneously cyclizes to cyclo-dopa (reaction ‘R3’ in Figure 1) [63] and 4,5-seco-dopa spontaneously cyclizes to betalamic acid (reaction ‘R5’) [64]. Also, the condensations of betalamic acid with cyclo-dopa, amino acids or other cellular amines seem to occur spontaneously (reactions ‘R6’ and ‘R8’) [65]. The latter is quite remarkable in being a bi-molecular reaction, although the high reactivity of free aldehydes might explain the spontaneity of this condensation [66]. Whilst in its natural context this may not be an issue, spontaneously occurring steps are likely to reduce the efficiency of engineered pathways. Firstly, spontaneous reactions, specifically those involving more than one reactant, can be slow, especially when taking place at conditions different from those prevalent in the native organism (e.g. pH). The cyclization of dopaquinone to cyclo-dopa (reaction ‘R3’) is an example of a spontaneous reaction that occurs at high rate. The rate constant for this reaction is 7.6 s1 [63,67]. Assuming that dopaquinone is present 1 mM, the actual rate would be 7.6  103 M s1. An enzyme that composes as much as 1% of the total E. coli proteome (is present at 40 mM [68]) would need to have a fairly high kcat (190 s1) and to operate near saturation (KM < 1 mM), to match the spontaneous rate. On the contrary, at pH > 6 (as in E. coli’s cytoplasm), the rate of spontaneous betanidin formation (condensation of betalamic acid with cyclo-dopa; reaction ‘R6’) is <0.01 nmol/ 100 ml/min [65], or <1.7  109 M s1. Here, an enzyme composing 0.1% of the E. coli proteome, having a kcat  1 s1, and operating at 50% saturation will drive the same reaction at a rate of 2  106 M s1, several www.sciencedirect.com

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orders of magnitude faster. Notably, the high concentrations of betanin in beets and mirabilis flowers suggest that in these plants, this spontaneous reaction occurs at a faster rate, possibly with a subcompartment of low pH: at pH 3, for example, the reaction is >40-old faster than in pH > 6 [64]. Spontaneous reactions are also problematic since they can divert a significant amount of the metabolic flux into waste products. For example, dopaquinone and cyclodopa can spontaneously react to give dopachrome, a precursor in the biosynthetic pathway of melanin [63]. The rate of this reaction is expected to be higher than the rate of dopaquinone conversion to cyclo-dopa [63]. As a result, the recombinant production of betalains may result in a significant accumulation of an undesired product [24]. Betalamic acid can also spontaneously react with various amino-acids, which under heterologous, overproduction conditions, may give rise to a large repertoire of undesired betaxanthins (reaction ‘R8’ in Figure 1). In addition, some spontaneous reactions involve highly reactive species that may be toxic if accumulated. A clear example is the aldehyde containing, highly electrophilic betalamic acid. Such reactive electrophiles are usually cleared out by glutathione S-transferases owing to their toxicity. Another example is 5,6-dihydroxyindole, a precursor of melanin that forms spontaneously from dopachrome, and is a potent cytotoxicant [69]. This may not be a problem with low fluxes, and/or if the pathway is compartmentalized to a specific organelle. It may, however, become problematic at high production rates in the cytoplasm of a host cell. Betalain biosynthesis may comprise an extreme example. However, other specialized metabolism pathways also include spontaneous reactions, as discussed in detail in Ref. [70]. Notable examples include several dehydration and isomerization steps in the biosynthesis of anthocyanins, for example, in the conversion of the leucoanthocyanidin to anthocyanidin 3-glucoside [71]. Another example involves strictosidine. Following its deglycosylation, strictosidine undergoes several spontaneous rearrangement and dehydration steps to yield 4,21dehydrogeissoschizine, a precursor for ajmaline and other indole alkaloids [72]. Similarly, the synthesis of the alkaloid Stephacidin B involves the presumed spontaneous dimerization of its precursor avrainvillamide [73].

Engineering opportunities Advances in enzyme engineering provide potent tools for optimizing natural enzymes for their original or for novel substrates and can even be applied for generating completely new enzymes. Describing the methodologies and achievements of enzyme engineering is beyond the scope of this review (see recent reviews [74–77] and references therein). For example, directed evolution www.sciencedirect.com

can be applied to optimize a computationally designed enzyme through the screening of libraries of random mutations [78]. Similarly, rational and/or computational design approaches, and different analytical approaches (phylogenetic and/or structural analyses), can be applied to create targeted libraries for screening [74]. Utilizing these tools we can now: (i) de novo engineer enzymes for unnatural substrates and reactions (e.g. [78]), and/or for substrates and reactions with which the starting point enzyme exhibits no detectable activity (e.g. [79,80]); (ii) augment weak, promiscuous activities by >104-fold (e.g. [81]); (iii) obtain engineered enzymes whose kinetic parameters rival those of natural enzymes (kcat/ KM  105 M1 s1) (e.g. [78,82]); (iv) dramatically reshape regio-selectivity and stereo-selectivity, including complete reversions of the latter (e.g. [82]); (v) engineer enzymes for high stability and resistance to various environments (e.g. [83]). There are several examples for enzymes engineered for specialized metabolism (e.g. [80,84,85]; reviewed in [86]). Yet, to our knowledge, the replacement within a grafted pathway of a natural enzyme by an engineered one is still quite rare (e.g. [87], see [86] for further examples). We surmise that metabolic engineering, and the engineering of natural product biosynthetic pathways with foreign hosts in particular, may greatly benefit from the ability to engineer individual enzymes, while aiming to increase fluxes, production rates and product homogeneity, and to decrease the metabolic burden of pathways and their effect on growth of the host organism. Other features of enzymes, including pH optimum and stability toward various insults such as high salt or temperature can be readily optimized, thereby assisting the operation of the newly recruited enzymes. Evolving de novo catalysts

Interesting opportunities are offered by natural transformations that may occur spontaneously in the original producing organism. The breadth of this phenomenon is unknown, and in some cases these transformations might turn out to be enzyme catalyzed. Nonetheless, the nature of these transformations is particularly appealing from an enzyme engineering point of view. The starting points for engineering enzymes toward unnatural reactions are either natural enzymes that promiscuously catalyze a reaction that they have never been selected for, or computational design leading to de novo active sites. In both cases, and particularly in computational design, success seems to be dependent on having inherently reactive substrates and transformations that occur with low activation energy barriers, just as is the case of spontaneous reactions. For example, an unnatural elimination reaction dubbed the Kemp Elimination was explored. Its spontaneous rate Current Opinion in Biotechnology 2013, 24:310–319

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with activated benzisoxazole substrates is relatively high (k  106 s1 at pH 7). Enzymes that promiscuously catalyze this reaction were readily found, even upon searching one proteome [88], and even non-catalytic proteins (serum albumins) exhibit appreciable rate accelerations [89]. However, the computationally designed Kemp eliminases, and the natural enzymes that promiscuously catalyze this reaction, exhibit low catalytic efficiency (kcat/KM  103 M1 s1) [90]. Their catalytic efficiency can, however, be dramatically enhanced by directed evolution, leading to enzymes with kcat/KM values of up to 6  105 M1 s1 [78]. The Kemp elimination comprises a model reaction of no practical utility (for deprotonation from an activated carbon and a subsequent isomerization). Many transformations in specialized metabolism, however, offer interesting chemistry and potential applications. The ease by which enzymes can evolve toward relatively simple reactions that proceed via low activation energy barriers, of the type often seen in specialized metabolism is also demonstrated by chalcone isomerase – an enzyme that diverged from a catalytically optimal, stereospecific enzyme that diverged from a non-catalytic fold within the time period of plant evolution [91]. The incorporation of such de novo engineered enzymes could substantially increase the pathway’s flux, and even more crucially, prevent other spontaneous reactions leading to undesired products. Optimizing catalytic efficiency and specificity

Directed evolution techniques can also be applied to enhance the catalytic efficiency of enzymes. Enzymes operating in specialized metabolism are characterized by kcat values that are typically >10-fold lower than their central metabolism counterparts [16]. One can therefore expect that many natural enzymes can be further optimized. In some cases, orthologous enzymes with considerably higher rates can be identified. The plant or fungi tyrosinases, for example, might be replaced by bacterial tyrosinases that typically exhibit much higher rates (kcat > 1000 s1 for some variants) [92]. In other cases, engineering the original enzyme may be the preferred option. Indeed, rate improvements of 104-fold, and beyond, have become a matter of routine in enzyme engineering [74]. Enzyme engineering can also play a major role in shaping specificity and thus decrease un-wanted and perhaps deleterious side reactions and products. This approach can be implemented to direct a single pathway from a variety of redundant alternative routes. We surmise that clear directionality can bypass spontaneous un-desired activity associated with some of the redundant alternatives. This approach can be further applied to select the metabolic route which involves intermediates that Current Opinion in Biotechnology 2013, 24:310–319

are more likely to accumulate within the cell without a deleterious effect [93]. Notably, increased substrate and/or product selectivity can be also readily achieved using current enzyme engineering technology (e.g. [45]). Increasing enzyme specificity can be especially useful for enzymes that catalyze two consecutive reactions on the same product. Using the example of tyrosinase again, evolving two enzymes, one which oxidizes tyrosine and the other dopa, can be highly beneficial since it will allow dopa to accumulate, lifting the kinetic barrier of betalamic acid synthesis. In addition, it is likely that dividing such consecutive steps between more than one enzyme may yield higher kinetic activity for all. In other instances it might be preferable to replace one enzyme with another type instead of applying long and costly enzyme evolution. For example, instead of evolving tyrosinase to catalyze only the oxidation of tyrosine, the enzyme tyrosine 3-monooxygenase, which already has the desired property, can be used instead (although requiring co-factor adaptation) [94]. Similarly, a hemedependent peroxidase can catalyze the ortho-hydroxylation of tyrosine to DOPA [95]. In fact, enzyme replacement can be applied multiple times to ‘re-construct’ the pathway from enzymes from various sources, a mix-and-match approach which was suggested for carbon fixation pathways [17]. This extensive pathway ‘rewiring’ can shortcut long pathways using existing enzymes, substantially increasing pathway flux. Enzyme evolution can be used in cases where shortcut enzymes are not available naturally but the biochemistry of such a shortcut is clear. Finally, plant secondary metabolism tends not to be optimized in terms of resources efficiency, spending more than the required ATP or NAD(P)H equivalents. In plants this is of minor importance since plants are generally not energy limited and since the flux through the secondary pathways is relatively low. Biotechnologically, however, this inefficiency can constrain pathway operation. Hence, using alternative reactions that spend fewer ATP or NAD(P)H equivalents can be highly beneficial [96]. For instance, replacing oxidases with dehydrogenases, when possible, would be energetically preferable, freeing more reducing equivalents for oxidative phosphorylation.

Acknowledgements We thank Asaph Aharoni, Dean DellaPenna, Natalia Dudareva, Viviana Izzo, Ron Milo, Joe Noel, Sarah O’Connor and Guy Polturak for helpful discussions and critique regarding the manuscript. D.S.T. is the Nella and Leon Benoziyo Professor of Biochemsitry, and acknowledges funding from the Meil de Botton Aynsley. www.sciencedirect.com

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References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of outstanding interest 1.

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