Evolutionary history and biotechnological future of carboxylases

Journal of Biotechnology 168 (2013) 243–251

Contents lists available at ScienceDirect

Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec

Evolutionary history and biotechnological future of carboxylases Lennart Schada von Borzyskowski, Raoul G. Rosenthal, Tobias J. Erb ∗ Institute of Microbiology ETH Zurich, Wolfgang Pauli Str. 10, 8093 Zurich, Switzerland

a r t i c l e

i n f o

Article history: Received 13 February 2013 Received in revised form 8 May 2013 Accepted 13 May 2013 Available online 20 May 2013 Keywords: CO2 -Fixation Synthetic biology Enzyme mechanisms Enzyme evolution RubisCO

a b s t r a c t Carbon dioxide (CO2 ) is a potent greenhouse gas whose presence in the atmosphere is a critical factor for global warming. At the same time atmospheric CO2 is also a cheap and readily available carbon source that can in principle be used to synthesize value-added products. However, as uncatalyzed chemical CO2 -fixation reactions usually require quite harsh conditions to functionalize the CO2 molecule, not many processes have been developed that make use of CO2 . In contrast to synthetical chemistry, Nature provides a multitude of different carboxylating enzymes whose carboxylating principle(s) might be exploited in biotechnology. This review focuses on the biochemical features of carboxylases, highlights possible evolutionary scenarios for the emergence of their reactivity, and discusses current, as well as potential future applications of carboxylases in organic synthesis, biotechnology and synthetic biology. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Carbon dioxide (CO2 ) is an atmospheric gas whose contribution to the greenhouse effect has been estimated as approximately 26% (Kiehl and Trenberth, 1997). Since the onset of large-scale industrialization in the late 18th century, the atmospheric concentration of CO2 has been increasing by more than 40% from 280 ppm preindustrial concentration to 394 ppm at the end of the year 2012 (Tans and Keeling, 2013). This anthropogenic increase of atmospheric CO2 concentration is believed to be a direct cause of the phenomenon of global warming, which was identified as one of the future challenges of mankind (King, 2004). Consequently, society and politics are working on different strategies and legal frameworks to limit and restrict anthropogenic CO2 -emissions from fossil fuels. Despite being a critical factor to climate change, CO2 is at the same time a very cheap and ubiquitous carbon source that should be in principle readily available for the synthesis of useful compounds. However, as CO2 is a highly oxidized and relatively inert molecule, its transformation by the chemical industry without suitable catalysts requires a large energy input and/or harsh conditions (Bell et al., 2007; Fujita et al., 2005; Glueck et al., 2010). So far, only a handful of processes are applied on industrial scale, the most prominent being the Kolbe-Schmitt reaction for the synthesis of salicylic acid, and the synthesis of urea. In total about 0.1 Gt/year

∗ Corresponding author. Tel.: +41 44 632 36 54; fax: +41 44 633 13 07. E-mail addresses: [email protected], [email protected] (T.J. Erb). 0168-1656/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jbiotec.2013.05.007

CO2 are converted into products by the chemical industry (Aresta, 2009; Glueck et al., 2010). Compared to industrial CO2 conversion, the biological transformation of CO2 into organic compounds accounts to approximately 100 Gt/year (Field et al., 1998), which demonstrates the huge catalytic potential of Nature. About 95% of the CO2 is estimated to be directly fixed by carboxylases, whereas the other 5% is fixed through reduction of CO2 , emphasizing the importance of biological carboxylation reactions. However, for a successful application of carboxylating reactions in organic chemistry, biotechnology and/or synthetic biology, the mechanistic principles of CO2 -fixation, as well as the evolutionary potential of the corresponding CO2 -fixing enzymes need to be understood. This review (i) summarizes the general mechanisms and biochemical constraints of CO2 -fixing enzymes, (ii) discusses the evolutionary history and flexibility of the active site of selected enzymes, and (iii) provides a brief outlook on current, as well as potential future applications of different carboxylases and carboxylating reactions in organic synthesis, biotechnology and synthetic biology.

2. Biochemical principles at the active site of carboxylases The challenge that carboxylases face is to catalyze the nucleophilic attack of a moderately activated carboxylation substrate onto a thermodynamically and kinetically stable CO2 -molecule. As a consequence, carboxylating enzymes employ both nucleophilic activation of substrates, as well as electrophilic activation of CO2 to enhance reactivity of the substrates and to promote catalysis (Lane, 1969). Although under physiological conditions CO2 is mostly present in its hydrated form as bicarbonate ion (HCO3 − ),

244

L. Schada von Borzyskowski et al. / Journal of Biotechnology 168 (2013) 243–251

carboxylases still use CO2 as reacting species at the active sites to form new C–C bonds. Consequently, Nature also employs mechanisms that raise the CO2 concentration at the active site of (or around) carboxylases to increase their carboxylation efficiency (e.g., conversion of HCO3 − into biotin-bound CO2 through ATP hydrolysis, see below). 2.1. Substrate activation: the creation of reactive intermediates A large number of enzymatic carboxylation reactions employ mechanisms that activate the carboxylation substrate by formation of an enol(ate), or in a few cases a corresponding enamin(at)e intermediate during catalysis. This is predicted from various mechanistic studies, as well as from the chemical structures and features of the different carboxylation substrates (e.g., enol phosphates, thioesters, ␣-keto acids) (Attwood et al., 1986; Hansen and Knowles, 1982; Stubbe et al., 1980). Formation of these enolate/enaminate intermediates serves to activate the individual substrates by putting a formal or partial negative charge on the atom attacking the CO2 molecule (usually a carbon atom), thereby increasing nucleophilicity and, hence, carboxylation reactivity. However, whereas formation of a enolate/enaminate intermediate is a central step in most, if not all carboxylases, different catalytic strategies to create this reactive species have been realized by Nature (Fig. 1). Substrate enol(ate)s have been reported to be formed by: (i) deprotonation of an acidic proton (most carboxylases, e.g., D-ribulose-1,5-bisphosphate carboxylase/oxygenase [RubisCO], pyruvate carboxylase (Attwood et al., 1986; Cleland et al., 1998)), (ii) dephosphorylation (phenylphosphate carboxylase, phosphoenolpyruvate [PEP] carboxylase (Hansen and Knowles, 1982; Janc et al., 1992; Schühle and Fuchs, 2004)), (iii) partial reduction of an enoyl double bond with NADPH as electron donor (crotonyl-CoA carboxylase/reductase [Ccr] (Erb et al., 2009a)) or (iv) reductive cleavage of C-S bonds with NADPH and coenzyme M as cofactors (2-ketopropyl-CoM carboxylase/oxidoreductase (Kofoed et al., 2011)). The latter mechanism presumably also operates in ferredoxin/thiamine-dependent enzymes, such as pyruvate synthase. In pyrrole-2-carboxylate (de)carboxylase, formation of the reactive enaminate carbanion is believed to proceed through deprotonation of the pyrrole ring that allows a subsequent nucleophilic attack on the CO2 molecule (Omura et al., 1998). In all these cases carboxylation is promoted by stabilizing the enolate/enaminate intermediate through charge complementation at the active site, conferred by active site residues, (e.g., the oxyanion hole in acyl-CoA carboxylase (Diacovich et al., 2004)), or metal ions (e.g., Mg2+ as in RubisCO (Lundqvist and Schneider, 1991) or PEP carboxylase (Ausenhus, 1993)). In summary, Nature makes use of reactive substrate carbanions that are conjugated with nitrogen or oxygen atoms as the nucleophilic species. However, the mechanisms that form and stabilize these intermediates are biochemically very diverse (Fig. 1). 2.2. Activation of CO2 and the problem of binding To facilitate the nucleophilic attack of the reactive enol(ate)/enamin(at)e intermediate on CO2 during the carboxylation reaction, the central carbon atom of the CO2 molecule needs also to be activated. This can be achieved by hydrogen bonding to the carbonyl oxygens of CO2 , which withdraws electron density from the C–O bond, increasing electrophilicity and thus reactivity of the central carbon of the CO2 molecule. With this in mind, it seems puzzling that enzymes that form novel C–C bonds by carboxylation do not always employ amino acids with the strongest hydrogen bonding capacities at the active site, as inferred from manual inspection of several crystal structures, but

rather ones with amide or hydroxyl side chains and often also show hydrophobic side chains close to the CO2 binding site. The rationale behind this active site architecture might be to avoid the strong binding of water (instead of CO2 ), in order to prevent the addition of an electrophilic proton to the nucleophilic carbanion. As many carboxylases have been reported to catalyze side reactions, it is apparent that a selective binding of CO2 at the active site cannot be realized easily. This finding is well known for RubisCO (D-ribulose-1,5-bisphosphate carboxylase/oxygenase) that is one of the most abundant enzymes in the biosphere (Ellis, 1979; Phillips and Milo, 2009). RubisCO catalyzes the key reaction of the Calvin-Benson-Bassham cycle, the major process of atmospheric CO2 -fixation on earth. However, the active site of RubisCO does not only accommodate CO2 , but to some extent also molecular oxygen (O2 ) (Tabita et al., 2008). Thus, O2 can act as alternative electrophile during the reaction, thereby causing an oxygenation side reaction that generates phosphoglycolate as a side product and leads to photorespiration, which can cause the loss of up to 25% of the fixed CO2 (Sharkey, 1988). Although there are RubisCO homologs that show a higher specificity for CO2 , such enzymes usually possess slower turnover numbers, so that in these “improved RubisCOs” higher specificity is traded for reaction velocity (see below) (Badger and Bek, 2008; Badger and Price, 2003; Tabita et al., 2008). A common mechanistic strategy of some carboxylases is to enhance the reactivity of CO2 by covalent activation. This increases not only affinity of the corresponding enzyme to the activated CO2 (or more precisely HCO3 − ) molecule, but also represses very effectively unwanted side reactions, which makes these carboxylases highly efficient. The prime example for this kind of activation strategy is PEP carboxylase that phosphorylates a bicarbonate ion with the phosphate group of PEP to generate a transient, highly reactive carboxyphosphate which is used to carboxylate the enol(ate)intermediate of pyruvate that has formed at the enzyme’s active site (Hansen and Knowles, 1982; Janc et al., 1992). Similarly, biotin dependent carboxylases also form a reactive carboxyphosphate from CO2 and ATP during catalysis that is used to generate carboxybiotin, the activated CO2 -carrier of this important class of CO2 -fixing enzymes that includes many physiologically relevant representatives, such as acetyl-CoA carboxylase, propionyl-CoA carboxylase, and pyruvate carboxylase ((Knowles, 1989; Ogita and Knowles, 1988), see below). 2.3. Increasing the effective CO2 -concentration at the active site Under physiological pH and temperature in equilibrium with the atmosphere only 10 ␮M dissolved CO2 is present, whereas the concentration of bicarbonate ion is 200 ␮M. Therefore, when the more electrophilic CO2 is used as substrate a clear benefit can be gained from raising its concentration near the active site. Raising the CO2 concentration increases the thermodynamic driving force towards the product and, at the same time, catalytic turnover. Nature uses different methods to achieve a higher effective CO2 concentration at the active site. One commonly used option is to ‘capture’ CO2 by covalently binding it to enzyme bound biotin at expense of one ATP. The carboxybiotin can then swivel to another domain of the enzyme where the ‘CO2 ’ is released in situ to carboxylate the substrate (Knowles, 1989; Ogita and Knowles, 1988). Another option was identified recently by molecular dynamics simulations on PEP carboxykinase, which showed that the enzyme has channels aligned with amino acids that interact and guide CO2 towards the active site (Drummond et al., 2012). Lastly, CO2 levels can also be raised “macroscopically” by localizing the carboxylating enzymes in carboxysomes, specialized cellular compartments with elevated CO2 concentrations (Kerfeld et al., 2010; Shively et al., 1973). In summary, Nature has evolved very different mechanistic strategies to capture CO2 into organic compounds. Even though the

L. Schada von Borzyskowski et al. / Journal of Biotechnology 168 (2013) 243–251

245

Fig. 1. The conserved mechanistic principle of carboxylating enzymes. All carboxylating enzymes generate either an enol(ate) or an enamin(at)e intermediate that can act as nucleophile to attack a CO2 or a CO2 equivalent. The different postulated mechanisms to generate an enolate are depicted: 1) removal of a proton by a base 2) dephosphorylation of a phospho-enol 3) reduction of an enoyl-thioester 4) cleavage of a C-S bond, and the proposed mechanism to generate an enamin(at)e 5) deprotonation of a pyrrole. The reactive enol/enamin(at)e intermediate can either react directly with a) CO2 or with a more activated CO2 equivalent like b) carboxyphosphate or c) carboxybiotin to form the carboxylation product.

carboxylation reactions vary greatly in their biochemistry, there are unifying themes such as generation of a carbanion conjugated with an oxygen or nitrogen atom, or the electrophilic activation of CO2 , which raises the question whether other carboxylation mechanism (e.g., radical reactions) are in principle not “biochemically feasible”, or whether they still need to be discovered. Further mechanistic studies on carboxylases and carboxylation reactions will help to answer this fundamental question. 3. Evolution of the carboxylase function in protein superfamilies: two case studies The occurrence of the carboxylating function in various enzyme superfamilies demonstrates that Nature has evolved the CO2 fixation trait several times independently in very different protein scaffolds (see below). In view of the general acceptance that “biochemical traits that are easy to acquire have been invented by Nature multiple times“,1 the capability of proteins to fix CO2 seems to be realized relatively easily. However, only very recent studies allow a more detailed understanding on the evolutionary processes that shape the emergence of different carboxylating enzymes. Such understanding is inevitable for any efforts that aim at improving the mechanisms of existing carboxylases or aim at designing CO2 -fixing enzymes de novo. 3.1. Evolution of RubisCO: a carboxylating enolase Given the importance of RubisCO for the global carbon cycle (see above), it is not surprising that there is much interest to under-

1

This concept has been introduced to the authors by R. Thauer, Marburg.

stand how the enzyme and its reaction might have emerged during evolution (Ashida et al., 2008, 2005; Tabita et al., 2008, 2007). The reaction of RubisCO is quite unique as it comprises (at least) three distinct mechanistic steps that are all catalyzed by the same active site. First, ribulose-1,5-bisphosphate is enolized, to form the reactive enol(ate) intermediate, which in a second step attacks the CO2 molecule to form the primary carboxylation product 2-carboxy-3ketoarabinitol-1,5-bisphosphate. This instable C6 -intermediate is then hydrolytically cleaved to yield the final reaction products: two molecules of phosphoglycerate. How can the evolution of such a complex carboxylation mechanism be rationalized? Part of the answer comes from recent sequencing efforts that identified close RubisCO-homologues (so-called “RubisCO-like proteins”) in a number of bacterial and archaeal genomes (Hanson and Tabita, 2001). However, in contrast to bona fide RubisCOs, these RubisCO-like proteins miss residues essential for the carboxylation reaction and consequently lack the ability to fix CO2 . This is also underlined by the fact that many of the organisms harboring a RubisCO-like protein are actually non-autotrophic (i.e., cannot fix CO2 for carbon supply). Based on sequence diversity a “RubisCO super-family” was proposed that can be divided in one sub-family of “true RubisCOs” and (at least) seven different RubisCO-like protein sub-families as shown in Fig. 2a (Erb et al., 2012; Tabita et al., 2007). Recent work has established the physiological function and the biochemistry of two RubisCO-like protein subfamilies. RubisCOlike proteins of the Bacillus subtilis subfamily are involved in a novel variation of the “methionine salvage pathway” that recycles 5 -methylthioadenosine (MTA), a dead-end product of S-adenosylmethionine (SAM)-dependent polyamine biosynthesis, into L-methionine (Ashida et al., 2003; Imker et al., 2007). For RubisCO-like proteins of Rhodospirillum rubrum subfamily, a function in MTA recycling was also demonstrated recently (Erb et al.,

246

L. Schada von Borzyskowski et al. / Journal of Biotechnology 168 (2013) 243–251

Fig. 2. Evolution of the carboxylation function in the RubisCO, as well as the MDR superfamily (as inferred from their phylogenies). (a) Phylogenetic tree of the RubisCO superfamily with its eight subfamilies, as defined by Erb (Erb et al., 2012). The seven, non-carboxylating subfamilies (RubisCO-like protein subfamilies) are highlighted in blue (true enolases) or gray (exact function unknown). The one subfamily of carboxylating enzymes (true RubisCO subfamily) is highlighted in red. (b) Phylogenetic tree of the MDR superfamily. The 14 largest subfamilies, as defined by Persson (Persson et al., 2008) are highlighted in blue (all reductases). The one subfamily of carboxylating enzymes (CCR subfamily) is highlighted in red. Phylogenetic trees were calculated from 333 (RubisCO superfamily) and 117 (MDR superfamily) protein sequences that were restricted to a length of 409 (RubisCO superfamily) and 378 (MDR superfamily) amino acids. Tree topographies and evolutionary distance are given by the neighbor-joining method. Numbers at nodes represent the percentage bootstrap values for the clades of this group in 500 (RubisCO superfamily), respective 50 (MDR superfamily) replications. Similar trees were obtained by using the minimum evolution and the maximum likelihood method. The sequence alignments used to create the trees are available as supporting online material.

2012). However, in contrast to B. subtilis, MTA recycling in R. rubrum follows a different route, the so-called “MTA-isoprenoid shunt”, in which the ribose moiety of MTA is scavenged into the isoprenoid precursor 1-deoxy-d-xyxlulose 5-phosphate (DXP) (Erb et al., 2012). Besides the interesting observation that all RubisCO-like protein subfamilies characterized so far seem to be linked to sulfurmetabolism (N.B.: for the C. tepidum RubisCO-like protein subfamily a sulfur phenotype was also established (Hanson and Tabita, 2003, 2001)), above findings allow for the very first time identification of common mechanistic features in the RubisCO superfamily: Apparently, all members of this superfamily are able to catalyze an enolization reaction. However, whereas proteins of the B. subtilis RubisCO-like protein subfamily acts as simple enolases (Imker et al., 2007), isomerases of the R. rubrum RubisCO-like protein subfamily appear to be more complex as they catalyze two subsequent enolization reactions (Imker et al., 2008). Finally, proteins of the “true RubisCO” subfamily combine substrate enolization with carboxylation and hydrolytic cleavage (i.e., catalyze the “classical” RubisCO-reaction). This apparent trend in mechanistic complexity might very well reflect evolutionary events in the RubisCO superfamily, according to which an ancestral enolase has mechanistically diversified into (at least) eight different functions (i.e., above mentioned eight subfamilies), among them one more complex subfamily of “carboxylating enolases” (i.e., true RubisCOs; Fig. 2a). In this sense, the RubisCO superfamily which has traditionally been considered a carboxylase superfamily containing some non-carboxylating members (i.e., RubisCO-like proteins) actually rather reflects an enolase superfamily, which contains some carboxylating members (i.e., true RubisCOs). However, although very reasonable, this evolutionary hypothesis needs of course to be re-evaluated upon functional assignment of the remaining RubisCO subfamilies that have not been characterized so far. 3.2. Evolution of Ccr: a carboxylating enoyl-CoA reductase The recently discovered Ccr (Crotonyl-CoA carboxylase/reductase) is a key enzyme in the central carbon metabolism of many ecologically relevant bacteria, where it

serves in the ethylmalonyl-CoA pathway, a novel acetyl-CoA assimilation strategy that represents an alternative to the wellknown glyoxylate cycle (Erb et al., 2009b, 2007). Moreover, Ccr and its closest homologues also supply precursor building blocks for the biosynthesis of many polyketide natural products, such as the antibiotic monensin and the immunosuppressant FK520 (Erb et al., 2007; Wallace et al., 1995; Wilson and Moore, 2012). The enzyme catalyzes the reductive carboxylation of crotonyl-CoA to (2S)-ethylmalonyl-CoA with NADPH and represents the prototype of a novel class of reductive carboxylases (Erb et al., 2009a, 2007), which now includes several homologs of different substrate specificity (Eustaquio et al., 2009; Mo et al., 2011; Rachid et al., 2011; Wilson et al., 2011; Xu et al., 2011; Yoo et al., 2011a,b). Most interestingly, Ccr not only catalyzes the reductive carboxylation of crotonyl-CoA to (2S)-ethylmalonyl-CoA, but also the “ordinary” reduction of crotonyl-CoA to butyryl-CoA, albeit only in the absence of CO2. The carboxylation reaction is clearly preferred over the reduction reaction, which is reflected by the enzyme turnover number, as well as the catalytic efficiency that are both approximately an order of magnitude higher for carboxylation (Erb et al., 2009a, 2007). This observation has evoked some interest, and raised the question as how (and why) this enzyme might have acquired such catalytic promiscuity. Phylogenetic analysis demonstrated that Ccr belongs to the superfamily of medium chain dehydrogenases/reductases that comprises various subfamilies, which all catalyze the reduction of different substrates, the only exception being the Ccr subfamily that strictly includes reductive carboxylases (Fig. 2b). Thus, it has been speculated that crotonyl-CoA carboxylase/reductase has emerged from ordinary reductases, to become a more complex “carboxylating enoyl-CoA reductase” and that the reduction side reaction might represent an evolutionary relict of this process (Erb et al., 2009a). Structural studies of the active site of Ccr indeed suggest that the enzyme is optimized to accommodate a CO2 molecule, to effectively promote carboxylation, while at the same time it reduces binding of water to minimize the competing reduction reaction without carboxylation (Quade et al., 2012), which strongly supports the proposed evolutionary scenario that resembles very much the evolutionary pattern of the RubisCO superfamily (see above).

L. Schada von Borzyskowski et al. / Journal of Biotechnology 168 (2013) 243–251

247

Table 1 Selected examples for the application of carboxylases (and modified CO2 -fixation pathways) in organic chemistry, biotechnology and synthetic biology. Application

Approach

Result

Reference

Single carboxylating enzymes in organic chemistry and biotechnology

Enzymatic carboxylation of phenol

High-yield synthesis of 4-hydroxybenzoate from phenylphosphate under mild conditions Highly regioselective carboxylation catalyzed by benzoate or phenolate decarboxylases Generation of carbapenem precursor 6-ethyl-t-carboxymethylproline in high purity Generation of a previously unknown polyketide, 6-desmethyl-6-ethylerythromycin A Generation of unnatural halogenated and methylated derivatives of salinosporamide, FK506, and cinnarabamide Generation of RubisCO mutants exhibiting higher folding and catalytic efficiency in an E. coli selection system Carbon fixation increased 6 to 12%; growth advantage for plants in early phase of development Functional expression of sub-pathways, but no autotrophic growth sustained

Aresta et al. (1998), Dibenedetto et al. (2006)

Enzymatic carboxylation of phenol and styrene derivatives Carboxylases in combinatorial biosynthesis

Use of Ccr to produce ethylmalonyl-CoA as precursor for carbapenem biosynthesis Overexpression of Ccr to provide ethylmalonyl-CoA as precursor in Saccharopolyspora erythrea Feeding of non-natural substrates to relaxed Ccr homologs to derive structurally modified polyketides

Improving CO2 fixation efficiency of existing pathways

Directed evolution of RubisCO to increase specific activity

Assembly of artificial CO2 fixation pathways

Overexpression of sedoheptulose-1,7-bisphosphatase in tobacco plants Realization of 3-hydroxypropionate CO2 -fixation pathway in E. coli

3.3. A generalized evolutionary scheme for carboxylases The case studies of RubisCO and Ccr suggest a common principle for the evolution of CO2 -fixing enzymes that has been observed for other mechanistically diverse superfamilies and that might hold true for the emergence of a number of (but not all) carboxylases: Dominance of the chemical mechanism thrives the evolution of novel reactions (Babbitt and Gerlt, 1997; Gerlt and Babbitt, 2001). According to this principle, an important prerequisite for the implementation of the carboxylation function into an enzyme scaffold is the presence of an active site that forms a nucelophilic enol(ate) intermediate during catalysis. Active site modifications that conserve formation of the reactive enolate species, but enable the binding of a CO2 molecule in close proximity, which could serve as electrophilic target for the enol(ate) intermediate, would allow a shift in reaction specificity towards substrate carboxylation. In this way, a primordial CO2 -fixing enzyme could emerge, which could subsequently undergo further rounds of selective evolution to optimize the carboxylation function, such as the stabilization of the negative charge developing at the carboxyl group, or the exclusion of other potential electrophiles besides CO2 in the active site (e.g., protons). For many modern day carboxylases such an evolutionary scenario sounds very reasonable; however, as other mechanisms have been identified that can lead to divergent evolution (Gerlt and Babbitt, 2001), some carboxylating enzymes (most notably biotin dependent enzymes) might have evolved differently.

4. Carboxylases in organic chemistry, biotechnology and synthetic biology The multitude of carboxylation reactions present in Nature makes them interesting targets for applications in organic chemistry, biotechnology and synthetic biology. Yet, due to the complex (cofactor-)requirements of most CO2 -fixing enzymes, only few of them have been successfully applied as “single enzymes” in organic chemistry or biotechnology. However, the discovery of novel enzymes and the development of novel strategies open new perspectives for biotechnology. In addition, some carboxylating enzymes have recently been used to alter the structure

Wuensch et al. (2012), Kirimura et al. (2010, 2011), Matsui et al. (2006a,b) Hamed et al. (2011)

Stassi et al. (1998)

Eustaquio and Moore (2008), Mo et al. (2011), Rachid et al. (2011)

Parikh et al. (2006), Greene et al. (2007)

Lefebvre et al. (2005)

Mattozzi et al., 2013

and biological activity of selected natural products by combinatorial diversification of secondary metabolite (bio)synthesis. Finally, additional (and future) applications of carboxylases range from the implementation of engineered carboxylases in natural CO2 -fixation pathways to enhance CO2 -uptake (and hence primary production) to their use in the assembly of completely artificial pathways for the fixation of CO2 . A short overview about different applications and novel trends is given in Table 1.

4.1. Applications in organic chemistry and biotechnology based on single enzyme systems Up to now, a practical application of CO2 -fixing enzymes in organic chemistry is limited by the availability of suitable carboxylases, because many carboxylating enzymes are highly substrate specific, oxygen sensitive, or require complex cofactors/cosubstrates, such as biotin, ATP, Ni2+ , or ferredoxin for catalysis (Erb, 2011). So far, the only “true” carboxylase that has been tested for application in larger scale is phenylphosphate carboxylase that yields 4-hydroxybenzoate, a value-added product, from carboxylation of phenylphosphate in the para-position. Phenylphosphate itself is synthesized chemically in a rapid and inexpensive way from phenol at ambient conditions (Aresta et al., 1998). This catalytical system has also been demonstrated to work with supercritical carbon dioxide, allowing for easier product recovery (Dibenedetto et al., 2006). Recent efforts to functionalize and apply enzymatic carboxylation reactions in biotechnology have focused on the use of aromatic acid (de)carboxylases, enzymes that usually catalyze the decarboxylation of substrates (Kirimura et al., 2011, 2010; Matsui et al., 2006a, 2006b; Wuensch et al., 2012). However, under conditions that shift the reaction equilibrium towards carboxylation, i.e., high bicarbonate ion concentrations (3 M) and an alkaline pH (8.5), conversions of up to 80% can be achieved. In a recent study, several (de)carboxylases have been investigated systematically for future biotechnological applications. These enzymes generally fall in two different functional classes (Fig. 3): (i) benzoic acid (de)carboxylases that perform CO2 -fixation on the aromatic ring of different phenols yielding ortho-hydroxybenzoic

248

L. Schada von Borzyskowski et al. / Journal of Biotechnology 168 (2013) 243–251

Fig. 3. Enzymatic carboxylations of phenol and styrene derivatives using benzoic and phenolic acid (de)carboxylases. While benzoic acid (de)carboxylases catalyze the ortho-carboxylation of the aromatic ring, phenolic acid (de)carboxylases catalyze the ␤-carboxylation of the aliphatic side chain. The respective maximum yields (c) and reaction products are indicated (scheme based on Wuensch (Wuensch et al., 2012)).

acid derivatives with very high conversion (up to 80%), which show a remarkably broad substrate spectrum; and (ii) phenolic acid decarboxylases that act exclusively at the beta-carbon atom of the side-chain of para-hydroxystyrenes forming (E)-cinnamic acid derivatives, which show moderate conversion of substrates (up to 40%) (Wuensch et al., 2012). Although very promising, application of these enzymes in larger scale needs to be validated. In this context it is interesting to note that the reverse reaction of phenolic acid decarboxylase (i.e., the decarboxylation of para-coumaric acid to yield para-hydroxystyrene) has already been demonstrated to operate in a small-scale packed reactor (Jung et al., 2012). 4.2. The role of carboxylases in combinatorial biosynthesis An interesting option for the application of carboxylases is generated by their ability to provide building blocks for the biosynthesis of secondary metabolites, most notably polyketide natural products. Biosynthesis of the “classical” polyketide backbone is dependent on acetyl-CoA carboxylase that supplies the “standard” building block malonyl-CoA. However, just very recently it has been realized that Ccr and its close homologs serve in providing more “exotic” building blocks for biosynthesis, such as ethylmalonyl-CoA, allylmalonyl-CoA, isobutyrylmalonylCoA, chloro-, or bromoethylmalonyl-CoA, as well as propyl-, hexyl-, or octanoylmalonyl-CoA (Chan et al., 2009; Erb et al., 2007; Wilson and Moore, 2012). Consequently, current efforts aim at functionalizing Ccr (and its homologs) to diversify the structural features and hence biological activities of natural (and semi-natural) products. In a recent example, Ccr was employed together with an engineered carboxymethylproline synthase to derive unprecedented precursors for the synthesis of new carbapenem antibiotics in vitro

(Hamed et al., 2011). Even more impressive than this in vitro approach is the potential of Ccr and its homologs to modify biosynthesis of polyketide natural products in vivo. It has already been shown that heterologous expression of Ccr can lead to synthesis of novel polyketide antibiotics in a “biocombinatorial way”. Overexpression of the enzyme in Saccharopolyspora erythrea provided ethylmalonyl-CoA to yield the previously unknown compound 6-desmethyl-6-ethylerythromycin A, an ethyl-substituted derivative of erythromycin (Stassi et al., 1998). Similar to this approach, the relaxed substrate specificity of some Ccr homologs has recently been functionalized through the means of “mutasynthesis” 2 . Feeding of non-natural precursors that serve as substrates for above mentioned “relaxed” Ccr homologs in the existing biosynthetic pathways of the secondary metabolites salinosporamide, FK506, and cinnabaramide resulted in the production of unnatural derivatives of these compounds. Most notably, some halogenated and methylated variants were derived (e.g., fluorosalinosporamide, 36-methyl-FK506, and 15-chlorocinnabaramide A) that showed superior bioactivities relative to their parent compounds (Eustaquio and Moore, 2008; Mo et al., 2011; Rachid et al., 2011). Consequently, it can be imagined that in future more Ccr homologs with diverse or relaxed substrate specificities, such as the recently characterized 2-octenoyl-CoA-carboxylase/reductase (Yoo et al., 2011a,b) might be used to further diversify the biosynthesis of other polyketides in a more systematic way.

2 Mutasynthesis, as defined by Rinehart in 1977 aims at creating diversity of natural products by intercepting their biosynthesis with structural analogs of the precursors.

L. Schada von Borzyskowski et al. / Journal of Biotechnology 168 (2013) 243–251

4.3. Engineering of existing CO2 fixation pathways to improve carboxylation efficiency Compared to the development of “single enzyme systems” or the biocombinatorial application of Ccr and its homologs as described above, more experimental effort has been put into increasing the activity of carboxylating enzymes to improve existing CO2 -fixation pathways. The prevalent approach has so far mostly consisted of attempts to enhance the specific activity of carboxylases and/or to free them of regulatory constraints. Considerable efforts have especially been undertaken to improve RubisCO, the carboxylase that has been identified as the rate-limiting enzyme in carbon fixation via the Calvin-BensonBassham cycle because it exhibits catalytic parameters far below the theoretically possible optimum for enzymes, as well as an oxygenation side reaction that causes the energetically “expensive” phenomenon of photorespiration (see above). This strategy of improving RubisCO is aimed at increasing the CO2 -fixation rate of crop plants by “supercharging photosynthesis”, allowing for a higher food/biomass production (Parry et al., 2013; Peterhansel et al., 2008). However, success in enhancing the catalytic efficiency of RubisCO (with concomitant suppression of the O2 -side activity) has so far fallen short of expectations. For example, although RubisCO mutants with increased activity could be isolated in an E. coli selection system, it was shown subsequently that the mutations mainly enhanced the folding efficiency of the protein in its heterologous host. This resulted in higher levels of correctly folded RubisCO and thus higher carboxylation activity in cell extracts, while the kinetic properties of the enzyme were only moderately improved (Greene et al., 2007; Parikh et al., 2006). A recent study has uncovered underlying reasons that account for the difficulties in enhancing RubisCO activity. The authors could show that the enzyme has actually evolved close to optimality in its net photosynthesis rate, highlighting the difficulty of improving this trait by variations on the sequence level (Savir et al., 2010). As a consequence, research focus has shifted towards increasing CO2 -concentration in organisms, e.g., by introduction of carboxysomes into chloroplasts, in order to limit the detrimental oxygenation reaction of RubisCO to a minimum (Zarzycki et al., 2013). The heterologous expression of ten proteins that constitute a proteobacterial carboxysome in E. coli has been reported recently. The resulting recombinant carboxysome was capable of fixing carbon dioxide in vivo as well as in vitro (Bonacci et al., 2012). However, due to frequent difficulties in the transformation of plants, it will certainly be much more challenging to introduce carboxysomes into chloroplasts, but this approach represents a promising alternative to the engineering of carboxylases. Another way of improving carbon fixation rates in autotrophic (“self-nourishing”) organisms is to overexpress non-carboxylating enzymes that limit the supply of carboxylation substrates or the downstream conversion of carboxylation products, and thus represent additional bottlenecks in CO2 -fixation pathways besides carboxylases (see above). According to this approach, sedoheptulose-1,7-bisphosphate, an enzyme of the reductive pentose phosphate pathway responsible for the regeneration of the CO2 acceptor ribulose-5-phosphate in the Calvin–Benson–Bassham cycle, was recently overexpressed in tobacco plants. This led to an increase in carbon fixation of 6–12% and resulted in growth advantages for plant leaves during early phases of development (Lefebvre et al., 2005). Similarly, the introduction of the bacterial pathway for glycolate metabolism into plants significantly reduced photorespiratory losses due to optimization of downstream metabolism of phosphoglycolate (Kebeish et al., 2007). These examples demonstrate that it is important to focus not exclusively on improving carboxylating enzymes and reactions, but also to consider additional, alternative solutions to increase and enhance carbon flux

249

through autotrophic pathways. This holistic strategy might become even more crucial when exploring possibilities to construct completely synthetic carbon fixation pathways (see below). 4.4. Applications in synthetic biology: design of artificial CO2 fixation pathways The most ambitious goal involving carboxylases in synthetic biology is to design and implement artificial pathways for CO2 fixation into suitable host organisms. This concept relies on the recombination of enzymes from various sources and different metabolic backgrounds to assemble pathways for autotrophy that are free of any conventional regulatory or evolutionary limits (Erb, 2011). A pioneering theoretical study has identified very recently potential candidate pathways for synthetic carbon fixation. Biochemical information on approximately 5000 metabolic enzymes known to occur in Nature was used to generate different autotrophic pathway variants in silico (Bar-Even et al., 2010). The authors list several optimized artificial CO2 -fixation pathways of which the most promising were all based on PEP carboxylase. As PEP carboxylase is one of most efficient carboxylating enzymes, these results are well in line with the observation that the total energetic costs of carbon fixation pathways are directly linked to their carboxylases (Erb, 2011), which emphasizes that the choice of the carboxylase is a critical factor in designing efficient synthetic CO2 -fixation pathways. However, the successful realization of artificial CO2 -fixation pathways in vivo presents numerous challenges, most importantly, the expression of active enzymes in a heterologous host, the finetuning of the de novo assembled enzymatic network, as well as the generation of sufficient pools of energy (ATP) and reduction equivalent (NAD(P)H) to allow an optimal flux of carbon through the synthetic pathway. These challenges require substantial insight into the carboxylating enzymes and all other proteins that are to be enhanced or employed in non-natural backgrounds. Without a detailed understanding of how the pieces and tools of a metabolic engineering approach work in vivo, even the best in silico concept is likely to fail due to practical problems. First experiments have demonstrated that it is indeed very tedious to assemble artificial CO2 -fixation pathways de novo. Realization of the 3-hydroxypropionate cycle for CO2 -fixation in E. coli has been only partly successful, as due to toxicity, single pathway modules, but not the complete pathway could be reconstructed in the heterologous host (Mattozzi et al., 2013). Thus, further investigation of the biochemical properties, mechanisms, and evolutionary constraints of carboxylases is indispensable for successful synthetic biology approaches in the future. 5. Conclusions (i) Most (if not all) enzymatic carboxylation reactions studied so far follow the same mechanistic principle: A carboxylation substrate is nucleophilically activated by formation of an enol/enamin(at)e intermediate. This intermediate attacks an electrophilically activated CO2 molecule to form a novel C–C bond. Electrophilic activation of CO2 can be achieved through active site geometry (e.g., hydrogen bonding) or by covalent modifications (e.g., carboxybiotin or carboxyphosphate). (ii) Carboxylases have evolved several times independently during evolution. Phylogenetic and mechanistic investigations of the RubisCO and MDR superfamilies exemplify how RubisCO and Ccr might have acquired their carboxylation functions and emerged within their enzyme superfamilies. (iii) Biotechnological application of carboxylases has been limited for a long time due to the complexity of most enzymes.

250

L. Schada von Borzyskowski et al. / Journal of Biotechnology 168 (2013) 243–251

However, the discovery of novel enzymes and the development of new catalytic strategies have opened exciting possibilities for practical applications of carboxylases. CO2 -fixation on “single enzyme level” has been realized by operating enzymes that usually catalyze decarboxylation reactions in the physiologically reverse direction. Identification and functionalization of Ccr as important enzyme in polyketide biosynthesis has enabled biocombinatorial and mutasynthetic modifications of natural products in vitro and in vivo, thus creating unprecedented “synthetic” natural products of superior bioactivity. Future applications of carboxylases will focus on improving naturally existing autotrophic pathways to enhance primary production, and on the realization of completely artificial (“synthetic”) CO2 -fixation pathways. Acknowledgments This work was supported by the Ambizione program of the Swiss National Science Foundation (SNF research grant PZ00P3 136828) and by an ETH grant (ETH-41 12-2) to T.J.E. Sections 1, 3 and 5 of the manuscript were written by T.J.E.; R.G.R. wrote section 2, and L.S.v.B. section 4 of the manuscript. The authors thank Ramon Weishaupt, Dominik Peter and Philipp Moosmann for critical reading of the manuscript. First authorship was determined by coin toss. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.jbiotec.2013.05.007. References Aresta, M., 2009. Carbon dioxide utilization: chemical, biological and technological applications. In: Buncel, E. (Ed.), Greenhouse Gases: Mitigation and Utilization, CHEMRAWN-XVII and ICCDU-IX Conference, July 8 to 12, 2007. Queen’s University, Kingston, ON, Canada, pp. 123–149. Aresta, M., Quaranta, E., Liberio, R., Dileo, C., Tommasi, I., 1998. Enzymatic synthesis of 4-OH-benzoic acid from phenol and CO2 : the first example of a biotechnological application of a carboxylase enzyme. Tetrahedron 54, 8841–8846. Ashida, H., Danchin, A., Yokota, A., 2005. Was photosynthetic RubisCO recruited by acquisitive evolution from RubisCO-like proteins involved in sulfur metabolism? Research in Microbiology 156, 611–618. Ashida, H., Saito, Y., Nakano, T., Tandeau de Marsac, N., Sekowska, A., Danchin, A., Yokota, A., 2008. RubisCO-like proteins as the enolase enzyme in the methionine salvage pathway: functional and evolutionary relationships between RubisCOlike proteins and photosynthetic RubisCO. Journal of Experimental Botany 59, 1543–1554. Ashida, H., Saito, Y., Kojima, C., Kobayashi, K., Ogasawara, N., Yokota Akiho, 2003. A functional link between RubisCO-like protein of Bacillus and photosynthetic RubisCO. Science 302, 286–290. Attwood, P.V., Tipton, P.A., Cleland, W.W., 1986. C-13 and deuterium-isotope effects on oxalacetate decarboxylation by pyruvate-carboxylase. Biochemistry 25, 8197–8205. Ausenhus, S.L., 1993. Phosphoenolpyruvate carboxylase from maize: function of the divalent metal ion in binding and catalysis. PhD thesis, University of Wisconsin, Madison, pp. 160. Babbitt, P.C., Gerlt, J.A., 1997. Understanding enzyme superfamilies – chemistry as the fundamental determinant in the evolution of new catalytic activities. Journal of Biological Chemistry 272, 30591–30594. Badger, M.R., Bek, E.J., 2008. Multiple RubisCO forms in proteobacteria: their functional significance in relation to CO2 acquisition by the CBB cycle. Journal of Experimental Botany 59, 1525–1541. Badger, M.R., Price, G.D., 2003. CO2 concentrating mechanisms in cyanobacteria: molecular components, their diversity and evolution. Journal of Experimental Botany 54, 609–622. Bar-Even, A., Noor, E., Lewis, N.E., Milo, R., 2010. Design and analysis of synthetic carbon fixation pathways. Proceedings of the National Academy of Sciences of the United States of America 107, 8889–8894. Bell, A.T., Gates, B.C., Ray, D., 2007. Basic research needs: Catalysis for energy. In: Report from the U.S. Department of Energy, Office of Basic Energy Sciences Workshop, Bethesda, MD, USA, August 6–8. Bonacci, W., Teng, P.K., Afonso, B., Niederholtmeyer, H., Grob, P., Silver, P.A., Savage, D.F., 2012. Modularity of a carbon-fixing protein organelle. Proceedings of the National Academy of Sciences of the United States of America 109, 478–483.

Chan, Y.A., Podevels, A.M., Kevany, B.M., Thomas, M.G., 2009. Biosynthesis of polyketide synthase extender units. Natural Products Reports 26, 90–114. Cleland, W.W., Andrews, T.J., Gutteridge, S., Hartman, F.C., Lorimer, G.H., 1998. Mechanism of RubisCO: the carbamate as general base. Chemical Reviews 98, 549–562. Diacovich, L., Mitchell, D.L., Pham, H., Gago, G., Melgar, M.M., Khosla, C., Gramajo, H., Tsai, S.C., 2004. Crystal structure of the beta-subunit of acyl-CoA carboxylase: structure-based engineering of substrate specificity. Biochemistry 43, 14027–14036. Dibenedetto, A., Lo Noce, R., Pastore, C., Aresta, M., Fragale, C., 2006. First in vitro use of the phenylphosphate carboxylase enzyme in supercritical CO2 for the selective carboxylation of phenol to 4-hydroxybenzoic acid. Environmental Chemistry Letters 3, 145–148. Drummond, M.L., Wilson, A.K., Cundari, T.R., 2012. Nature of protein–CO2 interactions as elucidated via molecular dynamics. Journal of Physical Chemistry B 116, 11578–11593. Ellis, R.J., 1979. The most abundant protein in the world. Trends in Biochemical Sciences 4, 241–244. Erb, T.J., 2011. Carboxylases in natural and synthetic microbial pathways. Applied and Environment Microbiology 77, 8466–8477. Erb, T.J., Berg, I.A., Brecht, V., Muller, M., Fuchs, G., Alber, B.E., 2007. Synthesis of C5dicarboxylic acids from C2-units involving crotonyl-CoA carboxylase/reductase: the ethylmalonyl-CoA pathway. Proceedings of the National Academy of Sciences of the United States of America 104, 10631–10636. Erb, T.J., Brecht, V., Fuchs, G., Muller, M., Alber, B.E., 2009a. Carboxylation mechanism and stereochemistry of crotonyl-CoA carboxylase/reductase, a carboxylating enoyl-thioester reductase. Proceedings of the National Academy of Sciences of the United States of America 106, 8871–8876. Erb, T.J., Evans, B.S., Cho, K., Warlick, B.P., Sriram, J., Wood, B.M., Imker, H.J., Sweedler, J.V., Tabita, F.R., Gerlt, J.A., 2012. A RubisCO-like protein links SAM metabolism with isoprenoid biosynthesis. Nature Chemical Biology 8, 926–932. Erb, T.J., Fuchs, G., Alber, B.E., 2009b. (2S)-Methylsuccinyl-CoA dehydrogenase closes the ethylmalonyl-CoA pathway for acetyl-CoA assimilation. Molecular Microbiology 73, 992–1008. Eustaquio, A.S., McGlinchey, R.P., Liu, Y., Hazzard, C., Beer, L.L., Florova, G., Alhamadsheh, M.M., Lechner, A., Kale, A.J., Kobayashi, Y., Reynolds, K.A., Moore, B.S., 2009. Biosynthesis of the salinosporamide A polyketide synthase substrate chloroethylmalonyl-coenzyme A from S-adenosyl-l-methionine. Proceedings of the National Academy of Sciences of the United States of America 106, 12295–12300. Eustaquio, A.S., Moore, B.S., 2008. Mutasynthesis of fluorosalinosporamide, a potent and reversible inhibitor of the proteasome. Angewandte Chemie International Edition (in English) 47, 3936–3938. Field, C.B., Behrenfeld, M.J., Randerson, J.T., Falkowski, P., 1998. Primary production of the biosphere: integrating terrestrial and oceanic components. Science 281, 237–240. Fujita, S., Bhanage, B.M., Arai, M., 2005. Chemical fixation of carbon dioxide: green processes to valuable chemicals. In: Bevy, L.P. (Ed.), Progress in Catalysis Research. Nova Science Publishers, Hauppauge, NY, USA, pp. 57–79. Gerlt, J.A., Babbitt, P.C., 2001. Divergent evolution of enzymatic function: mechanistically diverse superfamilies and functionally distinct suprafamilies. Annual Review of Biochemistry 70, 209–246. Glueck, S.M., Gumus, S., Fabian, W.M.F., Faber, K., 2010. Biocatalytic carboxylation. Chemical Society Reviews 39, 313–328. Greene, D.N., Whitney, S.M., Matsumura, I., 2007. Artificially evolved Synechococcus PCC6301 RubisCO variants exhibit improvements in folding and catalytic efficiency. Biochemical Journal 404, 517–524. Hamed, R.B., Gomez-Castellanos, J.R., Thalhammer, A., Harding, D., Ducho, C., Claridge, T.D.W., Schofield, C.J., 2011. Stereoselective C–C bond formation catalysed by engineered carboxymethylproline synthases. Nature Chemistry 3, 365–371. Hansen, D.E., Knowles, J.R., 1982. The stereochemical course at phosphorus of the reaction catalyzed by phosphoenolpyruvate carboxylase. Journal of Biological Chemistry 257, 4795–4798. Hanson, T.E., Tabita, F.R., 2001. A ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO)-like protein from Chlorobium tepidum that is involved with sulfur metabolism and the response to oxidative stress. Proceedings of the National Academy of Sciences of the United States of America 98, 4397–4402. Hanson, T.E., Tabita, F.R., 2003. Insights into the stress response and sulfur metabolism revealed by proteome analysis of a Chlorobium tepidum mutant lacking the Rubisco-like protein. Photosynthesis Research 78, 231–248. Imker, H.J., Fedorov, A.A., Fedorov, E.V., Almo, S.C., Gerlt, J.A., 2007. Mechanistic diversity in the RubisCO superfamily: the “enolase” in the methionine salvage pathway in Geobacillus kaustophilus. Biochemistry 46, 4077–4089. Imker, H.J., Singh, J., Warlick, B.P., Tabita, F.R., Gerlt, J.A., 2008. Mechanistic diversity in the RubisCO superfamily: a novel isomerization reaction catalyzed by the RubisCO-like protein from Rhodospirillum rubrum. Biochemistry 47, 11171–11173. Janc, J.W., Urbauer, J.L., Oleary, M.H., Cleland, W.W., 1992. Mechanistic studies of phosphoenolpyruvate carboxylase from Zea mays with (Z)-3fluorophosphenolpyruvate and (E)-3-fluorophosphoenolpyruvate as substrates. Biochemistry 31, 6432–6440. Jung, D.H., Choi, W., Choi, K.Y., Jung, E., Yun, H., Kazlauskas, R.J., Kim, B.G., 2012. Bioconversion of p-coumaric acid to p-hydroxystyrene using phenolic acid decarboxylase from Bacillus amyloliquefaciens in biphasic reaction system. Applied Microbiology and Biotechnology.

L. Schada von Borzyskowski et al. / Journal of Biotechnology 168 (2013) 243–251 Kebeish, R., Niessen, M., Thiruveedhi, K., Bari, R., Hirsch, H.J., Rosenkranz, R., Stabler, N., Schonfeld, B., Kreuzaler, F., Peterhansel, C., 2007. Chloroplastic photorespiratory bypass increases photosynthesis and biomass production in Arabidopsis thaliana. Nature Biotechnology 25, 593–599. Kerfeld, C.A., Heinhorst, S., Cannon, G.C., 2010. Bacterial microcompartments. Annual Review of Microbiology 64, 391–408. Kiehl, J.T., Trenberth, K.E., 1997. Earth’s annual global mean energy budget. Bulletin of the American Meteorological Society 78, 197–208. King, D.A., 2004. Climate change science: adapt, mitigate, or ignore? Science 303, 176–177. Kirimura, K., Gunji, H., Wakayama, R., Hattori, T., Ishii, Y., 2010. Enzymatic Kolbe–Schmitt reaction to form salicylic acid from phenol: enzymatic characterization and gene identification of a novel enzyme, Trichosporon moniliiforme salicylic acid decarboxylase. Biochemical and Biophysical Research Communications 394, 279–284. Kirimura, K., Yanaso, S., Kosaka, S., Koyama, K., Hattori, T., Ishii, Y., 2011. Production of p-aminosalicylic acid through enzymatic Kolbe–Schmitt reaction catalyzed by reversible salicylic acid decarboxylase. Chemistry Letters 40, 206–208. Knowles, J.R., 1989. The mechanism of biotin-dependent enzymes. Annual Review of Biochemistry 58, 195–221. Kofoed, M.A., Wampler, D.A., Pandey, A.S., Peters, J.W., Ensign, S.A., 2011. Roles of the redox-active disulfide and histidine residues forming a catalytic dyad in reactions catalyzed by 2-ketopropyl coenzyme M oxidoreductase/carboxylase. Journal of Bacteriology 193, 4904–4913. Lane, M.D., 1969. Comparison of enzymatic carboxylation mechanisms. In: Forster, R.E., Edsall, J.T., Otis, A.B., Roughton, F.J.W. (Eds.), CO2 : Chemical, Biochemical, and Physiological Aspects. NASA, Washington, D.C., pp. 195–206. Lefebvre, S., Lawson, T., Zakhleniuk, O.V., Lloyd, J.C., Raines, C.A., 2005. Increased sedoheptulose-1,7-bisphosphatase activity in transgenic tobacco plants stimulates photosynthesis and growth from an early stage in development. Plant Physiology 138, 451–460. Lundqvist, T., Schneider, G., 1991. Crystal structure of activated ribulose1,5-bisphosphate carboxylase complexed with its substrate, ribulose-1,5bisphosphate. Journal of Biological Chemistry 266, 12604–12611. Matsui, T., Yoshida, T., Hayashi, T., Nagasawa, T., 2006a. Purification, characterization, and gene cloning of 4-hydroxybenzoate decarboxylase of Enterobacter cloacae P240. Archives of Microbiology 186, 21–29. Matsui, T., Yoshida, T., Yoshimura, T., Nagasawa, T., 2006b. Regioselective carboxylation of 1,3-dihydroxybenzene by 2,6-dihydroxybenzoate decarboxylase of Pandoraea sp. 12B-2. Applied Microbiology and Biotechnology 73, 95–102. Mattozzi, M., Ziesack, M., Voges, M.J., Silver, P.A., Way, J.C., 2013. Expression of the sub-pathways of the Chloroflexus aurantiacus 3-hydroxypropionate carbon fixation bicycle in E. coli: toward horizontal transfer of autotrophic growth. Metabolic Engineering. Mo, S., Kim, D.H., Lee, J.H., Park, J.W., Basnet, D.B., Ban, Y.H., Yoo, Y.J., Chen, S.W., Park, S.R., Choi, E.A., Kim, E., Jin, Y.Y., Lee, S.K., Park, J.Y., Liu, Y.A., Lee, M.O., Lee, K.S., Kim, S.J., Kim, D., Park, B.C., Lee, S.G., Kwon, H.J., Suh, J.W., Moore, B.S., Lim, S.K., Yoon, Y.J., 2011. Biosynthesis of the allylmalonyl-CoA extender unit for the FK506 polyketide synthase proceeds through a dedicated polyketide synthase and facilitates the mutasynthesis of analogues. Journal of the American Chemical Society 133, 976–985. Ogita, T., Knowles, J.R., 1988. On the intermediacy of carboxyphosphate in biotindependent carboxylations. Biochemistry 27, 8028–8033. Omura, H., Wieser, M., Nagasawa, T., 1998. Pyrrole-2-carboxylate decarboxylase from Bacillus megaterium PYR2910, an organic-acid-requiring enzyme. European Journal of Biochemistry 253, 480–484. Parikh, M.R., Greene, D.N., Woods, K.K., Matsumura, I., 2006. Directed evolution of RubisCO hypermorphs through genetic selection in engineered E. coli. Protein Engineering Design and Selection 19, 113–119. Parry, M.A., Andralojc, P.J., Scales, J.C., Salvucci, M.E., Carmo-Silva, A.E., Alonso, H., Whitney, S.M., 2013. RubisCO activity and regulation as targets for crop improvement. Journal of Experimental Botany 64, 717–730. Persson, B., Hedlund, J., Jornvall, H., 2008. The MDR superfamily. Cellular and Molecular Life Sciences 65, 3879–3894.

251

Peterhansel, C., Niessen, M., Kebeish, R.M., 2008. Metabolic engineering towards the enhancement of photosynthesis. Photochemistry and Photobiology 84, 1317–1323. Phillips, R., Milo, R., 2009. A feeling for the numbers in biology. Proceedings of the National Academy of Sciences of the United States of America 106, 21465–21471. Quade, N., Huo, L.J., Rachid, S., Heinz, D.W., Muller, R., 2012. Unusual carbon fixation gives rise to diverse polyketide extender units. Nature Chemical Biology 8, 117–124. Rachid, S., Huo, L.J., Herrmann, J., Stadler, M., Kopcke, B., Bitzer, J., Muller, R., 2011. Mining the cinnabaramide biosynthetic pathway to generate novel proteasome inhibitors. Chembiochem 12, 922–931. Savir, Y., Noor, E., Milo, R., Tlusty, T., 2010. Cross-species analysis traces adaptation of RubisCO toward optimality in a low-dimensional landscape. Proceedings of the National Academy of Sciences of the United States of America 107, 3475–3480. Schühle, K., Fuchs, G., 2004. Phenylphosphate carboxylase: a new C–C lyase involved in anaerobic phenol metabolism in Thauera aromatica. Journal of Bacteriology 186, 4556–4567. Sharkey, T.D., 1988. Estimating the rate of photorespiration in leaves. Physiologia Plantarum 73, 147–152. Shively, J.M., Ball, F., Brown, D.H., Saunders, R.E., 1973. Functional organelles in prokaryotes – polyhedral inclusions (carboxysomes) of Thiobacillus neapolitanus. Science 182, 584–586. Stassi, D.L., Kakavas, S.J., Reynolds, K.A., Gunawardana, G., Swanson, S., Zeidner, D., Jackson, M., Liu, H., Buko, A., Katz, L., 1998. Ethyl-substituted erythromycin derivatives produced by directed metabolic engineering. Proceedings of the National Academy of Sciences of the United States of America 95, 7305–7309. Stubbe, J., Fish, S., Abeles, R.H., 1980. Are carboxylations involving biotin concerted or non-concerted? Journal of Biological Chemistry 255, 236–242. Tabita, F.R., Hanson, T.E., Li, H.Y., Satagopan, S., Singh, J., Chan, S., 2007. Function, structure, and evolution of the RubisCO-like proteins and their RubisCO homologs. Microbiology and Molecular Biology Reviews 71, 576–599. Tabita, F.R., Satagopan, S., Hanson, T.E., Kreel, N.E., Scott, S.S., 2008. Distinct form I, II, III, and IV RubisCO proteins from the three kingdoms of life provide clues about RubisCO evolution and structure/function relationships. Journal of Experimental Botany 59, 1515–1524. Tans, P., Keeling, R., 2013. http://www.esrl.noaa.gov/gmd/ccgg/trends/ or http://scrippsco2.ucsd.edu/ (accessed 04.02.13). Wallace, K.K., Bao, Z.Y., Dai, H., Digate, R., Schuler, G., Speedie, M.K., Reynolds, K.A., 1995. Purification of crotonyl-CoA reductase from Streptomyces collinus and cloning, sequencing and expression of the corresponding gene in Escherichia coli. European Journal of Biochemistry 233, 954–962. Wilson, M.C., Moore, B.S., 2012. Beyond ethylmalonyl-CoA: the functional role of crotonyl-CoA carboxylase/reductase homologs in expanding polyketide diversity. Natural Products Reports 29, 1480. Wilson, M.C., Nam, S.J., Gulder, T.A.M., Kauffman, C.A., Jensen, P.R., Fenical, W., Moore, B.S., 2011. Structure and biosynthesis of the marine Streptomycete ansamycin Ansalactam A and its distinctive branched chain polyketide extender unit. Journal of the American Chemical Society 133, 1971–1977. Wuensch, C., Glueck, S.M., Gross, J., Koszelewski, D., Schober, M., Faber, K., 2012. Regioselective enzymatic carboxylation of phenols and hydroxystyrene derivatives. Organic Letters 14, 1974–1977. Xu, Z.L., Ding, L., Hertweck, C., 2011. A branched extender unit shared between two orthogonal polyketide pathways in an endophyte. Angewandte Chemie International Edition (in English) 50, 4667–4670. Yoo, H.G., Kwon, S.Y., Kim, S., Karki, S., Park, Z.Y., Kwon, H.J., 2011a. Characterization of 2-octenoyl-CoA carboxylase/reductase utilizing pteB from Streptomyces avermitilis. Bioscience, Biotechnology, and Biochemistry 75, 1191–1193. Yoo, H.-G., Kwon, S.-Y., Kim, S., Karki, S., Park, Z.-Y., Kwon, H.-J., 2011b. Characterization of 2-Octenoyl-CoA carboxylase/reductase utilizing pteB from Streptomyce avermitilis. Bioscience, Biotechnology, and Biochemistry 75, 1191–1193. Zarzycki, J., Axen, S.D., Kinney, J.N., Kerfeld, C.A., 2013. Cyanobacterial-based approaches to improving photosynthesis in plants. Journal of Experimental Botany 64, 787–798.