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Letter
A Catalytically Competent Terpene Synthase Inferred Using Ancestral Sequence Reconstruction Strategy Daniele G. Guzzetti, Aurélien Lebrun, Maeva Subileau, Estelle Grousseau, Eric Dubreucq, and Jullien Drone ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.6b01332 • Publication Date (Web): 12 Jul 2016 Downloaded from http://pubs.acs.org on July 16, 2016
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ACS Catalysis
A Catalytically Competent Terpene Synthase Inferred Using Ancestral Sequence Reconstruction Strategy Daniele Guzzetti,1 Aurélien Lebrun,2 Maeva Subileau,1 Estelle Grousseau,1 Eric Dubreucq,1 Jullien Drone1,3* 1 Ingénierie des Agropolymères et Technologies Emergentes, UMR IATE, SupAgro / INRA / CIRAD / UM, 2 place Pierre Viala, 34060 Montpellier, France 2 Laboratoire de Mesures Physiques, Place Eugène Bataillon, 34095 Montpellier, France 3 École Nationale Supérieure de Chimie Montpellier, 8 rue de l’École Normale, 34296 Montpellier, France
Supporting Information Placeholder ABSTRACT: On the basis of bioinformatic analysis of 137 putative and characterized bacterial terpene synthases (TS), we applied ancestral reconstruction strategy to design a biosynthetic protein sequence (AncCL1). We biochemically confirmed its catalytic competence and product profile. This enzyme not only catalyzes epi-isozizaene (minor product) but also epi-zizaene (major product) formation. We compared AncCL1 with two related TS to allow a more detailed insight into the product specificity determinants. In the future, the presented method will benefit for engineering of biosynthetic TS but also for function elucidation of unknown enzymes.
KEYWORDS: Biocatalysis, Protein engineering, Ancestral sequence reconstruction, Terpene synthase, Metabolic engineering.
Terpene synthases (TS) are key enzymes involved into the terpenoid biosynthetic pathways. They trigger and chaperone the cyclization of the universal linear polyprenyl precursors, such as geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP) or geranylgeranyl pyrophosphate (GGPP) leading respectively to the monoterpene, sesquiterpene or 1 diterpene scaffolds found in Nature. More than 55,000 different terpenoids from plant, fungal and animal species have 1,2 been identified to date. Even bacteria, that have long been a neglected source of terpenoids, are now pointed out as effective producers of unique molecules from this class and several bacterial TS have been experimentally characterized 3–8 to date. High-throughput sequencing of bacterial genomes is a valuable source of putative TS genes and new insigths into bacterial terpene biosynthesis have been gathered dur3 ing the last decade.
In the present study we collected TS sequences based, not on the BLAST algorithm, but on profile-hidden Markov Models (profile-HMM). A sample of 137 bacterial sequences (putative and characterized) was used to construct a phylogenetic tree. From this tree, a cluster of sequences was subjected to probabilistic ancestral sequence reconstruction (ASR). The inferred sequence (AncCL1) was then expressed into a farnesyl pyrophosphate-overproducing strain of Escherichia coli to demonstrate its catalytic competence. Subsequently, we used high-cell-density cultivation (HCDC) to produce the corresponding sesquiterpenes in sufficient amounts to establish their chemical structures by nuclear magnetic resonance (NMR) and mass spectrometry (MS) analysis. We identified a mixture of epi-isozizaene (1, 15%) and epi-zizaene (2, 85%). We compared the catalytic activitiy, sequence and 3D-model of AncCL1 with the epiisozizaene synthase (EIZS) from Streptomyces coelicolor 5 A3(2) (SCO5222) and the epi-zizaene synthase from Strep8 tomyces sp. Tü6071 (EZS), two catalytically and phylogenetically related TS. All these results suggested that the enzymatic cluster of which AncCL1 would be at the origin is likely isofunctional, producing either 1 or 2. Finally, we showed that, in this case, the relaxed specificity of AncCL1 was ruled by residues outside a sphere of radius ≈ 7.5 Å around the substrate. Recently, authors have suggested that profile-HMM algorithm could be used instead of BLAST local alignment algorithm for mining TS protein sequences in silico in order to 3,9 circumvent low overall sequence similarity between them. Using the Pfam entry PF03906 (terpene synthase family, 10 metal binding domain) and the HMMER Web server to search UniProtKB and NCBI public databases, we obtained approximately 20,000 protein sequences related to TS from all species including 277 from bacteria. Expected values (E) -73 -6 were comprised between 1.1 × 10 and 8.6 × 10 for these 277 bacterial sequences. Lower E thresholds are more stringent, leading statistically to fewer chance-matches being reported. -73 The 100 first bacterial protein sequences (1.1 × 10 ≤ E ≤ 4 ×
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10 ) were selected. Extremely divergent sequences (2-methyl isoborneol synthases) and duplicate records were deleted (21 sequences). Bacterial TS experimentally characterized (18) or 6 identified by homology (40) were manually added to finally establish a databank of 137 protein sequences. These sequences were aligned and the corresponding phylogenetic tree was built using a maximum likelihood (ML) algorithm 11 by FastML server (see Supporting Information). Three clusters could be clearly distinguished (Figure S1): cluster 1 with 26 sequences (identity 57-87%; mean 351 aa; standard deviation (σ) = 22 aa), cluster 2 with 68 sequences (identity 4776%; mean 749 aa, σ = 35 aa) and cluster 3 with 17 sequences (identity 28-78%; mean 336 aa; σ = 10 aa). In cluster 1, eight characterized members were present (Figure S2). Two of them have been previously cloned and expressed: the EIZS from S. coelicolor A3(2) (NP_629369; SCO5222; 361 aa), a functional type I sesquiterpene synthase fully characterized 5,12 and crystallized and the EIZS from S. avermitilis 7 (NP_824208; SAV3032; 363 aa; 82% identity with SCO5222). The remaining six of them were held responsible for the biosynthesis of volatile terpenes produced by microorgan8 isms and detected by headspace analysis. It was the epizizaene synthase from S. sp Tü6071 (EZS; ZP_08452581; 353 aa) and five EIZS (ZP_06593440, ZP_06528571, ZP_06576746, ZP_7310844 and ZP_07306383). Among cluster 2, two bifunctional enzymes containing two class I terpenoid synthase domains (germacradienol/geosmin synthase from S. coelicol13 or A3(2); GI:81625580) and germacradienol/geosmin syn14 thase from S. avermitilis; Q82L49) were reported into the literature. They both convert FPP into geosmin through the intermediary of germacradienol. Finally, cluster 3 comprised 5 experimentally characterized TS out of 17, with pentalenene 4 synthase from S. exfoliatus (GI:2833457), pentalenene syn15 thase from S. avermitilis (Q82IY4), germacradien-4-ol syn16 thase from S. citricolor SC1 (GI:357527174), avermitilol syn17,18 thase from S. avermitilis (GI:29826616), and epi-α16 bisabolol synthase from S. citricolor SC2 (GI:357527176). Inference of extinct proteins can be performed using ASR 19,20 strategies. Basically, ASR integrates primary sequences, corresponding alignment and phylogenetic tree of presentday homologous proteins. For each internal node of the phylogenetic tree, the ASR algorithm builds up a statistical model for the ancestral primary sequence for which each amino acid is evaluated in terms of likelihood. Hence, it is an in silico approach and Web servers have been developed to facilitate ASR. It is a valuable tool for evolutionary studies and successful reconstructions have been applied for example to GFP-like proteins, steroid receptors or enzymes like 21–29 alcohol dehydrogenase. It has also proven useful for protein engineering to design more soluble variants of a hydro30 phobic, aggregation-prone, phosphate-binding protein. In our case, we tested the hypothesis whether it was possible to use ASR for creating a biosynthetic and catalytically compe11 tent TS. FastML was used to generate the most probable ancestor of the phylogenetic cluster showing the highest identity range, namely cluster 1. The most probable sequence (AncCL1) was made of 395 aa and had 59% identity with SCO5222, 60% identity with SAV_3032 and 89% identity with EZS from S. sp Tü6071 (Figure S3B). Sequence alignments 99 103 240 248 showed the same D DRHD / N DLCSLPKE motifs
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between SCO5222, SAV_3032, EZS and AncCL1 (numbering with respect to SCO5222) (Figure S3A). The DDXXD and NSE consensus sequences are implicated in the binding of 2+ Mg into the active site and are characteristic of type I TS. The 4 residues involved into the interaction with the pyrophosphate moiety of FPP (R194, K247, R338 and Y339) were also 100% identical. A glycine-rich gap made up of 9 residues was found in both AncCL1 and EZS while it was absent from both SCO5222 and SAV_3032. Amino terminus was longer for AncCL1, SCO5222 and SAV_3032 than for EZS (42-44 supplementary residues). A longer carboxy terminus section (25 supplementary residues) was found for AncCL1 and EZS compared to SCO5222 and SAV_3032. It is difficult to establish whether such differences were acquired by individual TS rather than being originally present in the ancestral sequence. This might be a limitation for some evolutionary studies but in the context of protein engineering and development of methods to create novel enzymes, it might allow the exploration of a larger sequence space and thus become 31 an advantage. A gene encoding for AncCL1 was deduced from the reconstructed protein sequence, codon optimized for expression in E. coli, synthesized and cloned into the pET-Duet1 vector (pAncCL1) (Figure S4). Relying on transferal of the mevalonate-dependent isoprenoid precursor pathway from yeast into E. coli as implement32 ed by the Keasling group, in an approach that has been 33–35 pioneered by Peters and Croteau groups, the strain BL21(DE3) of Escherichia coli was transformed with the 36 plasmid pJBEI-2999 to yield strain BL2999. This plasmid allows the optimized expression of a parallel mevalonate pathway in E. coli to overproduce FPP, the universal linear 36 precursor of sesquiterpenes. Cultivation of BL2999 was performed and expression of the engineered mevalonate pathway was induced by the addition of isopropyl thiogalactoside (IPTG, 1 mM) during the exponential growth phase. Concomitantly, dodecane (10% v/v) was added to recover terpenoids synthesized during the cultivation without ham36 pering cell growth. After 24 hours, the organic overlay was harvested, analysed by GC/MS and farnesol was detected (retention time 10.89 min, Figure 1a). The presence of farnesol in the docecane overlay is due to the rapid hydrolysis of FPP after its biosynthesis in vivo and subsequent release into the external medium. BL2999, our FPP-producing plateform, was then co-transformed either with pAncCL1 or with 5 pET28a(+)/SCO5222 , the plasmid used by the team of Christianson for the expression of EIZS from S. coelicolor A3(2), hereafter pEIZS. Cultivation of both strains was performed identically as above. For the BL2999/pEIZS strain, a single product (retention time 6.60 min) having the characteristic m/z 189 and 204 ions was found (Figure 1b). The mass spectrum of this molecule was nearly identical to the fragmentation pattern of epi-isozizaene (1) reported previously (Figure 12 S5). For the BL2999/pAncCL1 strain, two molecules (retention times 6.60 and 6.97 min) with m/z 189 and 204 ions were obtained (Figure 1c). This result clearly demonstrated that AncCL1 was enzymatically active promoting the cyclization of FPP into 1 (retention time 6.60 min, 15%) and another sesquiterpene product (2) (retention time 6.97 min, C15H24, m/z 204, 85%). At this stage the exact structure of 2 could
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not be elucidated unambiguously based only on its fragmentation pattern (Figure S6). While functional in vivo, preliminary experiments to harvest and purify AncCL1 were unsuccessful since the recombinant protein appeared to be unstable in vitro. In order to produce amounts of sesquiterpenes 1 and 2 suitable for structure confirmation and determination by NMR spectroscopy, we performed high-cell-density cultivations (HCDC) of the strains BL2999/pEIZS and BL2999/pAncCL1. Once cell density around 60 g of dry-cell weight per liter of culture was reached, IPTG (1 mM) was added to induce the expression of the entire biosynthetic pathway as well as C18-grafted silica powder (Si-C18) to adsorb hydrophobic compounds such as C15H24 hydrocarbons directly during the cultivation. After 24 hours, the resulting suspension of cells and Si-C18 was centrifuged. The pellet was extracted with HPLC-grade pentane and the crude extracts purified by silica gel chromatography.
sesquiterpene produced by AncCL1 (2). The structure and relative stereochemistry of 2 were determined by a combina1 13 1 1 1 13 1 13 1 tion of H, C, H- H COSY, H- C HSQC, H- C HMBC, H13 1 1 C HSQC-TOCSY and H- H NOESY NMR experiments (Figures S9-S16, Scheme S2 and Table S3) and were fully consistent with epi-zizaene (2) (Scheme 1). The presence of an olefinic methylene group in 2 was indicated by the presence of two protons signals at high frequencies (H-12, δ 4.75 and 4.57). HSQC NMR data showed that these protons correlate 1 with olefinic carbon (C-12) in J at 105.0 ppm. The quaternary olefinic carbon (C-11) was assigned based on its typical chemical shift at 157.5 ppm. Three methyl groups were readily identified: a geminal singlet pair (H-13, δ 1.06; C-13, 26.3 ppm and H-15, δ 1.09; C-15, 28.9 ppm) bound to quaternary carbon (C-3, 40.5 ppm). The last methyl group (H-14, δ 0.86, d, J = 6.5 Hz; C-14, 14.5 ppm) was attached to a C-H (H-7, δ 1.91 1 13 ppm; C-7, 39.3 ppm). H- C HSQC-TOCSY spectrum with mixing time delay of 60 ms was used to identify unambiguously both spin systems thanks to non-overlapped signals on 13 C dimension. Long-range correlation was observed from methyl carbon C-14 (14.5 ppm) to methine C-10 (51.7 ppm; H10, δ 2.37). Complementary COSY information allowed for clear identification of C-8 (31.4 ppm; H-8, δ 1.87-1.11) and C-9 (23.8 ppm; H-9, δ 1.61-1.56). On the other spin system, carbons C-1 (23.0 ppm; H-1, δ 1.42), C-2 (48.7 ppm; H-2, δ 1.72), C-4 (38.2 ppm; H-4, δ 1.60-1.48) and C-5 (21.2 ppm; H-5, δ 1.12-0.84) were assigned by the use of combinated COSY and HSQC. The configuration of the secondary methyl group was unambigously assigned as anti to the C-10 methine, based on weak NOE cross-peak between H-7 (δ 1.91) and H-10 (δ 2.37). Both protons correlated with strong NOE with H-1a (δ 1.60). Another strong NOE was also observed between H-10 and the methyl group H-15 (δ 1.09, strong NOE). On the other hand, strong NOE cross-peaks were identified between H-1b (δ 1.48) and methyl group (δ 0.86) and also methine H-2 (δ 1.72); the latter had a strong NOE correlation with the methyl 1 group (δ 1.06). The H shifts and coupling constants de37 scribed by Hanayama for epi-zizaene (2) synthetically prepared from epi-zizanoic acid were fully consistent with those we found (Table S4).
Scheme 1. Sesquiterpene hydrocarbons produced by the ancestral bacterial TS AncCL1 from farnesyl pyrophos-
Figure 1. GC-MS analysis of the organic overlays obtained after 24 hours of induction with the strains a) BL2999, b) BL2999/pEIZS, c) BL2999/pAncCL1. Detection was performed using selective ion monitoring at m/z 189 and 204. 1
phate (FPP) where epi-isozizaene (1) was the less abundant (15%) and epi-zizaene (2) the most abundant (85%).
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H and C NMR data (Scheme S1, Figures S7, S8 and Tables S1, S2) confirmed that epi-isozizaene (1) was the only detectable sesquiterpene produced by the strain BL2999/pEIZS. For the strain BL2999/pAncCL1, we isolated the most abundant
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Scheme 2. From FPP, the zizaenyl cation is generated and subsequent deprotonation could occur either at C-10 to yield epi-isozizaene (1) or from the methyl group at C-12 to produce epi-zizaene (2).
Cyclization mechanisms were not investigated here but given the structures of 1 and 2 (the two products only differ in double bond placement) it can be assumed that the formation of both sesquiterpenes follows essentially the same 12 pathway. Divergence probably takes place during the very last step where deprotonation can occur at two different positions on the zizaenyl cation, their common precursor (Scheme 2). Indeed, deprotonation at the tertiary position C10 would lead to 1 while deprotonation at the primary position C-12 would produce 2. In the absence of an enzyme, deprotonation at C-12 would be kinetically favored over deprotonation at C-10. Indeed, sterics, statistics and relative stabilization of negative charge at the transition state would all seem to prefer exocyclic versus endocyclic elimination. While EIZS guides the zizaenyl cation intermediate toward the most stable product 1, EZS and our biosynthetic TS (AncCL1) favor the formation of the kinetic product 2. From the 1.60 Å resolution X-ray crystal structure of re2+ combinant SCO5222 in complex with three Mg ions, inorganic pyrophosphate and the carbocation analogue benzyltriethyl ammonium cation (BTAC; Protein Data Bank 5 entry 3KB9), we identified 17 amino acids within 5 Å of each
atom of BTAC (first-shell residues). Second-shell residues were identified as residues within 2.5 Å of the first-shell residues, encompassing 24 supplementary residues. In order to determine the residues important for AncCL1 function, homology structure models for AncCL1 and EZS were built 38 using the 3D-structure of SCO5222 as a template. Indeed, the respective 56 and 59% amino acid sequence identities between EZS and AncCL1 with SCO5222 enabled accurate protein modeling. The models were then energy-minimized and superimposed onto the SCO5222 structure. The firstshell residues of AncCL1 (Figure 2A) and EZS were identified and it appeared that the 17 first-shell residues were identical for the 3 proteins. The 24 second-shell residues were identical between AncCL1 and EZS (Figure 2B). However, we found SCO5222 had 6 residues different from AncCL1 and EZS in the second-shell (Figure 2B). Among these 6 aa, only 2 of them lined the active site cavity (M73 / Y91 for SCO5222, V73 / F91 for AncCL1 and V31 / F49 for EZS). With the ability to produce both 1 and 2, AncCL1 showed a 8 12 more "relaxed" selectivity than EZS and SCO5222. An important implication of the catalytic activity of AncCL1 and extant members of cluster 1 would be that this enzymatic
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cluster is likely isofunctional, producing selectively either 1 or 2. EZS and AncCL1 have exactly the same 41 residues within a sphere of radius ≈ 7.5 Å from the substrate analog, thus it is logical that AncCL1 had a clear preference for epi-zizaene formation. On the other hand, it appeared very unlikely that the second-shell changes we noticed between AncCL1 and SCO5222 were sufficient to explain the inversion of specificity between them. Greenhagen et al. previously demonstrated that, for phylogenetically related TS from plants, alteration of the product profile could be due to residues above the se39 cond-shell. Our results showed that it was the case here. As a consequence, we sustain the hypothesis that residues outside a sphere of radius ≈ 7.5 Å around the substrate analog are responsible for explaining the specificity for 1 or 2. Further experiments are actually under progress to identify these residues. The Schmidt-Dannert group studied the role of the active site's H-1α loop on product profile and selectivi40 ty of fungal TS. The residues of the respective H-1α loops of
AncCL1, SCO5222 and EZS were 100% identical. Thus this structural element can not be held responsible for the product profile differences. In summary, we have shown that it was possible to apply the ASR strategy to create a catalytically competent terpene synthase with original catalytic properties. Using a metabolically engineered strain of E. coli was a reliable way to determine the product profile of AncCL1. Also, it could be used in 33,34 the future to characterize unknown and/or difficult to purify TS as well as to understand some of the possible evolutionary relationships between these biocatalysts.
Figure 2. The homology structural model for the biosynthetic terpene synthase AncCL1 active site. (A) Using the 1.60 Å resolution X-ray crystal structure of recombinant SCO5222 in complex with three Mg2+ ions (clear blue), inorganic pyrophosphate (not shown), and the carbocation analogue benzyltriethyl ammonium cation (BTAC; ball and stick; brown) as a template (pdb 3KB9), the homology structural model of AncCL1 was built. The 17 residues of the first-shell (within 5 Å from BTAC) are shown as sticks and labeled. (B) The 24 residues of the second-shell (within 2.5 Å from first-shell residues) are listed for SCO5222, AncCL1 and EZS. Six second-shell differences were observed between these three synthases and only 2 of them are lining the active site (bold).
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AUTHOR INFORMATION
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Corresponding Author (14)
[email protected] (15)
Notes The authors declare no competing financial interests.
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ASSOCIATED CONTENT Supporting Information
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Contents of material supplied as Supporting Information: Bioinformatics, protein squence of AncCL1, protocols for shake flask cultivations at low cell density, for high-celldensity cultivation, for GC-MS analysis and for purification of the terpenoids as well as NMR spectroscopy data. Protein sequences of the 137 putative and characterized TS are available as a separate compressed folder. This material is available free of charge via the Internet at http://pubs.acs.org.
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ACKNOWLEDGMENT
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The authors are grateful to J. Arens and A. Sacre for assistance with GC/MS instrumentation, high-cell-density cultivations, sesquiterpene purification and fruitful discussions. This work was supported by the Joint Research Unit IATE. GC-MS analyses were performed using the LipPolGreen platform (www.supagro.fr/plantlippol-green). We thank Dr D. W. Christianson (University of Pennsylviana, USA) for generously providing us with a genetical sample of wild-type EIZS.
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