University of Groningen
Nonribosomal peptide synthetases
Zwahlen, Reto Daniel
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Zwahlen, R. D. (2018). Nonribosomal peptide synthetases: Engineering, characterization and biotechnological potential. University of Groningen.
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CHAPTER III
A golden gate based
system for convenient
assembly of chimeric
nonribosomal peptide
synthetases
Reto D. Zwahlen,1 Roel A.L. Bovenberg,2,3 and Arnold J.M. Driessen1,4
1Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands
2Synthetic Biology and Cell Engineering, Groningen
Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands
3DSM Biotechnology Centre, Delft, The Netherlands 4Kluyver Centre for Genomics of Industrial Fermentations,
Abstract
Antimicrobial compounds have been increasingly put into the spotlight of modification and novel discovery approaches, espe-cially since the emergence of various multi-resistant pathogenic strains. These compounds are frequently complex in structure, bearing multi-ring structures and various side branches in their scaffold. This complexity is mainly caused by its nonribosomal peptide production route, guided through nonribosomal peptide synthetases (NRPS) and associated factors, such as MbtH-like pro-teins (MLP). NRPS are large multi-modular enzymes, composed of modules which incorporate a distinct substrate into a growing peptide-compound. Due to its complexity and significant size, en-gineering has always been a delicate task, however site, domain and module directed efforts have led to feasible new NRPS de-signs exhibiting novel activities. On basis of the Nocardia lactam-durans ACVS in combination with a series of L-hydroxyphenyl-glycine (hpg) incorporating modules, we here seek to establish a golden gate based system, for the exchange of NRPS parts in a dedicated manner. Therefore, a small part library was constructed, allowing for the assembly of chimeric NRPS, which were subse-quently co-transformed with their dedicated MLP, expressed in E. coli HM0079 and utilized in an in vitro peptide formation assay. Ultimately all samples were analyzed on LC/MS, and screened for the proposed novel hpg-cysteine-valine (DLD-hpgCV) tripeptide as well as other potentially interesting compounds. The configured system, allowed for the formation of a multitude of hybrid NRPS constructs in a medium throughput manner. However, we could not confirm the production of DLD-hpgCV or any other related compound. Nonetheless, we have successfully demonstrated, how a system can be implemented, which allows for the shuffling, over-expression and subsequent compound analysis of chimeric NRPS or other modular enzymes. It does not only allow for the fast cre-ation of an expandable library of domains, modules and hybrid NRPS, but also for their subsequent characterization in a medium throughput manner.
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Introduction
Nonribosomal peptide synthetases are large and complex multi-modular sys-tems. As key enzymes in the production of important, pharmacologically rel-evant compounds, they gradually moved into the spotlight of combinatorial engineering efforts [1–3]. However, since these multi-modular assembly lines contain a variety of under-investigated parts, previous trials on targeted engi-neering yielded only moderate success. Even though there have been success-ful trials on exchanging sub-domains [4], domains [5] and modules, respec-tively, the resulting chimeric enzymes predominantly function at low activities. In order to enable and explore the full potential of those enzymes, a more systematic approach for the exchange of modules, domains and sub- domains is desired. However, different bottlenecks, intrinsic to NRPS must be taken into account. Certain NRPS characteristics can be predetermined thorough bioinformatics, such as domain and module boundaries, and adenylation do-main specificities based on conserved sequence motifs [6]. Though as for many large proteins, the major bottleneck is represented by the sheer size of the underlying genes, at a size of up to 65 kb [7]. Even the ever- decreasing costs of DNA synthesis, as well as increasingly elaborate synthesis tech-niques, do not allow for the inexpensive construction of comprehensive hybrid NRPS libraries. This is a direct consequence of the number of NRPS which are derived from rather uncharacterized or uncultivated organisms [8], bearing overruling complexities, such as very high GC content (>80 %). Es-sentially, a system is required, which allows for the exchange and incorpo-ration of various numbers of DNA parts of different sizes, in an extendable, convenient and specific manner.
In the era of synthetic biology, different alternative cloning systems have been established, which enable the precise recombination of DNA. In addi-tion to in vivo systems, such as yeast recombinaaddi-tion [9–10], there is a variety of in vitro techniques, which omit the utilization of an intermediate host. All these techniques allow for the recombination of DNA, based on either short homologous sequences or rely on the activity of the unusual type IIS class of restriction enzymes, in combination with a ligase. The latter reaction or golden gate reaction [11], has advantages over homology based methods, as it allows for a one step assembly. Due to the activity of type IIS enzymes, which cleave outside of their recognition sequence, a considerable number of dedicated overhangs, which ultimately allow for the utilization of a high number of potential fragments. Furthermore, no additional oligonucleotides are required for this reaction, giving it an important advantage in terms of ease to handle as well as time consumption.
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Materials and methods
A system based on the golden gate principal, adjusted for the incorpora-tion of differently sized DNA fragments, would not only allow for the shuf-fling of larger genes, but may enable the reprogramming of enzymes rele-vant for the production of bioactive compounds. For instance, the alteration of natural compounds routes such as that of the NRPS derived compounds penicillin or cephalosporine has a long history [12]. To overcome resistance, the production of a variety of novel compounds has been realized, includ-ing cephaloglycin or cephalexin from cephalosporin [13–15], and ampicillin or amoxicillin from penicillin [16]. Unfortunately, all of these compounds are still produced in semi-synthetic manner, creating vast amounts of economi-cally and ecologieconomi-cally non-beneficial side-products.
The following research portrays an attempt at creating a novel NRPS which catalyzes the formation of the tripeptide hydroxyphenyl-L-cysteinyl- D-valine (DLD-hpgCV), a precursor to amoxicillin. Therefore, we make use of the Nocardia lactamdurans L-δ-(α-aminoadipyl)-L-cysteinyl-D-valine synthe-tase (ACVS), a NRPS responsible for the production of the L-α-aminoadipyl- L-cysteinyl-D-valine (LLD-ACV) penicillin precursor. By exchanging the first L-α-aminoadipic acid (aaa) activating module of the three modular NRPS, with either L-phenylglycine (pg) or L-4-hydroxyphenylglycine (hpg) specific parts, we intend to shift the native LLD-ACV specificity towards the desired DLD-hpgCV. The parts required for the first module, a series of hpg activat-ing modules, have been identified in chapter I and will serve as a fundament for the creation of a part library, intended for the assembly of DLD-hpgCV specific chimeric NRPS. This study describes attempt to generate such a chi-meric NRPS using the golden gate system in a medium throughput manner.
Materials and methods
Bacterial strains, plasmids and culture conditions
Cloning was performed using E. coli DH5α. Selection was with 25 µg/ml zeo-cin for pBAD destination plasmids, 100 µg/ml ampicillin for pMAL-c5x donor plasmids and 15 µg/ml for pACYCtac-MLP plasmids, respectively. E. coli BL21 (DE3) strain was used for expression of domains and modules. All cultures were grown using 2xPY (bacto-tryptone 15 g/l, yeast extract 10 g/l, NaCl 10 g/l, pH 7.0) at 37 °C and 200 rpm (innova shaker).
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Domain prediction and separation
Domain borders prediction on the sequences of all 12 putative hpg activat-ing modules along with the ACV synthetase were done usactivat-ing HMMMR (ref). The Modules, consisting of the domain set A-T-(E)-C were divided into three
constructs, A, T(E) and C respectively. Inter-domain linkers were determined by up- and downstream predicted domains and equally placed on the corre-sponding constructs. Constructs were either amplified using gDNA obtained from their corresponding strains (supplementary Table 1) or ordered as syn-thetic genes (Geneart). All donor constructs were cloned into pMAL-c5x vector at HindIII, EcoRI or EcoRV according to compatibility. Required BsrDI restriction sites were introduced as part of primer sequences (Supplemen-tary table 1). Correctness of the constructs was confirmed by means of re-striction analysis and subsequent DNA sequencing (MacroGen). The accep-tor plasmid pBAD-Nl-ACVS, represents a truncated version of the full length pBAD-Nl-ACVS-6×his plasmid, containing a gateway cloned version of the
Nocardia lactamdurans ACV synthetase and was kindly provided by DSM
Bio-technology, Delft.
Golden Gate reaction
Donor and destination plasmids were added in an equimolar ratio to a total volume of 4 µl along with 1 µl T4 DNA ligase (HC 5 U/µl), 1 µl T4 DNA ligase buffer 10x, 0.5 µl BsrDI (5 U/µl) and adjusted to 10 µl using MilliQ water. All enzymes were obtained from Thermo scientific. The reactions were in-cubated in a Thermocycler PCR machine (BioRad) using the program: 5 min 37 °C; (2 min 37 °C; 5 min 16 °C) × 50; 5 min 37 °C; 20 min 80 °C; ∞ 4 °C. Re-actions were transformed and correct clones confirmed using restriction analysis. Correct constructs were transformed together with their corre-sponding pACYCtac-MbtH variant into E. coli HM0079.
Expression and purification of hybrid NRPS and hpg modules
Cultures were grown to an OD600 of 0.6, transferred to 18 °C and 200 rpm for 1 h and subsequently induced using 0.5 mM IPTG and 0.2 % L-arabinose. Har-vest 18 h after induction was done by spinning at 3500 g for 15 minutes. After resuspension in lysis buffer (HEPES 50 mM pH 7.0, NaCl 300 mM, DTT 2 mM, cOmpleteTM EDTA free protease inhibitor; Roche No. 04693159001), cells
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were disrupted using sonication (6 s/15 s; on/off, 50×, 10 µm amplitude) and cell-free lysate obtained by centrifugation at 4 °C, 13000 g, 15 minutes. Puri-fied enzyme was extracted by means of Ni-NTA bead (Qiagen) supported his-tag affinity purification using gravity flow. Wash steps were performed using wash buffer (HEPES 50 mM pH7.0, NaCl 300 mM, imidazole 20 mM) and a one-step elution using elution buffer (HEPES 50 mM pH7.0, NaCl 300 mM, imidazole 250 mM). Imidazole was removed, while simultaneously concen-trating the sample with Amicon-100 spin filters (Amicon). Final concentra-tion was determined using A280 (OD 1 = 1 mg/ml).
In vitro product formation assay
Isolated enzymes were subjected to in vitro assays, in order to determine product formation properties. Assay conditions include HEPES 50 mM pH 7.0, NaCl 300 mM, ATP 5 mM pH 7.0, CoA 100 µM, sfp (NEB) 0.2 µM, L- cysteine 2 mM, L-valine 2 mM, MgCl2 5 mM, DTT 2 mM, hybrid NRPS 0.3–0.5 µM and either L- or D-hydroxyphenylglycine (L/D-hpg) 5 mM. Negative controls in-cluded reactions omitting hybrid NRPS or L/D-hpg. Full length Nocardia
lact-amdurans ACVS served as positive control, using 5 mM L-α-aminoadipic acid
instead of L/D-hpg. Reactions were run at 30 °C and sampling took place 0, 60, 120 and 240 minutes after initiation. 0.1 M NaOH was used to stop the reactions. Samples were subsequently reduced with 10 mM DTT at 4 °C for 16 h and stored upon analysis at −80 °C.
Liquid chromatographic and masspectrometric analysis (LC/MS)
Samples (50 µl) obtained from an in vitro reaction, was subjected to LC/MS analysis. Two technical replicates were run per sample at 5 µl each. Analysis was performed using a LC/MS Orbitrap (Thermo Scientific) in combination with a RP-C18 column (Shimadzu Shim pack XR-ODS 2.2; 3.0×75 mm). Scan range was set at 80–1600 M/Z in positive Ion (4.2 kV spray, 87.5 V capillary and 120 V of tube lens) mode, with capillary temperature set at 325 °C. A gradient program with miliQ water (A), Acetonitrile (B) and 2 % Formic acid (C) was run; 0 min; A 90 %, B 5 %, C 5 %; 4 min, A 90 %, B 5 %, C 5 %; 13 min, A 0 %, B 95 %, C 5 %; 16 min A 0 %, B 95 %, C 5 %; 16 min, A 90 %, B 5 %, C 5 %; 21 min A 90 %, B 5 %, C 5 % at a flow rate of 0.3 ml min-1. Standards of LLD-ACV; LLD- and DLD-hpgCV obtained at Bachem were used to identify peaks according to retention time and accurate mass.
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Results
To establish a domain-targeted swapping mechanism for the reshuffling of NRPS, a series of different Initiation modules was used in combination with two ACVS derived di-modules. The specificities of the chosen enzymes was revealed in previous studies (chapter II) and the resulting chimeric NRPS are projected to produce hpgCV as well as potentially pgCV tripeptides, the ul-timate precursor molecules to the production of amoxicillin and ampicillin, respectively. Therefore, we adapted a plasmid based system, analogue to a golden gate reaction, which is expendable and allows for the dedicated ex-change of previously determined NRPS domains (Figure 1). Domain bound-aries were determined using a combinatorial approach focusing on the uti-lization of the NCBI conserved domain database (CDD). Subsequently, all obtained sequences were screened for the presence of type IIS restriction enzyme sites, ultimately leading to the choice of BsrDI, the least abundantly occurring site. With respect to this choice, two plasmids, a donor pMAL-c5x and an acceptor, pBAD, were developed and the remaining BsrDI sites re-moved (Supplementary table 2). Following the BsrDI site removal, the 33 do-nor plasmids and the acceptor plasmid were cloned, using either PCR ampli-fication or synthetic DNA fragments (Supplementary table 1). In addition to the golden gate relevant plasmids, the 6 corresponding MbtH-like proteins (MLP) were cloned, completing the list of all required parts for the subse-quent hybrid NRPS assembly.
Golden gate reaction optimization and chimeric NRPS assembly
The obtained donor plasmids in combination with the acceptor vector, allow for the assembly of a maximum of 40 novel chimeric NRPS using either three or four NRPS parts, respectively. To achieve an efficient and specific reaction, the conditions were optimized, using the ACVS as a model. Taking ACVS; A, T and C constructs and the destination pBAD-CV plasmid, conditions as de-scribed in [11] were tested initially. The initial reaction volume of 30 µl, lead to an overall efficiency of >50 % of correctly assembled constructs. Further adjustments, increasing concentration of T4 DNA ligase and BsrDI, did not lead to a consistent nor significant increase of correct constructs or total number of transformants (Data not shown). However, a comparison of reac-tion volumes of 10 µl, 20 µl and 30 µl, lead to an improvement of correct con-structs to >80 %, using a final volume of 10 µl (Figure 2). Using this as a pro-tocol, all remaining golden gate reactions were carried out. Thereby, 34 out
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of 40 possible hybrid constructs could be obtained. To exclude the possibility of sequence anomalies due to BsrDI or ligase activity, constructs were se-quenced at the hypothetical ligation sites. No alterations were determined
E A T C A T C A T C A T E Te A E T E A T Te A T C T C C 1 2 3 4 +BsrDI +T4 lig HPG ACVS Control Donor Acceptor A D A B B C B C C D C D GCAATGCCNN CGTTACGGNN
A B GCAATGAGNNCGTTACTCNN C GCAATGCANNCGTTACGTNN DGCAATGTTNNCGTTACAANN
BsrDI C E A T Te A T C E A T Te A T C E A T Te A T C E A T Te A T C A A A A C T T E T E T C C I II III IV A B C M1 M2 M3
Figure 1 — Experimental setup, part library and resulting hybrid NRPS.
(A) Domain borders of hpg modules and ACVS were determined and transferred to the
re-spective vectors. (B) The created donor (1–3) and acceptor vectors (4) contain BsrDI restric-tion sites which allow for the dedicated joining of generated overhangs (A–D). The reacrestric-tions will ultimately lead to the production of one out of four dedicated hybrids (C).
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and the constructs were subsequently co-transformed with their respective MLP into E. coli HM0079.
Expression, purification and in vitro assessment of hybrid NRPS
All 34 constructs were co-transformed with their respective MLP and se-quentially expressed using screening volumes of 100 ml. For this set of constructs, 23 hybrids showed traceable amounts of expression following SDS-PAGE analysis in combination with coomassie brilliant blue staining (Figure 3). Constructs containing Hpg13 or Hpg26 did not reveal any expres-sion. A mixed pattern, allowing for the expression of certain hybrid variants is seen for Hpg11, 21, 24 and 27. The observed levels however, remain con-sistently beneath 1 mg l-1 culture, requiring the utilization of cultures at a 1000 ml volume for the following in vitro analysis. This resulted in a final
3000 60008000 10000 1000 10 uL 20 uL 30 uL 9/10 8/10 7/10 bp
Figure 2 — Optimization of golden gate assembly.
The reaction conditions of the golden gate reactions were optimized, based on the original setup. A decrease in the reaction volume to 10 µl had the most beneficial impact, though no improvements were achieved through the variation of BsrDI, T4 ligase or buffer conditions.
. + VEG 8 22 I ACVS 404 - VEG 8 22 II 22 III 22 IV 22 I 22 II 22 III 22 IV 22 I + VEG 8 conc E I E II CFL
Figure 3 — Sample of NRPS hybrid yield.
(A) Expression of Hpg22 hybrids (I–IV) with and without the MLP VEG8. Hybrids including
epimerization domains appear at 410 kD (I + II) and without E domain (III + IV) at 370 kD. (B) Expression, purification and enrichment of hybrid 22.I based on a 1000 ml culture.
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concentration of up to 0.5 mg/ml NRPS concentrate at a purity of >50 %. The concentrated chimeric NRPS were sequentially used in an in vitro
prod-uct formation assay and the samples were ultimately analyzed using LC/MS. Table 1 — overview of NRPS assembly and assessment.
Module A 1M1 parts (Hpg)T-E 2 C 3 M2 parts (ACVS) Assembly Expression ProductT C 11 I x - x x - - - -II x - - x x ü ü -III x x x - - ü - -IV x x - - x ü - -13 I x - x x - - - -II x - - x x ü - -III x x x - - ü - -IV x x - - x - - -21 I x - x x - ü - -II x - - x x ü - -III x x x - - ü ü -IV x x - - x ü ü -22 I x - x x - ü ü -II x - - x x ü ü -III x x x - - ü ü -IV x x - - x ü ü -23 I x - x x - ü ü -II x - - x x ü ü -III x x x - - ü ü -IV x x - - x ü ü -24 I x - x x - ü ü -II x - - x x ü - -III x x x - - ü - -IV x x - - x ü - -26 I x - x x - - - -II x - - x x ü - -III x x x - - ü - -IV x x - - x - - -27 I x - x x - - - -II x - - x x ü ü -III x x x - - ü ü -IV x x - - x ü ü -1* I x - x x - ü ü -II x - - x x ü ü -III x x x - - ü ü -IV x x - - x ü ü -4** III xx -- -x -- -x üü üü - -Tcp9** III xx -- x- -- -x üü üü - -ACVS*** x - - x x ü ü
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The resulting chromatographic profiles were screened for a series of di- andtripeptide compounds, including the desired hpgCV. However, none of the analyzed reactions, independent of substrate stereochemistry revealed any traces of LLD- nor DLD-hpgCV formation. In fact, no tripeptidic compound was found in any of the chimeric NRPS setups. However, the dipeptide con-taining cysteine and valine, CV, was formed in significant amounts, in all re-actions. Furthermore, the reconstituted ACVS exhibits a strong production of LLD-ACV in a linear fashion over the full experimental course of 4 h in addition to minor amounts of a cysteine-valine dipeptide (Data not shown).
Discussion
To establish a tool for the construction of large, multi-modular, chimeric NRPS, we initiated the development of a golden gate based method, allowing for a virtually scar-free synthesis of novel NRPS. As a fundament, we used a variety of hydroxyphenylglycine activating modules including their associated MLPs (chapter II) alongside the two C-terminal modules of the Nocardia
lactamdu-rans ACVS. Due to the high abundance of the classic type IIS restriction site
BsaI within the chosen genes, a series of alternative enzymes were evaluated and BsrDI, a 2-N overhang generating RE, was used subsequently for the pro-jected Donor and Acceptor plasmids. The proposed system is thus expandable for the utilization of novel parts within the boundaries of the dedicated nu-cleotide overhangs, intrinsic to the plasmid system. The determined and gen-erated NRPS parts were subsequently subjected to shuffling reactions and the obtained chimeric NRPS constructs, co-transformed with their respective MLP, expressed and screened for the production of hpgCV and related com-pounds using an in vitro assay in combination with a LC/MS analysis.
The first step taken involved the assembly of the NRPS, was the imple-mentation and refinement of the original golden gate method [11]. T4 ligase, BsrDI and buffer adjustments did not improve on the conducted reactions. In contrast, decreasing the reaction volume, without further variation in component concentration significantly increased the reactions efficiency with respect to the share of correctly assembled constructs. A further bot-tleneck was elucidated concerning the short 212 bp ACVS T domain, which appeared to be a shared factor in a series of lower efficiency reactions (Data not shown). However, by simply increasing the partial molarity of this com-ponent, we ultimately managed to obtain almost all desired constructs. The subsequent expression trials revealed a very low amounts of protein, though exclusively in soluble form (Supplementary data). Increasing the overall
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culture volume was sufficient to bypass this problem for over half of the constructs. The remaining constructs did not show any traceable amounts of chimeric NRPS. Due to the radical reshuffling of parts originated in a variety of organisms, as well as their partial codon optimization, this appears to be a logical consequence, since translational pauses and tRNA availability play a crucial role in NRPS expression [17–19]. Furthermore, an expression pro-file analysis showed overall equally low expression probability values across all hybrid constructs, which is also reflected in the overall low expression levels of the hybrids (Data not shown). The subsequent in vitro analysis of the isolated and partially purified protein revealed a series of noteworthy observations. First of all, it appears, that the assay conditions allow for LLD-ACV formation through the LLD-ACVS. This supports that the NRPS is in its active
holo state, thus the conditions should be applicable for the chimeric NRPS
as well. However, the intended DLD-hpgCV tripeptide was not detected in any of the analyzed samples, though we did detect significant amounts of a cysteine-valine di-peptide. This is also seen to a lesser degree with the ACVS and has been reported in previous studies (DSM). This suggests, that the novel NRPS must be present in a minimal functionally folded state, however not allowing for the incorporation of hpg. Due to the complexity of the en-zyme it is difficult to pinpoint what exactly prohibits the chimeric NRPS from properly performing, only additional experiments focused on the functional characterization could ultimately unravel this information.
Conclusion and outlook
In this study, we propose a combinatorial approach for the shuffling of NRPS including the downstream in vitro characterization and mass spectromet-ric analysis in a streamlined fashion. Despite a lack of anticipated activity in the constructed NRPS, we still show how the adapted golden gate method can be used for the efficient shuffling of NRPS parts. If paired with the lat-est information concerning structural and dynamical properties of NRPS as-sembly lines, this system could be further improved by determining additional non-conserved regions, thereby increasing the number of potential shuffling sites. Furthermore, in an attempt to create functional chimeric enzymes, rel-evant NRPS sub-parts [4] could be considered for the creation of novel parts, rather than domains or full modules, representing a more gentle and less inva-sive approach that likely maintains subdomain interactions. Overall, given the correct adjustments to the system, alongside the decreasing costs of synthetic genes, this approach could serve as a scaffold for the development of new
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