Directed evolution of a cyclodipeptide synthase with new activities via label-free mass spectrometric screening

Directed evolution is a powerful approach to engineer enzymes via iterative creation and screening of variant libraries. However, assay development for high-throughput mutant screening remains challenging, particularly for new catalytic activities. Mass spectrometry (MS) analysis is label-free and well suited for untargeted discovery of new enzyme products but is traditionally limited by slow speed. Here we report an automated workflow for directed evolution of new enzymatic activities via high-throughput library creation and label-free MS screening. For a proof of concept, we chose to engineer a cyclodipeptide synthase (CDPS) that synthesizes diketopiperazine (DKP) compounds with therapeutic potential. In recombinant Escherichia coli, site-saturation mutagenesis (SSM) and error-prone PCR (epPCR) libraries expressing CDPS mutants were automatically created and cultivated on an integrated work cell. Culture supernatants were then robotically processed for matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) MS analysis at a rate of 5 s per sample. The resulting mass spectral data were processed via custom computational algorithms, which performed a multivariant analysis of 108 theoretical mass-to-charge (m/z) values of 190 possible DKP molecules within a mass window of 115–373 Da. An F186L CDPS mutant was isolated to produce cyclo(l-Phe–l-Val), which is undetectable in the product profile of the wild-type enzyme. This robotic, label-free MS screening approach may be generally applicable to engineering other enzymes with new activities in high throughput.

A robotic workflow for directed evolution of new enzymatic activities via high-throughput library creation and label-free MS screening.

Mimicking random mutagenesis and natural selection in the laboratory,^1–3 directed enzyme evolution is valuable in improving a variety of properties of biocatalysts (i.e., catalytic activity, stability, substrate specificity, and enantioselectivity).^4–6 While it is straightforward to generate thousands of enzyme variants using established methods such as error-prone PCR (epPCR), site-saturation mutagenesis (SSM), and DNA shuffling, phenotypic screening remains a major bottleneck due to the substrate and product versatility of biocatalysts.^7,8 Optical assays are widely applied in high-throughput screening (HTS) during directed evolution, often requiring a surrogate substrate or a coupling assay with a colorimetric or fluorogenic readout.^9,10 However, it is very challenging to apply such approaches to discover new catalytic activities. On the other hand, mass spectrometry (MS) is label-free and suitable for untargeted molecular profiling.¹¹ The high sensitivity and chemical resolution of MS further highlight its potential in discovering new products, because new catalytic activities are often weak when first emerging from enzyme promiscuity.¹²We and others have recently developed a range of high-throughput MS screening methods for directed protein evolution.^13–16 However, the label-free advantage of MS has not been fully demonstrated in engineering new enzymatic activities, possibly due to the difficulties in untargeted MS screening. Particularly, it requires careful standardization and optimization of sample preparation, MS acquisition, and data processing, which are necessary to minimize experimental noise and spot the weak signals of a new product. On the other hand, biofoundries provide an emerging infrastructure to assist the design–build–test–learn (DBTL) cycles in biological engineering via robotic standardization and parallelization.^17–19 Using an integrated biofoundry, here we report a workflow for unlabeled MS screening of recombinant libraries to rapidly isolate enzyme mutants that catalyze the formation of new products. This new workflow extends our previous matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) MS-based screening approach from agar colonies¹⁶ to liquid cultures in industry-standard microplates for better uniformity. Unlike colony biomass, liquid culture media often contain high concentrations of nonvolatile components, which interfere with MALDI matrix crystallization and cause severe ion suppression during MS analysis. Therefore, sample preparation steps, such as liquid–liquid extraction and solid-phase extraction, need to be incorporated and automated.Cyclodipeptide synthases (CDPSs) are a family of enzymes that catalyze the formation of a diketopiperazine (DKP) from two aminoacyl-tRNA substrates (aa-tRNAs), which is an important pharmacophore for modern drug development^20–22 (Fig. 1A and S1^†). To produce new DKP derivatives for therapeutic and industrial applications, the catalytic mechanism of CDPSs has been studied^23,24 to guide engineering. AlbC (239 aa) is the first identified CDPS protein, taking phenylalanyl-tRNA^Phe (Phe-tRNA^Phe) and leucyl-tRNA^Leu (Leu-tRNA^Leu) as substrates to synthesize cyclo(l-Phe–l-Leu) (cFL) in its native host Streptomyces noursei.^25,26 Recombinant E. coli with AlbC overexpression is able to produce more cyclodipeptide derivatives in addition to cFL.²⁷ To date, eight CDPSs have been structurally characterized,^26,28–32 and mutagenesis studies reveal that the residues within the P1 and P2 catalytic pockets are the key determinants of substrate specificity. However, the detailed mechanism of substrate recognition and catalysis is still elusive, and CDPSs are recognized as recalcitrant targets for rational engineering.^33,34 To our knowledge, large-scale screening of CDPS variants has not been reported, possibly due to the lack of applicable HTS assays.Open in a separate windowFig. 1(A) Scheme of diketopiperazine (DKP) biosynthesis catalyzed by cyclodipeptide synthase (CDPS). (B) Workflow of the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS)-based high-throughput screening method for directed evolution of AlbC protein in E. coli.Here we develop and apply untargeted MS screening on a biofoundry for directed evolution of AlbC mutants that form new CDP products. The workflow consists of strain library creation, colony picking, microtiter cultivation, organic solvent extraction, transfer of the sample to a MALDI target and acquisition of mass spectra, followed by data processing and visualization (Fig. 1B). For library creation, AlbC variants were generated by either SSM or epPCR approaches and expressed from a pET28a plasmid under control of a T7 promoter. Upon genetic transformation of E. coli Rosetta(DE3), individual clones were transferred by a colony picker into microtiter plates. Subsequent cultivation, inducible production by addition of isopropyl β-d-1-thiogalactopyranoside (IPTG), ethyl acetate extraction, and MS sample preparation were performed using an integrated robotic workcell in the Shenzhen Synthetic Biology Infrastructure. This workcell integrates common instruments for synthetic biology, including a liquid-handling station, shaking incubators, centrifuge, plate reader, and so on. Organic extracts of the liquid cultures were spotted onto a MALDI target and overlaid with 4-CHCA matrix solution. MS targets were then manually transferred to a stand-alone MALDI mass spectrometer in this study, although a robotic configuration has been previously reported to automate this step.³⁵We first applied the workflow to analyze the wild-type (WT) AlbC-expressing strain, Rosetta(DE3)/pET28-AlbC(WT). After cell cultivation, inducible production, and sample preparation, MS analysis was performed using an Autoflex MALDI-ToF mass spectrometer in the reflector positive ion mode (Fig. 1). In the resulting MALDI mass spectra, we observed [M + H]⁺ ion peaks with m/z values corresponding to cFL (m/z 261.17), cFY (m/z 311.15), cFM (m/z 279.12), cYL (m/z 277.16), cFF(cYM) (m/z 295.15), cLL (m/z 227.16) and cMM (m/z 263.12), all of which were absent from the control strain Rosetta(DE3)/pET28 (Fig. S2^†). Production of these CDP molecules was further confirmed by examining tandem MS results using LC-MS in the multiple reaction monitoring (MRM) mode (Table S3^†), where ion fragmentation patterns were consistent with the literature.^26,27 Moreover, the observed production profile was also consistent with previous studies.^26,27 These results validated our workflow to detect CDP products from microplate cultures of AlbC-expressing E. coli using MALDI-ToF MS.To engineer AlbC mutants that synthesize new products, we first focused on the key residues in the substrate-binding pockets. AlbC employs a “ping-pong” catalytic mechanism: the initial step transfers the aminoacyl moiety of the first aa-tRNA onto a conserved serine, leading to the formation of an aminoacyl enzyme intermediate; then, the aminoacyl enzyme reacts with the aminoacyl moiety of the second aa-tRNA to form a dipeptidyl enzyme.³² Previous biochemical experiments demonstrated that two binding pockets P1 and P2 of AlbC accommodate the aminoacyl moieties of the two aa-tRNA during the biosynthesis²⁴ (Fig. 2). Only a limited number of mutations were examined on P1 and P2 residues,^25,26 possibly due to the lack of HTS assays for large-scale analysis. Instead, we applied our workflow to create and screen the SSM libraries of select residues, including 10 residues in the binding pockets (L33, V65, L119, L185, L200, M152, M159, I204, T206 and P207) and 4 residues (R99, R101, R102 and D205) outside the pockets which have interactions of the tRNA moiety reported in the literature (Fig. 2A).Open in a separate windowFig. 2(A) Structural analysis of the AlbC (PDB ID, 3OQV). Enlarged view of the catalytically active pocket. The possible catalytic residues are shown in green (pocket-1) and orange (pocket-2) and basic residues on helix α4 are labelled in cyan. (B) Heatmap of the relative activity change in the cFL production levels of mutation of 14 amino acid residues in AlbC. WT residues are labeled with a black dashed rectangle. Dark blue boxes indicate that a specific mutant was not covered in randomly picked clones (activity assigned as −1).For SSM libraries, we adopted the “22c-trick” strategy to design degenerative primers, and 94 library clones were randomly picked for each residue library to reach a >98.6% probability of full library coverage of the 20 canonical amino acids.³⁶ In addition to library variants, the WT and control strains were also included in the same 96-well plate. Overall, the abovementioned workflow (Fig. 1B) takes approximately 5 s for sample preparation and MS analysis for each mutant culture. Tentative ion peaks of cyclodipeptides were assigned based on theoretical m/z values (ESI Section 1^†) using the MetaboAnalyst webserver.³⁷ For each ion peak, one-way analysis of variance (ANOVA) was used to evaluate statistical differences between the mean peak areas of a library variant and those of the control group. AlbC gene mutations were revealed by Sanger sequencing for all 14 SSM library members.To visualize the MALDI-ToF MS screening results, a heatmap was generated based on the cFL production of library members relative to that of WT (Fig. 2B), and we observed a general consistency between our results and literature data (Fig. 3A), respectively, which was similar to a previous study.²⁶ Also, mutagenesis of basic residues (R99, R101 and R102) outside the pockets led to the decline of cFL production in the mutants, although R101 was more tolerant to mutations than R99 and R102. Furthermore, the substitution of D205 in the β6-α8 loop to alanine enhanced AlbC catalytic activity as observed previously.²⁵ On the other hand, some previously unnoticed phenomena were observed. For example, in the D205 SSM library, relative ion intensities of cFL (m/z 261.17) in many mutants are higher than that of WT (Fig. S3^†). The top 3 mutants, D205M, D205K and D205R, were subsequently analyzed by LC-MS/MS. The results not only confirmed augmented synthesis of cFL, but also revealed increased production of other leucine-containing products including cYL, cLL and cML (Fig. 3B). Also interestingly, the discovery that most mutations of the T206 residue in the P2 pocket abolished the biosynthesis of the main product cFL was unprecedented (Fig. S3 and ESI Section 2^†). When examining MALDI mass spectra in detail, we noticed that although the T206F mutation greatly reduced cFL production, cFF and cFY products were largely not affected. Replacing the small threonine side chain with a bulky aromatic phenylalanine side chain could affect the activity of WT, because the phenyl ring structure increased the steric hindrance of T206F, thus impairing binding with its substrate and causing loss of its enzyme activity for cFL (Fig. 5A). These results suggested that T206 is also a key residue for the selectivity of the second Leu-tRNA substrate binding in the pocket. Unfortunately, we did not observe any new CDP molecules produced from the 14 SSM library mutants.Impact of AlbC mutations on the main product cFL

Synthesis of polyethers containing cyclodipeptide moiety in the main chain

The dichlorodihydropyrazines derived from 2,5‐diketopiperazine were treated with diols in a basic condition to afford polyethers containing cyclodipeptide moiety in the main chain, which were found to be degraded into amino acid components in an acidic condition. © 2001 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 39: 927–933, 2001     

Solid phase peptide synthesis: Fluoride ion release of peptide from the resin

A linkage to anchor the growing peptide chain has been designed in order to allow swift release of the peptide product by Bu₄N+F. Preparation of the linker (4) is described.     

Comparative investigation of the cleavage step in the synthesis of model peptide resins: implications for Nalpha-9-fluorenylmethyloxycarbonyl-solid phase peptide synthesis

Based on our studies of the stability of model peptide-resin linkage in acid media, we previously proposed a rule for resin selection and a final cleavage protocol applicable to the Nalpha-tert-butyloxycarbonyl (Boc)-peptide synthesis strategy. We found that incorrect choices resulted in decreases in the final synthesis yield, which is highly dependent on the peptide sequence, of as high as 30%. The present paper continues along this line of research but examines the Nalpha-9-fluorenylmethyloxycarbonyl (Fmoc)-synthesis strategy. The vasoactive peptide angiotensin II (AII, DRVYIHPF) and its [Gly8]-AII analogue were selected as model peptide resins. Variations in parameters such as the type of spacer group (linker) between the peptide backbone and the resin, as well as in the final acid cleavage protocol, were evaluated. The same methodology employed for the Boc strategy was used in order to establish rules for selection of the most appropriate linker-resin conjugate or of the peptide cleavage method, depending on the sequence to be assembled. The results obtained after treatment with four cleavage solutions and with four types of linker groups indicate that, irrespective of the circumstance, it is not possible to achieve complete removal of the peptide chains from the resin. Moreover, the Phe-attaching peptide at the C-terminal yielded far less cleavage (50-60%) than that observed with the Gly-bearing sequences at the same position (70-90%). Lastly, the fastest cleavage occurred with reagent K acid treatment and when the peptide was attached to the Wang resin.     

A new cyclodipeptide from the cultures of Geotrichum candidum

A new cyclodipeptide,cyclo(phenylalanyl-N-methyltyrosyl)1,was isolated from the mycelial solid cultures of Geotrichum candidum.Its structure was elucidated by spectroscopic methods.The antifungal activity of the new compound against Peronophythora litchii was assessed by microplate Alamar blue assay(MABA).     

Nonribosomal cyclic peptides: specific markers of fungal infections

Some cyclic peptides and depsipeptides are synthesized in microorganisms by large multienzymes called nonribosomal peptide synthetases. The structures of peptide products originating in this way are complex and diverse and are microorganism-specific. This work proposes the use of fungal cyclic peptides and depsipeptides as extremely specific markers of fungal infections. Since a reliable molecular tool for diagnosing fungal infections at an early stage is still missing, we present mass spectrometry as a new, modern, broadband (with respect to fungal strain) and specific tool for clinical mycologists. More than 40 different fungal species can be rapidly characterized according to specific families of cyclic peptides, and in some cases, a particular fungal strain can be identified on the basis of its cyclopeptide profile. This paper is also aimed at initiating discussion on the biological role of these secondary metabolites, especially of those synthesized by medically important strains. Proven cytotoxic, anti-inflammatory or immunosuppressive activities of some cyclic peptides indicate that these molecules may contribute to the synergistic array of fungal virulence factors and support microbial invasion during fungal infection. In addition to an overview on recent mass spectrometric protocols for cyclic peptide sequencing, the structures of new peptides from Paecilomyces and Pseudallescheria are presented.     

Enzyme-catalyzed peptide synthesis in ice

A new method for increasing product yield in kinetically controlled enzymatic peptide synthesis catalyzed by serine and cysteine proteases is proposed - freezing the reaction mixture. The use of the method in preparative synthesis has been demonstrated.     

Use of enzymes in peptide synthesis

Recent experiments in several laboratories have emphasized the benefits of proteolytic enzymes as effective catalysts for the formation of peptide bonds for synthesis and semisynthesis. This review summarizes successful applications in both stepwise synthesis for small peptides and fragment condensation to produce large polypeptides and proteins.     

Protection of histidine in peptide synthesis: A Reassessment of the trityl group

The trityl (Trt) group is ideally suited for the side-chain protection of His in peptide syntheses, in combination with 9-fluorenylmethyloxycarbonyl (Fmoc) in N^α- and protecting groups cleavable by mild acidolysis in other positions of the peptide. 2,4,5-trichlorophenyl (Tcp)- and pentafluorophenyl (Pfp)-esters of Fmoc-His(Trt)-OH and Trt-His(Trt)-OH are strongly activated, but stable compounds. N^α-Trt is selectively removable in the presence of N^Im-Trt.     

Diastereoselectivity in the solid-phase synthesis of peptide heterocycle hybrids

Sixteen compounds containing the bicyclic moiety (3,8,10-trisubstituted 2,9-dioxo-5-thia-1,8-diazabicyclo[4.4.0]decane) were produced via solid-phase synthesis. Differing substitution at the 3- and 10-positions was used. These were analyzed using 2-D NMR techniques (ROESY) to determine the stereoselectivity of ring formation in the core heterocycle. Conformational analysis of the proposed transition state structure using Sybyl 6.8^® was used to rationalize the stereochemical outcome of ring formation.     

Reaction mechanisms in peptide synthesis. Part 2. Tautomerism of the peptide bond

Summary We had concluded in previous work that ring opening of a 2-alkyl-5(4H)-oxazolone by water or ammonia leads to transient high-energy imidol intermediates which instantly tautomerize to the native amides. Using the MOPAC molecular orbital program, detailed geometric and energetic characteristics of the tautomerism of a peptide bond have been determined on the AM1 level. The results demonstrate that tautomerism of a peptide bond comprises a three-stage process involving three successive transition states and a bimolecular mechanism: (i) EZ peptide bond isomerization followed by dimerization, (ii) concerted double-hydrogen exchange leading to an -hydroxyimine (imidic acid) followed by splitting of the dimer, and (iii) ZE N-methylimine inversion. While pathway (iiiiii) is predicted as a feasible route terminating in the formation of a peptide bond, the inverse route (iiiiii) is excluded as a possible initial step in the generation of a 5(4H)-oxazolone intermediate.     

Microwave heating in solid-phase peptide synthesis

The highly refined organic chemistry in solid-phase synthesis has made it the method of choice not only to assemble peptides but also small proteins - mainly on a laboratory scale but increasingly also on an industrial scale. While conductive heating occasionally has been applied to peptide synthesis, precise microwave irradiation to heat the reaction mixture during coupling and N(α)-deprotection has become increasingly popular. It has often provided dramatic reductions in synthesis times, accompanied by an increase in the crude peptide purity. Microwave heating has been proven especially relevant for sequences which might form β-sheet type structures and for sterically difficult couplings. The beneficial effect of microwave heating appears so far to be due to the precise nature of this type of heating, rather than a peptide-specific microwave effect. However, microwave heating as such is not a panacea for all difficulties in peptide syntheses and the conditions may need to be adjusted for the incorporation of Cys, His and Asp in peptides, and for the synthesis of, for example, phosphopeptides, glycopeptides, and N-methylated peptides. Here we provide a comprehensive overview of the advances in microwave heating for peptide synthesis, with a focus on systematic studies and general protocols, as well as important applications. The assembly of β-peptides, peptoids and pseudopeptides are also evaluated in this critical review (254 references).     

多肽合成中几种形成二硫键方法的介绍

综述了多肽合成中几种形成二硫键的方法。介绍了经典的空气氧化法、铁氰化钾氧化法和碘氧化法及近年来发展起来的二甲亚砜氧化法、三氟乙酸铊氧化法和氯硅烷-亚砜氧化法等方法的特点,同时还介绍了形成两对或多对二硫键的策略。     

Incorporation of peptide bonds in the synthesis of polyazamacrocyclic complexes

Summary A new series of 15- and 16-membered dioxotetraamine macrocyclic complexes, [ML¹X₂] and [ML²X₂] (M = Mn^II, Co^II, Ni^II, Cu^II or Zn^II; X = Cl or NO₃), respectively, were prepared by reacting o-aminobenzoic acid, diaminoethane or 1,3-diaminopropane with 2,4-pentanedione in the presence of metal ions. The complexes were identified by analytical data, i.r., ¹H-n.m.r., e.p.r. and u.v.-vis. spectroscopies, and by conductivity and magnetic moment measurements. They exhibit an octahedral geometry leaving the peptide oxygen uncoordinated. They are air stable and insoluble in water and most organic solvents, but soluble in DMSO and DMF. The low conductivity suggests that the complexes are non-ionic.