Co-reporter:Ingy I. Abdallah, Magdalena Czepnik, Ronald van Merkerk, and Wim J. Quax
Journal of Natural Products 2016 Volume 79(Issue 10) pp:2455-2463
Publication Date(Web):September 27, 2016
DOI:10.1021/acs.jnatprod.6b00236
Amorphadiene synthase (ADS) is known for its vital role as a key enzyme in the biosynthesis of the antimalarial drug artemisinin. Despite the vast research targeting this enzyme, an X-ray crystal structure of the enzyme has not yet been reported. In spite of the remarkable difference in product profile among various sesquiterpene synthases, they all share a common α-helical fold with many highly conserved regions especially the bivalent metal ion binding motifs. Hence, to better understand the structural basis of the mechanism of ADS, a reliable 3D homology model representing the conformation of the ADS enzyme and the position of its substrate, farnesyl diphosphate, in the active site was constructed. The model was generated using the reported crystal structure of α-bisabolol synthase mutant, an enzyme with high sequence identity with ADS, as a template. Site-directed mutagenesis was used to probe the active site residues. Seven residues were probed showing their vital role in the ADS mechanism and/or their effect on product profile. The generated variants confirmed the validity of the ADS model. This model will serve as a basis for exploring structure–function relationships of all residues in the active site to obtain further insight into the ADS mechanism.
Co-reporter:Zheng Guan;Dan Xue;Ingy I. Abdallah
Applied Microbiology and Biotechnology 2015 Volume 99( Issue 22) pp:9395-9406
Publication Date(Web):2015 November
DOI:10.1007/s00253-015-6950-1
Terpenoids are the largest group of small-molecule natural products, with more than 60,000 compounds made from isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). As the most diverse group of small-molecule natural products, terpenoids play an important role in the pharmaceutical, food, and cosmetic industries. For decades, Escherichia coli (E. coli) and Saccharomyces cerevisiae (S. cerevisiae) were extensively studied to biosynthesize terpenoids, because they are both fully amenable to genetic modifications and have vast molecular resources. On the other hand, our literature survey (20 years) revealed that terpenoids are naturally more widespread in Bacillales. In the mid-1990s, an inherent methylerythritol phosphate (MEP) pathway was discovered in Bacillus subtilis (B. subtilis). Since B. subtilis is a generally recognized as safe (GRAS) organism and has long been used for the industrial production of proteins, attempts to biosynthesize terpenoids in this bacterium have aroused much interest in the scientific community. This review discusses metabolic engineering of B. subtilis for terpenoid production, and encountered challenges will be discussed. We will summarize some major advances and outline future directions for exploiting the potential of B. subtilis as a desired “cell factory” to produce terpenoids.
Co-reporter:Dan Xue;Ingy I. Abdallah;Ilse E. M. de Haan
Applied Microbiology and Biotechnology 2015 Volume 99( Issue 14) pp:5907-5915
Publication Date(Web):2015 July
DOI:10.1007/s00253-015-6531-3
Creating novel biosynthetic pathways and modulating the synthesis of important compounds are one of the hallmarks of synthetic biology. Understanding the key parameters controlling the flux of chemicals throughout a metabolic pathway is one of the challenges ahead. Isoprenoids are the most functionally and structurally diverse group of natural products from which numerous medicines and relevant fine chemicals are derived. The well-characterized and broadly used production organism Bacillus subtilis forms an ideal background for creating and studying novel synthetic routes. In comparison to other bacteria, B. subtilis emits the volatile compound isoprene, the smallest representative of isoprenoids, in high concentrations and thus represents an interesting starting point for an isoprenoid cell factory. In this study, the effect of systematic overexpression of the genes involved in the methylerythritol phosphate (MEP) pathway on isoprenoid production in B. subtilis was investigated. B. subtilis strains harboring a plasmid containing C30 carotenoid synthetic genes, crtM and crtN, were combined with pHCMC04G plasmids carrying various synthetic operons of the MEP pathway genes. The levels of produced carotenoids, diaponeurosporene and diapolycopene, were used as indication of the role of the various enzymes on the flux of the MEP pathway. It was shown that the production of carotenoids can be increased significantly by overexpressing the MEP pathway enzymes. More broadly, the strains developed in this study can be used as a starting point for various isoprenoid cell factories.
Co-reporter:Marcel Bokhove;Bauke W. Dijkstra;Gudrun Koch;Robbert H. Cool;Wim J. Quax;Pol Nadal-Jimenez;Carlos R. Reis;Remco Muntendam;Elena Melillo
PNAS 2014 Volume 111 (Issue 4 ) pp:1568-1573
Publication Date(Web):2014-01-28
DOI:10.1073/pnas.1311263111
The use of enzymes to interfere with quorum sensing represents an attractive strategy to fight bacterial infections. We used
PvdQ, an effective quorum-quenching enzyme from Pseudomonas aeruginosa, as a template to generate an acylase able to effectively hydrolyze C8-HSL, the major communication molecule produced by
the Burkholderia species. We discovered that the combination of two single mutations leading to variant PvdQLα146W,Fβ24Y conferred high activity toward C8-HSL. Exogenous addition of PvdQLα146W,Fβ24Y dramatically decreased the amount of C8-HSL present in Burkholderia cenocepacia cultures and inhibited a quorum sensing-associated phenotype. The efficacy of this PvdQ variant to combat infections in vivo
was further confirmed by its ability to rescue Galleria mellonella larvae upon infection, demonstrating its potential as an effective agent toward Burkholderia infections. Kinetic analysis of the enzymatic activities toward 3-oxo-C12-L-HSL and C8-L-HSL corroborated a substrate switch.
This work demonstrates the effectiveness of quorum-quenching acylases as potential novel antimicrobial drugs. In addition,
we demonstrate that their substrate range can be easily switched, thereby paving the way to selectively target only specific
bacterial species inside a complex microbial community.
Co-reporter:Luis F. Godinho;Carlos R. Reis;Ronald van Merkerk;Gerrit J. Poelarends ;Wim J. Quax
Advanced Synthesis & Catalysis 2012 Volume 354( Issue 16) pp:3009-3015
Publication Date(Web):
DOI:10.1002/adsc.201200211
Abstract
The Escherichia coli esterase YbfF displays high activity towards 1,2-O-isopropylideneglycerol (IPG) butyrate and IPG caprylate, and prefers the R-enantiomer of these substrates, producing the S-enantiomer of the IPG product in excess. To improve the potential of the enzyme for the kinetic resolution of racemic esters of IPG, an enhancement of the activity and enantioselectivity would be highly desirable. Molecular docking of the R-enantiomer of both IPG esters into the active site of YbfF allowed the identification of proximal YbfF active site residues. Four residues (25, 124, 185 and 235) were selected as targets for mutagenesis, in order to enhance YbfF activity and enantioselectivity towards IPG esters. Random mutagenesis at positions 25, 124, 185 and 235 yielded several best YbfF variants with enhanced activity and enantioselectivity towards IPG esters. The best YbfF mutant, W235I, exhibited a 2-fold higher enantioselectivity than wild-type YbfF, with an E=38 for IPG butyrate and an E=77 for IPG caprylate. Molecular docking experiments further support the enhanced enantioselectivity shown experimentally and the structural effects of this amino acid substitution on the active site of YbfF are provided. The engineered W235I mutant is an attractive catalyst for practical applications in the kinetic resolution of IPG esters.
Co-reporter:C R Reis, A M van der Sloot, A Natoni, E Szegezdi, R Setroikromo, M Meijer, K Sjollema, F Stricher, R H Cool, A Samali, L Serrano and W J Quax
Cell Death & Disease 2010 1(10) pp:e83
Publication Date(Web):2010-10-01
DOI:10.1038/cddis.2010.61
The tumour necrosis factor family member TNF-related apoptosis-inducing ligand (TRAIL) selectively induces apoptosis in a variety of cancer cells through the activation of death receptors 4 (DR4) and 5 (DR5) and is considered a promising anticancer therapeutic agent. As apoptosis seems to occur primarily via only one of the two death receptors in many cancer cells, the introduction of DR selectivity is thought to create more potent TRAIL agonists with superior therapeutic properties. By use of a computer-aided structure-based design followed by rational combination of mutations, we obtained variants that signal exclusively via DR4. Besides an enhanced selectivity, these TRAIL-DR4 agonists show superior affinity to DR4, and a high apoptosis-inducing activity against several TRAIL-sensitive and -resistant cancer cell lines in vitro. Intriguingly, combined treatment of the DR4-selective variant and a DR5-selective TRAIL variant in cancer cell lines signalling by both death receptors leads to a significant increase in activity when compared with wild-type rhTRAIL or each single rhTRAIL variant. Our results suggest that TRAIL induced apoptosis via high-affinity and rapid-selective homotrimerization of each DR represent an important step towards an efficient cancer treatment.
Co-reporter:Carlos R. Reis, Almer M. van der Sloot, Eva Szegezdi, Alessandro Natoni, Vicente Tur, Robbert H. Cool, Afshin Samali, Luis Serrano and Wim J. Quax
Biochemistry 2009 Volume 48(Issue 10) pp:
Publication Date(Web):February 23, 2009
DOI:10.1021/bi801927x
Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent and selective inducer of apoptosis in various tumor types, raising enthusiasm for TRAIL as a potential anticancer agent. TRAIL-induced apoptosis is mediated by death receptors 4 (DR4) and DR5. The design of rhTRAIL variants either with improved affinity or selectivity toward one or both death-inducing receptors is thought to enhance the therapeutical potential of TRAIL. Here we demonstrate that a single amino acid mutation at the position of glycine 131 to lysine or arginine in wild-type rhTRAIL significantly improved the affinity of rhTRAIL toward its death receptors, with the highest affinity increase observed for the DR4 receptor. These variants were able to induce higher in vitro levels of apoptosis in cancer cells responsive to only DR4 or to both death receptors and could therefore increase the potential use of rhTRAIL as an anticancer therapeutic agent.
Co-reporter:Ferno López-Gallego;Lorena Betancor;CharlesF. Sio;CarlosR. Reis;Pol Nadal Jimenez;JoseM. Guisan;Wim.J. Quax;Roberto Fernez-Lafuente
Advanced Synthesis & Catalysis 2008 Volume 350( Issue 2) pp:343-348
Publication Date(Web):
DOI:10.1002/adsc.200700320
Abstract
2-Oxoadipoyl-7-ACA is an intermediate in the conversion of cephalosporin C (CPC) to 7-aminocephalosporanic acid (7-ACA) when using a new route involving D-amino acid oxidase, catalase and glutaryl acylase. A key point in the reaction design is to avoid the accumulation of hydrogen peroxide in the reaction medium as the yields of 7-ACA decrease in the presence of this compound due to its low stability. Looking for an enzyme with improved activity towards 2-oxoadipoyl-7-ACA, different mutants of glutaryl acylase from Pseudomonas SY-77 with an improved activity towards adipoyl-7-ACA were evaluated. The best results on 2-oxoadipoyl-7-ACA hydrolysis were found with the double mutant Y178F+F375H, which showed a Kcat increase of 6.5-fold and a Km decrease of 3-fold compared to the wild-type (wt) enzyme. When this enzyme was tested in the tri-enzymatic system to convert CPC into 7-ACA, this mutant permitted us to reach more than an 80 % yield of 7-ACA using a 3-fold mass excess compared to DAAO; while the wt enzyme gave only a 40 % yield. Therefore, the application of this new mutant to the one-pot conversion of CPC to 7-ACA gives very good result in terms of efficiency, yield and rate of the process.
Co-reporter:Ykelien L. Boersma, Tjaard Pijning, Margriet S. Bosma, Almer M. van der Sloot, Luís F. Godinho, Melloney J. Dröge, Remko T. Winter, Gertie van Pouderoyen, Bauke W. Dijkstra, Wim J. Quax
Chemistry & Biology 2008 Volume 15(Issue 8) pp:782-789
Publication Date(Web):25 August 2008
DOI:10.1016/j.chembiol.2008.06.009
Lipases are successfully applied in enantioselective biocatalysis. Most lipases contain a lid domain controlling access to the active site, but Bacillus subtilis Lipase A (LipA) is a notable exception: its active site is solvent exposed. To improve the enantioselectivity of LipA in the kinetic resolution of 1,2-O-isopropylidene-sn-glycerol (IPG) esters, we replaced a loop near the active-site entrance by longer loops originating from Fusarium solani cutinase and Penicillium purpurogenum acetylxylan esterase, thereby aiming to increase the interaction surface for the substrate. The resulting loop hybrids showed enantioselectivities inverted toward the desired enantiomer of IPG. The acetylxylan esterase-derived variant showed an inversion in enantiomeric excess (ee) from −12.9% to +6.0%, whereas the cutinase-derived variant was improved to an ee of +26.5%. The enantioselectivity of the cutinase-derived variant was further improved by directed evolution to an ee of +57.4%.
Co-reporter:Ykelien L. Boersma Dr.;Melloney J. Dröge Dr.;Almer M. van der Sloot Dr.;Tjaard Pijning;Robbert H. Cool Dr.;Bauke W. Dijkstra Dr.;Wim J. Quax Dr.
ChemBioChem 2008 Volume 9( Issue 7) pp:1110-1115
Publication Date(Web):
DOI:10.1002/cbic.200700754
Abstract
In directed evolution experiments, success often depends on the efficacy of screening or selection methods. Genetic selections have proven to be extremely valuable for evolving enzymes with improved catalytic activity, improved stability, or with altered substrate specificity. In contrast, enantioselectivity is a difficult parameter to select for. In this study, we present a successful strategy that not only selects for catalytic activity, but for the first time also for enantioselectivity, as demonstrated by the selection of Bacillus subtilis lipase A variants with inverted and improved enantioselectivity. A lipase mutant library in an aspartate auxotroph Escherichia coli was plated on minimal medium that was supplemented with the aspartate ester of the desired enantiomer (S)-(+)-1,2-O-isopropylidene-sn-glycerol. To inhibit growth of less enantioselective variants, a covalently binding phosphonate ester of the opposite (R)-(−)-1,2-O-isopropylidene-sn-glycerol enantiomer was added as well. After three selection rounds in which the selection pressure was increased by raising the phosphonate ester concentration, a mutant was selected with an improved enantioselectivity increased from an ee of −29.6 % (conversion 23.4 %) to an ee of +73.1 % (conversion 28.9 %) towards the (S)-(+)-enantiomer. Interestingly, its amino acid sequence showed that the acid of the catalytic triad had migrated to a position further along the loop that connects β7 and αE; this shows that the position of the catalytic acid is not necessarily conserved in this lipase.
Co-reporter:Melloney J. Dröge Dr.;Ykelien L. Boersma Drs.;Gertie van Pouderoyen Dr.;Titia E. Vrenken Drs.;Carsten J. Rüggeberg Dr.;Manfred T. Reetz Dr.;Bauke W. Dijkstra Dr.;Wim J. Quax Dr.
ChemBioChem 2006 Volume 7(Issue 1) pp:
Publication Date(Web):12 DEC 2005
DOI:10.1002/cbic.200500308
Phage display can be used as a protein-engineering tool for the selection of proteins with desirable binding properties from a library of mutants. Here we describe the application of this method for the directed evolution of Bacillus subtilis lipase A, an enzyme that has important properties for the preparation of the pharmaceutically relevant chiral compound 1,2-O-isopropylidene-sn-glycerol (IPG). PCR mutagenesis with spiked oligonucleotides was employed for saturation mutagenesis of a stretch of amino acids near the active site. After expression of these mutants on bacteriophages, dual selection with (S)-(+)- and (R)-(−)-IPG stereoisomers covalently coupled to enantiomeric phosphonate suicide inhibitors (SIRAN Sc and Rc inhibitors, respectively) was used for the isolation of variants with inverted enantioselectivity. The mutants were further characterised by determination of their Michaelis–Menten parameters. The 3D structures of the Sc and Rc inhibitor–lipase complexes were determined and provided structural insight into the mechanism of enantioselectivity of the enzyme. In conclusion, we have used phage display as a fast and reproducible method for the selection of Bacillus lipase A mutant enzymes with inverted enantioselectivity.
Co-reporter:Putri Dwi Utari, Wim J. Quax
Trends in Microbiology (July 2013) Volume 21(Issue 7) pp:315-316
Publication Date(Web):1 July 2013
DOI:10.1016/j.tim.2013.04.006
The susceptibility of Caenorhabditis elegans to different virulent phenotypes of Pseudomonas aeruginosa makes the worms an excellent model for studying host–pathogen interactions. Including the recently described liquid killing, five different killing assays are now available offering superb possibilities for performing high-throughput screens for novel antibiotics using a whole-body infection system.
Co-reporter:Elena Melillo, Remco Muntendam, Wim J. Quax, Oliver Kayser
Journal of Biotechnology (31 October 2012) Volume 161(Issue 3) pp:302-307
Publication Date(Web):31 October 2012
DOI:10.1016/j.jbiotec.2012.05.024
For the first time, the pentalenene synthase (PSS) gene from Streptomyces UC5319 was expressed in Xanthophyllomyces dendrorhous, a native producer of astaxanthin. For the expression of the gene and the concurrent knock out of the native crtE or crtYB genes, two new vectors were engineered and used for the transformation of the wild-type strain of X. dendrorhous. The transformations resulted in white colonies, showing a complete shutdown of the carotenoid production. Furthermore, an additional vector was constructed for the insertion of the PSS gene in the rDNA of the yeast. All the mutant strains produce the sesquiterpene pentalenene and show no difference in growth when compared to the wild-type strain. In this report, we demonstrate that X. dendrorhous is a suitable host for the expression of heterologous terpene cyclases and for the production of foreign terpene compounds.Highlights► Two new vectors for integration of genes in Xanthophyllomyces dendrorhous are provided.► X. dendrorhous mutants expressing pentalenene synthase produce pentalenene.► X. dendrorhous can be used to produce non-native terpenes.► The total terpene pool in X. dendrorhous shows high upward potential for pentalenene production.
Co-reporter:Luis F. Godinho, Carlos R. Reis, Henriëtte J. Rozeboom, Frank J. Dekker, Bauke W. Dijkstra, Gerrit J. Poelarends, Wim J. Quax
Journal of Biotechnology (31 March 2012) Volume 158(Issues 1–2) pp:36-43
Publication Date(Web):31 March 2012
DOI:10.1016/j.jbiotec.2011.12.026
Previously studied Bacillus subtilis carboxylesterases (CesA and CesB) have potential for the kinetic resolution of racemic esters of 1,2-O-isopropylideneglycerol (IPG). CesA exhibits high activity but low enantioselectivity towards IPG-butyrate and IPG-caprylate, while the more enantioselective CesB does not process IPG-butyrate and exhibits several-fold lower activity than CesA towards IPG-caprylate. A sequence and structure comparison allowed us to identify active site residues that may cause the difference in (enantio)selectivities of CesA and CesB towards these IPG esters. This structure-based approach led to the identification of two active site residues in CesA (F166 and F182), as promising candidates for mutagenesis in order to enhance its enantioselectivity. Mutagenesis of positions 166 and 182 in CesA yielded novel variants with enhanced enantioselectivity and without significant loss of catalytic activity. For IPG-butyrate, a CesA double mutant F166V/F182 C (ER = 13) was generated showing a ∼13-fold increased enantioselectivity as compared to wild-type CesA (E = 1). For IPG-caprylate, we designed a CesA double mutant F166 V/F182Y (ER = 9) displaying a ∼5-fold increased enantioselectivity as compared to the wild-type enzyme (ER = 2). These findings, combined with the results of molecular docking experiments, demonstrate the importance of residues at positions 166 and 182 for the enantioselectivity of CesA, and may contribute to the development of efficient biocatalysts.Highlights► Critical amino acids responsible for the large difference in enantioselectivity between two related esterases have been identified. ► Targeted mutagenesis of these enantioselectivity determining residues has resulted in the isolation of carboxylesterase variants with superior enantioselectivity. ► The novel esterases can be used for the synthesis of S-IPG, the major building block for the pharmaceutically important class of β-blockers.
Co-reporter:Joanna Krzeslak, Peter Braun, Rome Voulhoux, Robbert H. Cool, Wim J. Quax
Journal of Biotechnology (15 July 2009) Volume 142(Issues 3–4) pp:250-258
Publication Date(Web):15 July 2009
DOI:10.1016/j.jbiotec.2009.05.005
Penicillin G acylase (PGA) is a widely studied bacterial enzyme of great industrial importance. Since its overproduction in the original organisms is mostly limited to the intracellular bacterial spaces which may lead to aggregation and cell toxicity, we have set out to explore the host organism Pseudomonas aeruginosa that possesses the Xcp machinery for secretion of folded proteins to the extracellular medium. We have made fusion proteins, consisting of Pseudomonas Sec- or Tat-specific signal peptides, the elastase propeptide and the mature penicillin G acylase. With all constructs we obtained production of PGA in P. aeruginosa, but we observed that processing of the PGA was temperature dependent and that the active enzyme could only be found after growth at 25 °C or lower temperatures. Remarkably, the mature protein, expressed from a TatProPGA hybrid, was not only found in the extracellular medium and the periplasm, but also in the cytoplasm as assessed by comparison to the reporter beta-lactamase protein. The unusual cytoplasmic localization of the mature protein strongly suggests that processing of PGA can also occur in the cytoplasm of P. aeruginosa. The extracellular localization of the TatProPGA hybrid was found not to be dependent on the tatABC-genes. The elastase signal sequence/propeptide combination appeared to be an inadequate carrier for transporting penicillin G acylase across the outer membrane of P. aeruginosa.
Co-reporter:Mariana Wahjudi, Senthil Murugappan, Ronald van Merkerk, Anko C. Eissens, Marinella R. Visser, Wouter L.J. Hinrichs, Wim J. Quax
European Journal of Pharmaceutical Sciences (12 March 2013) Volume 48(Issues 4–5) pp:637-643
Publication Date(Web):12 March 2013
DOI:10.1016/j.ejps.2012.12.015
In the lungs of cystic fibrosis (CF) patients, Pseudomonas aeruginosa commonly causes chronic infections. It has been shown that the P. aeruginosa quorum sensing (QS) system controls the expression of virulence factors during invasion and infection to host cells. PvdQ is an acyl–homoserine lactone (AHL) acylase able to degrade the signal molecule of P. aeruginosa QS. The role of PvdQ in inhibiting the QS and its successive virulence determinants has been established in in vitro as well as in in vivo, the latter in a Caenorabdhitis elegans model. For the treatment of pulmonary P. aeruginosa infections, we propose that PvdQ can be best administered directly to the lungs of the patients as a dry powder because this is expected to give specific advantages in delivery as compared to nebulizing. Therefore in this study we investigated the production of a PvdQ powder by spray-freeze drying using mannitol, trehalose and inulin as excipient. The activity of PvdQ in the powder was determined immediately after production and after subsequent storage during 4 weeks at 20 °C and 55 °C. We found that the enzymatic activity of PvdQ is fully maintained during spray-freeze drying using mannitol, trehalose or inulin as excipient. However, mannitol was not able to stabilize the protein during storage, while PvdQ incorporated in trehalose or inulin was fully stabilized even during storage at 55 °C for at least 4 weeks. The poor stabilizing capacities of mannitol during storage could be related to its crystalline nature while the excellent stabilizing capacities of trehalose and inulin during storage could be related to their amorphous nature. The trehalose and inulin-based particles consisted of porous spheres with a volume average aerodynamical diameter of ∼1.8 μm implying that they are suitable for pulmonary delivery. This is the first study in which an AHL-degrading enzyme is processed into spray-freeze-dried powder suitable for inhalation.Download high-res image (121KB)Download full-size image
