Research Article ChemBioChem doi.org/10.1002/cbic.202200635 www.chembiochem.org Very Important Paper Myxococcus xanthus as Host for the Production of Benzoxazoles Lea Winand,[a] Lucia Lernoud,[a] Saskia Anna Meyners,[a] Katharina Kuhr,[a] Wolf Hiller,[b] and Markus Nett*[a] Benzoxazoles are important structural motifs in pharmaceutical benzoxazole pathway and the endogenous myxochelin path- drugs. Here, we present the heterologous production of 3- way led to the combinatorial biosynthesis of benzoxazoles hydroxyanthranilate-derived benzoxazoles in the host bacte- featuring a 2,3-dihydroxybenzoic acid (2,3-DHBA) building rium Myxococcus xanthus following the expression of two genes block. Subsequent in vitro studies confirmed that this crosstalk from the nataxazole biosynthetic gene cluster of Streptomyces is not only due to the availability of 2,3-DHBA in M. xanthus, sp. Tü 6176. The M. xanthus expression strain achieved a rather, it is promoted by the adenylating enzyme MxcE from benzoxazole titer of 114.6�7.4 mgL 1 upon precursor supple- the myxochelin pathway, which contributes to the activation of mentation, which is superior to other bacterial production aryl carboxylic acids and delivers them to benzoxazole biosyn- systems. Crosstalk between the heterologously expressed thesis. Introduction dehydration to give a benzoxazole (Figure 1A).[6] Natural products such as the antibiotics caboxamycin and A-33853 or Several pharmaceutical drugs incorporate one or more hetero- the anticancer agent nataxazole are synthesized in this way cyclic substructures. These moieties often contribute to the (Figure 1B).[6,7] A distinct assembly strategy is pursued in the biological activity of the drug as part of the pharmacophore. biosynthesis of the closoxazoles, which were recently identified Furthermore, they influence the physicochemical properties as from the anaerobic bacterium Clostridium cavendishii well as the bioavailability.[1] For that reason, the synthesis of DSM21758.[8] The closoxazole pathway represents the first heterocyclic compounds and their functionalization has been example of a benzoxazole biosynthetic pathway that utilizes a addressed in an impressive number of studies.[2] 3,4-disubstituted aryl carboxylic acid as a building block and, Nature has developed diverse biosynthetic routes for the hence, generates meta-substituted benzoxazoles. synthesis of heterocycles by way of enzymes such as polyketide Benzoxazole-containing compounds are used in different synthases, nonribosomal peptide synthetases, cyclodipeptide therapeutic areas, as exemplified by the approved drugs synthases or Pictet-Spenglerases.[3] Recently, some members of chlorzoxazone (muscle relaxant), tafamidis (transthyretin stabil- the amidohydrolase superfamily were reported to catalyze izer) or suvorexant (treatment of insomnia).[9] Their potent heterocyclizations. In the biosynthesis of the anti-inflammatory biological activities combined with the understanding of natural product pseudochelin A, the amidohydrolase MxcM benzoxazole biosynthesis have stimulated the biotechnological condenses a β-aminoethyl amide residue to generate an production of benzoxazole analogs.[6–8,10–12] Recently, the genes imidazoline moiety.[4,5] A similar mechanism was described in involved in nataxazole biosynthesis were expressed in the the biosynthesis of benzoxazoles. Upon enzymatic linkage of 3- bacterium Escherichia coli and, upon feeding of 3-HAA together hydroxyanthranilic acid (3-HAA) with another aryl carboxylic with other aryl carboxylic acids, various benzoxazoles were acid via an ester bond, an amidohydrolase catalyzes the generated, albeit in low titers.[12] formation of a hemiorthoamide intermediate and a subsequent In this study, we describe an alternative heterologous production system for benzoxazoles. As production organism, [a] L. Winand, L. Lernoud, S. A. Meyners, K. Kuhr, Prof. Dr. M. Nett we chose the myxobacterium Myxococcus xanthus. Unlike E. coli, Department of Biochemical and Chemical Engineering M. xanthus possesses an endogenous pathway to the benzox- Laboratory of Technical Biology, TU Dortmund University azole building block 3-HAA according to an analysis of the Emil-Figge-Str. 66, 44227 Dortmund (Germany) [13] E-mail: markus.nett@tu-dortmund.de KEGG database. This suggested that the heterologous [b] Prof. Dr. W. Hiller production of benzoxazoles in M. xanthus would not depend on Department of Chemistry and Chemical Biology precursor feeding. Furthermore, M. xanthus is known to be NMR Laboratory, TU Dortmund University Otto-Hahn-Str. 4a, 44227 Dortmund (Germany) highly amenable to secondary metabolite biosynthesis. By Supporting information for this article is available on the WWW under means of metabolic engineering considerable product titers can https://doi.org/10.1002/cbic.202200635 be achieved with this host,[14] which was also demonstrated to © 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH. This is outcompete E. coli in the heterologous production of structur- an open access article under the terms of the Creative Commons Attribution ally complex secondary metabolites.[15] More recently, a plas- Non-Commercial NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non- mid-based expression system has been developed, which commercial and no modifications or adaptations are made. facilitates the expression of foreign genes in M. xanthus.[5] This ChemBioChem 2023, 24, e202200635 (1 of 8) © 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH Wiley VCH Donnerstag, 16.02.2023 2305 / 281988 [S. 60/67] 1 Research Article ChemBioChem doi.org/10.1002/cbic.202200635 Figure 1. Amidohydrolase-mediated benzoxazole biosynthesis. A) General reaction mechanism. B) Examples of benzoxazole-containing natural products.[6,7] system was already successfully used not only for the expression of the imidazoline-forming amidohydrolase MxcM, but also for the recombinant production of alkaloids in M. xanthus.[16,17] In sum, it was expected that the advantages of M. xanthus would outweigh its slower growth in comparison to E. coli. Results and Discussion Previous studies indicated that only two enzymes are required for benzoxazole biosynthesis from 3-HAA, namely an ATP- dependent ligase and an amidohydrolase.[6] The two genes natL2 and natAM from the nataxazole biosynthetic gene cluster of Streptomyces sp. Tü 6176, which code for the aforementioned enzymes, were inserted into a myxobacterial expression plasmid (Figure S3 in the Supporting Information).[17,18] After transferring the resulting vector pMEX14 into M. xanthus NM[19] and cultivation of the expression strain, LC–MS analysis of the Figure 2. LC–MS chromatograms of A) the raw extract from M. xanthus NM: bacterial raw extract indicated the presence of an amide shunt pMEX14, B) the plasmid-free M. xanthus NM control strain, and C) the in vitro product (1’a)[6] as well as the benzoxazole product (2a; reaction with isolated NatL2 and NatAM. Black: BPC, blue: EIC of 1’a (m/z 289.0819), green: EIC of 2a (m/z 271.0713). Figure 2). The identity of 2a was confirmed by LC–MS/MS and NMR analyses, respectively (Figures S9 and S10). With the benzoxazole-producing strain at our disposal, we further investigated the influence of substrate feeding on the As genomic analyses indicated that M. xanthus synthesizes titer of 2a (Figure 3A). Without supplementation of any 3-HAA from l-tryptophan via the kynurenine pathway, we also biosynthetic precursors, a product titer of 10.7�1.8 mgL 1 was evaluated the effect of supplementing this amino acid to 1 mL obtained in shake flasks. Feeding of 3-HAA to the M. xanthus cultures in a microbioreactor (Figure S13). While the growth of cultures positively affected the production of 2a in a concen- the myxobacterial host was only slightly affected, the produc- tration-dependent manner, although the molar yield coefficient tion of 2a increased with the concentration of l-tryptophan. decreased. This finding suggested that the amount of biocata- Without feeding of the amino acid, a titer of 0.3�0.1 mgL 1 lyst is a limiting factor under the given conditions or that was obtained. In presence of 1 gL 1 l-tryptophan, the produc- substrate- or product-inhibitory effects occur. It is further tion was roughly increased by a factor of 65 to 15.5� noteworthy that the growth of M. xanthus NM: pMEX14 was 1.7 mgL 1. These values are significantly lower than the titers inhibited with increasing amounts of 3-HAA (Figure 3B). In the from the shake flask experiment, which probably is caused by presence of 480 mgL 1 3-HAA, the growth was completely the different cultivation condition and extraction method. To suppressed. The highest product titer (114.6�7.4 mgL 1) was verify this assumption, we tested the effect of l-tryptophan achieved after the addition of 320 mgL 1 3-HAA, with a yield supplementation in a shake flask experiment. When M. xanthus coefficient of 40.6�2.6%. NM: pMEX14 was grown in the standard cultivation medium ChemBioChem 2023, 24, e202200635 (2 of 8) © 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH Wiley VCH Donnerstag, 16.02.2023 2305 / 281988 [S. 61/67] 1 14397633, 2023, 5, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cbic.202200635 by Technical University Dortmund, Wiley Online Library on [16/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Research Article ChemBioChem doi.org/10.1002/cbic.202200635 In vivo incorporation of endogenous and supplemented aromatic carboxylic acids into benzoxazoles In the chemical analysis of M. xanthus NM: pMEX14, we consistently detected a low abundance peak (2b) that was not present in the plasmid-free control strain. The m/z value and the retention time of this peak suggested that it might belong to a structural analog of 2a, but its low titer precluded an unequivocal structural identification. Foregoing studies had already indicated a possible crosstalk between benzoxazole biosynthesis and other natural product pathways. In particular, aromatic carboxylic acids, such as salicylic acid and 2,3- dihydroxybenzoic acid (2,3-DHBA), had been proposed to be diverted from siderophore pathways into benzoxazole biosynthesis.[7c,10,11] For that reason, we assumed that there might also be a crosstalk between the heterologously expressed benzoxazole biosynthesis enzymes and the native myxochelin pathway in M. xanthus, in which 2,3-DHBA represents an intermediate.[20] To test if 2,3-DHBA is indeed used by M. xanthus NM: pMEX14 for benzoxazole assembly, a culture of the expression strain was supplemented with additional 2,3- DHBA and its metabolic profile was recorded. In the corre- sponding chromatogram, the intensity of the previously observed low abundance peak (2b) was increased (Figure 4). LC–MS/MS suggested that 2b represents a benzoxazole made from 3-HAA and 2,3-DHBA (Figure S14). Upon re-examination of the chromatogram a peak was detected, of which the [M+H]+ ion is consistent with the amide shunt product 1’b. To assess the substrate tolerance of benzoxazole biosyn- thesis in M. xanthus, we fed our expression strain with other aryl carboxylic acids. Because an in vitro characterization of NatL2 Figure 3. Influence of feeding M. xanthus NM: pMEX14 with 3-HAA. A) Pro- and NatAM had indicated that these enzymes are capable of oduction level of 2a and molar substrate-specific yield. Product titers were accepting 3-hydroxybenzoic acid (3-HBA) and its derivatives as determined after extraction of 50 mL cultures from shake flasks. B) Growth curves recorded in a microbioreactor system (BioLector, m2p-labs). substrates, we initially evaluated the combination of 3-HAA and 3-HBA.[6] After extraction of the bacterial culture, we detected the masses of the amides 1’a and 1’c as well as of the respective benzoxazoles 2a and 2c by LC–MS analysis with addition of 1 gL 1 l-tryptophan, a product titer of 57.3 mgL 1 was observed. Production of benzoxazoles in E. coli was only possible after feeding of 3-HAA and 6-methylsalicylic acid.[12] The highest product titers that were reported from recombinant E. coli cells are 3.5 mgL 1 in case of a methylated caboxamycin derivative and 4 mgL 1 in case of AJI9561 (Figure 1).[12] These titers were obtained following a two-day incubation, whereas the cultiva- tion of M. xanthus took three days. However, care must be taken in the comparison of space-time yields (2 mgL 1d 1 in E. coli and 38.2 mgL 1d 1 in M. xanthus), as more benzoxazole biosynthesis genes were heterologously expressed in E. coli, which also led to a different product spectrum. Nevertheless, the present data indicate that M. xanthus is a promising host for recombinant benzoxazole production. Figure 4. Incorporation of 2,3-DHBA into benzoxazoles. A) LC–MS chromato- gram of the raw extract from M. xanthus NM: pMEX14 fed with 50 mgL 1 3- HAA and 2,3-DHBA. B) In vitro reaction with isolated MxcE, NatL2 and NatAM. Blue: EIC of 1’a (m/z 289.0819), green: EIC of 2a (m/z 271.0713), yellow: EIC of 1’b (m/z 290.0659), red: EIC of 2b (m/z 272.0553). ChemBioChem 2023, 24, e202200635 (3 of 8) © 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH Wiley VCH Donnerstag, 16.02.2023 2305 / 281988 [S. 62/67] 1 14397633, 2023, 5, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cbic.202200635 by Technical University Dortmund, Wiley Online Library on [16/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Research Article ChemBioChem doi.org/10.1002/cbic.202200635 (Figure S15). Interestingly, the signal corresponding to 2c has a was carried out. For this, we expressed natL2 and natAM as well larger peak area than that of 2a, indicating a preferred as mxcE in E. coli BL21(DE3) as hexahistidyl-tagged recombinant conversion. After HPLC purification, we obtained 1.3 mg of 1’c proteins using pET28a(+)-derived plasmids. The enzymes were and 2.1 mg of 2c from a 50 mL culture. The structures of the purified via Ni-NTA affinity chromatography. Subsequently, two compounds were verified by NMR and MS analyses in vitro reactions were performed with different enzyme (Figures S16–S26). Subsequently, salicylic acid (SA), benzoic acid combinations using 0.5 mM 3-HAA and 0.5 mM of another aryl (BA) and 3-chlorobenzoic acid (3-ClBA) were successfully used carboxylic acid (2,3-DHBA, 3-HBA, SA, BA, or 3-ClBA) as as building blocks for the synthesis of benzoxazoles 2d-f substrates. The exclusive combination of MxcE and NatAM did (Figures S27, S29, and S41). While 2d corresponds to the known not lead to any product formation in absence of NatL2. This natural product caboxamycin,[7c] the BA- and 3-ClBA-derived shows that MxcE is not able to entirely substitute the activity of benzoxazoles (2e and 2f) have not been reported in the NatL2. Subsequently, we compared the product profiles from literature before. Compounds 1’e, 2e, 1’f and 2f were purified reactions including the enzymes MxcE, NatL2 and NatAM with by HPLC. From 1 L cultures, we collected 18.8 mg of 1’e, 3.4 mg those of NatL2 and NatAM only (Table 1). In presence of MxcE, of 2e, 6.5 mg of 1’f and 1.1 mg of 2f. The purified derivatives the areas of all detected amide and product peaks were were fully structurally characterized by spectroscopic analyses increased. As expected from a previous study,[6] only NatL2 and (Figures S30–S40 and S42–S52). NatAM were needed for the in vitro synthesis of the 3-HBA- derived benzoxazole (2c). Still, the presence of MxcE improved the production level of 2c by 46% (Table S3). While it was not Involvement of a M. xanthus enzyme in combinatorial possible to produce the 2,3-DHBA- and SA-derived benzox- benzoxazole assembly azoles in reactions with NatL2 and NatAM alone, the addition of MxcE allowed the synthesis of both 3-hydroxycaboxamycin (2b) Next, we turned our attention to clarify the enzymatic basis of and caboxamycin (2d). The BA- and 3-ClBA-derived benzox- combinatorial benzoxazole assembly. Two scenarios were azoles (2e, 2f) could not be generated in vitro. Although the conceivable. Either the substrate flexibility of NatL2 and NatAM addition of MxcE positively affected the production of 1’e and was sufficient to enable the synthesis of the benzoxazoles 2b–f 1’f, which can be expected to derive from the relevant ester in M. xanthus depending on the availability of appropriate intermediates 1e and 1f, no heterocyclization was observed. precursors or the participation of one or more host enzymes We assume that this is due to comparatively low titers of 1e was additionally necessary. The question of which scenario and 1f. The NatAM reaction is likely not favored in an aqueous applies to the observed crosstalk could not be answered on the phase for thermodynamic reasons. Higher concentrations of the basis of previous investigations.[6,7c,10,11] ester intermediate would thus be necessary to promote In the second scenario, the M. xanthus enzyme MxcE was benzoxazole formation. This is also supported by the formation the most likely candidate for an involvement in benzoxazole of 2b after addition of MxcE (Table 1). In case of 1e and 1f, it assembly. In the biosynthesis of myxochelins, the ligase MxcE cannot be excluded that they occur in higher concentrations activates 2,3-DHBA as adenylate before its incorporation.[21] in vivo, as M. xanthus enzymes other than MxcE could also Moreover, MxcE is known to exhibit a broad substrate tolerance, contribute to the activation of their building blocks. which can be exploited for the biosynthesis of myxochelin Consolidating our results with observations made by other analogs incorporating different aryl carboxylic acids.[22] We thus groups, it is now possible to deduce a model for the hypothesized that MxcE had possibly contributed to the combinatorial biosynthesis of benzoxazoles in M. xanthus and in outcome of our feeding experiments. In order to probe this other bacteria (Figure 5).[6,7c,10,11] According to this model, a possibility, an in vitro testing of the aforementioned enzymes NatL2-type enzyme is essential for benzoxazole formation, Table 1. Benzoxazoles produced by in vitro biotransformation of two substrates with NatL2 and NatAM in the absence or presence of MxcE. Substrate Substrate Amide shunt Peak area of amide shunt product in Benzoxazole Peak area of benzoxazole product in #1 #2 product relation to 1’a [%] product relation to 2a [%] after reaction w/o after reaction with after reaction w/o after reaction with MxcE MxcE MxcE MxcE 3-HAA 2,3-DHBA 1’b 8.7% 9.6% 2b 0.0% 0.1% 3-HAA 3-HBA 1’c 24.3% 24.0% 2c 25.5% 26.4% 3-HAA SA 1’d 0.0% 0.4% 2d 0.0% 1.6% 3-HAA BA 1’e 0.2% 0.4% 2e 0.0% 0.0% 3-HAA 3-ClBA 1’f 0.3% 0.3% 2 f 0.0% 0.0% ChemBioChem 2023, 24, e202200635 (4 of 8) © 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH Wiley VCH Donnerstag, 16.02.2023 2305 / 281988 [S. 63/67] 1 14397633, 2023, 5, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cbic.202200635 by Technical University Dortmund, Wiley Online Library on [16/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Research Article ChemBioChem doi.org/10.1002/cbic.202200635 Figure 5. Proposed reaction mechanism for benzoxazole biosynthesis in M. xanthus including spontaneous conversion of the ester intermediate into an amide shunt product. The mxcE gene is naturally present in the chromosome of M. xanthus, whereas the genes natL2 and natAM originate from Streptomyces sp. Tü 6176 and were heterologously expressed in M. xanthus. because of its ability to link 3-HAA with other aryl carboxylic the yield previously reported from E. coli (2 mgL 1d 1), which is acid adenylates. When it comes to the adenylation reaction, not able to produce benzoxazoles without the addition of 3- however, there is some redundancy, in that other enzymes can HAA.[12] However, care must be taken in the comparison of activate utilizable building blocks in a manner similar to NatL2. these values, as the reconstruction of benzoxazole biosynthesis Such enzymes (e.g., MxcE) might even expand the product in E. coli involved the heterologous expression of a total of five spectrum depending on their substrate tolerance. It is thus genes, which also led to a different product spectrum. evident that benzoxazole biosynthesis not only draws metabo- In M. xanthus, various benzoic acid-derived building blocks lites from other pathways, but that it also capitalizes on ligases activated by the adenylating enzyme MxcE can be incorporated of other pathways. This finding has important implications for into the benzoxazole scaffold, representing crosstalk between the biotechnological production of benzoxazoles. In brief, the the native myxochelin pathway and the heterologously ex- heterocyclic scaffold can be furnished with different substitu- pressed benzoxazole pathway. This biocombinatorial concept ents by combining benzoxazole biosynthesis pathways with opens new opportunities for the assembly of benzoxazole other adenylating enzymes.[23] The 4-halobenzoate-coenzyme A analogs, considering the large diversity of adenylating enzymes ligase from Pseudomonas sp. CBS-3 or the naphthoic acid- in natural product pathways. In the future, it will be interesting coenzyme A ligase NcsB2 from Streptomyces carzinostaticus to evaluate the production of structurally more complex ATCC15944 are examples of characterized adenylation enzymes benzoxazoles in M. xanthus and also to tune the expression of that exhibit a broad substrate specificity.[24] Other alternatives the biosynthesis genes in order to further increase the product might be found in adenylation domains of nonribosomal titers. Promoter engineering would be one promising approach peptide synthetases that can be engineered for modification of for the latter task.[27] the substrate specificity, as exemplified by the DhbE adenyla- tion domain from enterobactin biosynthesis.[25] One must take into account, however, that the substrate tolerance of the Experimental Section heterocycle-forming amidohydrolase NatAM might become a Strains, nucleic acids, and plasmids: The bacterial strains and limiting factor for the combinatorial biosynthesis of benzox- plasmids used in this study are described in Table S1. The azoles, which should be further investigated in future. nonmotile strain M. xanthus NM[19] was purchased from the American Type Culture Collection (ATCC). The genes natL2 and natAM originating from the nataxazole gene cluster (GenBank Conclusions accession number LN713864) were codon-optimized for M. xanthus and subsequently synthesized by Life Technologies GmbH (Thermo Fisher Scientific, see Supporting Information). For E. coli, the codon- Beside synthetic approaches for derivatization, concepts of optimized genes published by Ouyang et al. were used.[12] bioengineering that include semisynthesis, combinatorial bio- Growth conditions and nucleic acid extraction: E. coli TOP10 was synthesis, precursor-directed biosynthesis, mutasynthesis or cultured in liquid or solidified lysogeny broth (LB) medium at 37 °C. in vitro biocatalysis have been established for natural product Liquid cultures were shaken at 180 rpm. M. xanthus was grown in derivatization over the past decades.[26] In this study, we CYE medium (10 gL 1 casitone, 5 gL 1 yeast extract, 2.1 gL 1 MOPS, evaluated M. xanthus as an alternative host organism for the 1 gL 1 MgSO4·7H2O, 0.5 mgL 1 vitamin B12; pH 7.4) at 30 °C. For generation of benzoxazole-containing natural products. The liquid cultures, an agitation speed of 130 rpm was applied. The antibiotic kanamycin (50 μgL 1myxobacterium M. xanthus naturally produces the required ) was used as selection marker. Plasmid DNA was obtained from E. coli cultures using the building block 3-hydroxyanthranilic acid (3-HAA), facilitating the NucleoSpin Plasmid (NoLid) Mini Kit (Macherey-Nagel). Nucleic acids synthesis of benzoxazoles upon heterologous expression of embedded in agarose gels were isolated using the NucleoSpin Gel only two genes from the nataxazole biosynthetic pathway. and PCR Clean-up Mini Kit (Macherey-Nagel). Additional supplementation of 3-HAA led to competitive space– time yields in M. xanthus of up to 38 mgL 1d 1. This exceeds ChemBioChem 2023, 24, e202200635 (5 of 8) © 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH Wiley VCH Donnerstag, 16.02.2023 2305 / 281988 [S. 64/67] 1 14397633, 2023, 5, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cbic.202200635 by Technical University Dortmund, Wiley Online Library on [16/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Research Article ChemBioChem doi.org/10.1002/cbic.202200635 General cloning procedure: Plasmid construction was generally (Figure S11). The following HPLC conditions were applied for performed by making use of the Gibson assembly method.[28] For product quantification: Flow rate: 1 mL/min. Mobile phases: this purpose, the insert DNA fragments were amplified by overhang acetonitrile and water with 0.1% (v/v) trifluoroacetic acid. Column: PCR using the Phusion High-Fidelity DNA polymerase (Thermo- EC 250/4 Nucleodur C18 Isis, 5 μm (Macherey–Nagel). Column oven: Scientific). Circular plasmid DNA was linearized with FastDigest 30 °C. Gradient: 0–5 min: 30% ACN; 5–16 min: 30–81% ACN; 16– restriction enzymes (ThermoScientific) and dephosphorylated with 18 min 81–100% ACN; 18–20 min: 100% ACN; 20–21 min: 100–30% the FastAP alkaline phosphatase (ThermoScientific) to avoid ACN; 21–25 min: 30% ACN. Retention time of 2a: 12.6 min. recircularization. For DNA assembly, 2x GeneArt Gibson Assembly HiFi Master Mix (Invitrogen) was mixed with 50 ng linear plasmid Feeding experiments: Growth curves of M. xanthus NM: pMEX14 DNA in a reaction volume of 10 μL. The amount of insert DNA was were recorded using the microbioreactor BioLector I (m2p-labs) and adjusted to different insert/vector ratios (1 : 1, 3 : 1, 5 : 1). The 48 well flower-shaped microtiter plates (FlowerPlates, m2p-labs). A reaction mixture was incubated for 60 min at 50 °C. Subsequently, defined volume of CYE medium containing kanamycin (50 μgmL 1) the assembled plasmids were introduced into chemically compe- was inoculated with a seed culture to an OD600 nm of 0.1. Each well tent E. coli TOP10 cells. was filled with 990 μL of this cell suspension or with sterile CYE medium, alternatively. 10 μL of 3-HAA or l-tryptophan stock Construction of M. xanthus NM: pMEX14: The E. coli-M. xanthus solutions (60% (v/v) DMSO) were added. The microcultures were shuttle vector pMEX03[17] carrying the pilA-promoter (PpilA) and the incubated at 30 °C, 1000 rpm and a humidity of 85%. The light first 15 codon of the pilA-gene was linearized using the restriction scattering was measured every hour. Each experiment was enzyme ScaI. The synthetically prepared genes natL2 and natAM conducted in triplicate. If necessary, the cultures from the BioLector were amplified with the primer pairs P01/P02 or P03/P04 (Table S2) experiments were transferred to 2 mL-Eppendorf tubes and to add Gibson overhangs. Both genes were cloned into the ScaI- extracted two times with ethyl acetate. The ethyl acetate was restriction site of the linearized pMEX03 via Gibson assembly to evaporated in a vacuum concentrator (Concentrator plus, Eppen- create the vectors pMEX12 and pMEX13 (Figures S1 and S2). dorf) and the dried extract was dissolved in 100 μL methanol for Subsequently, the construct PpilA-natAM was amplified with the HPLC-UV analysis. Additionally, the 3-HAA feeding experiment was primers P05/P06 and integrated into the ScaI-site of pMEX12 to repeated in shake flasks. For this, 50 mL CYE medium containing give the expression plasmid pMEX14 (Figure S3). All plasmids were 50 μgmL 1 kanamycin were inoculated with a preculture of transferred into chemically competent E. coli TOP10 cells and M. xanthus NM: pMEX14 to an OD600 nm of 0.05. The cultures were validated via colony PCR (primer pair P07/P08) and sequencing. The supplemented with 0, 20, 80, 160 and 320 mgL 1 3-HAA and plasmid pMEX14 was introduced into electrocompetent M. xanthus incubated for 3 days at 30 °C and 130 rpm. Product isolation was NM cells according to a previously published protocol.[16] Successful performed with the adsorber resin Amberlite XAD7HP as described plasmid uptake was confirmed by colony PCR using the primer above. Each experiment was performed in three replicates. pairs P09/P10 and P08/P11, respectively (Figure S4). Spectroscopic analyses: LC–MS measurements were conducted in Production of benzoxazoles in M. xanthus: Well grown seed positive mode using an Agilent 1260 Infinity HPLC system cultures of M. xanthus NM: pMEX14 were inoculated into 50 mL CYE combined with a Bruker Daltonics Compact quadrupole time of medium with 50 μgmL 1 kanamycin to an OD600 nm of 0.05. For the flight mass spectrometer. The HPLC was operated with Nucleoshell production of 2a, 20 mgL 1 3-HAA were added as a supplement. RP 18 ec column (100×2 mm, 2.7 μm; Macherey–Nagel) at the For the generation of 2b–f, the cultures were individually fed with following conditions: Flow rate: 0.4 mL/min. Column oven: 40 °C. 50 mgL 1 3-HAA and 50 mgL 1 of another aryl carboxylic acid (2,3- Mobile phases: acetonitrile and water with 0.1% (v/v) formic acid. DHBA, 3-HBA, SA, BA or 3-ClBA). After three days of incubation at Gradient: 0–10 min: 2–98% ACN; 10–15 min: 98% ACN; 15–17 min: 30 °C and 130 rpm, 3% (w/v) of the adsorber resin Amberlite 98–5% ACN; 17–20 min: 5% ACN; 27–28 min: 100–30% ACN; 28– XAD7HP (Sigma–Aldrich) were added, and the cultures were 32 min: 30% ACN. The MS analyses were performed at a capillary incubated for two additional hours. Afterwards, the adsorber resin voltage of 4.5 kV, a desolvation gas (N2) temperature of 220 °C and was collected by filtration and washed with 100 mL water. Elution a dry gas (N2) flow rate of 12 L/min. LC–MS/MS measurements were of adsorbed compounds was performed by adding 100 mL performed with collision energies of 18, 23 or 30 eV. NMR measure- methanol. The bacterial raw extract was concentrated using a rotary ments were carried out at ambient temperature using a Bruker AV evaporator (Heidolph) and analyzed via LC–MS. For isolation of the 700 Avance III HD (CryoProbe) spectrometer, which is equipped compounds 1’e and 2e, and 1’f and 2f, the cultivation was with a 5 mm helium-cooled inverse quadrupol resonance cryop- repeated and upscaled to a volume of 1 L CYE medium. The robe. The NMR spectra were recorded with deuterated chloroform compounds 1’c and 2c, 1’e and 2e, and 1’f and 2f were purified (CDCl3) or methanol (MeOD) as solvent and internal standard via HPLC (Shimadzu) by applying the following chromatographic (chloroform-d: δH 7.24 ppm and δC 77.0 ppm; methanol-d4: δH conditions: Flow rate: 4 mL/min. Mobile phases: acetonitrile (ACN) 3.31 ppm and δC 49.0 ppm). and water with 0.1% (v/v) trifluoroacetic acid. Column: VP250/10 1 Nucleodur C 2-(2,3-Dihydroxybenzamido)-3-hydroxybenzoic acid (1’c): H NMR18 Isis, 5 μm (Macherey-Nagel). Gradient: 0–5 min: 30% ACN; 5–20 min: 30–50% ACN; 20–25 min: 50–100% ACN; 25– (700 MHz, MeOD, 300 K): δ=7.66 (dd, J=7.5, 1.8 Hz, 1H, CH-6), 7.54 27 min: 100% ACN; 27–28 min: 100–30% ACN; 28–32 min: 30% (ddd, J=7.7, 1.8, 0.9 Hz, 1H, CH-13), 7.48 (dd, J=2.5, 1.8 Hz, 1H, CH- ACN. Retention times: 1‘c–14 min; 2c–15 min; 1‘e–19.2 min; 2e– 9), 7.36 (t, J=8.0, 7.8 Hz, 1H, CH-12), 7.21 (t, J=8.3, 7.5 Hz, 1H, CH- 21 min; 1‘f–23.8 min; 2f–24.5 min. 5), 7.18 (dd, J=8.3, 1.8 Hz, 1H, CH-4), 7.04 (ddd, J=8.1, 2.5, 0.9 Hz, 1H, CH-11); 13C NMR (175 MHz, MeOD, 300 K): δ=172.0 (C-7), 169.0 Quantification of 2a produced by M. xanthus: For preparative (C-14), 159.2 (C-10), 151.6 (C-3), 135.9 (C-8), 131.0 (C-12), 129.0 (C-2), isolation of 2a, M. xanthus NM: pMEX14 was cultured in 100 mL 127.1 (C-5), 124.7 (C-4), 124.4 (C-1), 124.2 (C-6), 120.7 (C-11), 119.8 CYE medium supplemented with 50 μgmL 1 kanamycin and (C-13), 115.8 (C-9); HRMS (ESI): m/z calcd for C14H11NO5: 274.0710 [M 20 mgL 1 3-HAA. Product isolation and purification was conducted +H]+; found: 274.0703. as described above. The retention time of 2a was 17.2 min under the chosen HPLC conditions. After purification, compound 2a was 2-Benzamido-3-hydroxybenzoic acid (1’e): 1H NMR (700 MHz, subjected to NMR analysis (Figure S10). To quantify the amount of MeOD, 300 K): δ=8.07 (dd, J=8.3, 1.3 Hz, 2H, CH-9, CH-13), 7.66 2a in bacterial raw extracts, a calibration curve between the UV (dd, J=7.2, 2.2 Hz, 1H, CH-6), 7.63 (dt, J=7.5, 1.3 Hz, 1H, CH-11), peak area at 320 nm and the injected mass of 2a was recorded 7.55 (t, J=8.3, 7.5 Hz, 2H, CH-10, CH-12), 7.22 (t, J=8.0, 7.2 Hz, 1H, ChemBioChem 2023, 24, e202200635 (6 of 8) © 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH Wiley VCH Donnerstag, 16.02.2023 2305 / 281988 [S. 65/67] 1 14397633, 2023, 5, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cbic.202200635 by Technical University Dortmund, Wiley Online Library on [16/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Research Article ChemBioChem doi.org/10.1002/cbic.202200635 CH-5), 7.21 (dd, J=8.0, 2.2 Hz, 1H, CH-4); 13C NMR (175 MHz, MeOD, 100 mL/L glycerol, pH 8). The cells were lysed by ultrasonication (5 300 K): δ=171.4 (C-7), 169.0 (C-14), 151.9 (C-3), 134.5 (C-8), 133.7 cycles of 30 s, 4 °C, 10% amplitude) and the cell debris was (C-11), 129.9 (C-10), 129.9 (C-12), 128.9 (C-9), 128.9 (C-13), 128.8 (C- removed via centrifugation (ThermoScientific Sorvall RC6+ centir- 2), 127.4 (C-5), 124.9 (C-4), 124.2 (C-6), 123.9 (C-1); HRMS (ESI): m/z fuge, rotor F13-14x50cy, 13000 rpm, 20 min, 4 °C). The supernatant calcd for C14H11NO4: 258.0761 [M+H] +; found: 258.0772. was subjected to Ni-nitrilotriacetic acid (NiNTA) affinity chromatog- 1 raphy. For this, 2 mL Protino NiNTA agarose (Macherey–Nagel) were2-(3-Chlorobenzamido)-3-hydroxybenzoic acid (1’f): H NMR transferred into a polypropylene column and equilibrated with (700 MHz, MeOD, 300 K): δ=8.06 (s, 1H, CH-9), 7.99 (d, J=8.0 Hz, 10 mL lysis buffer. Afterwards, the supernatant containing the His- 1H, CH-13), 7.64 (dd, J=8.0, 1.5 Hz, 1H, CH-6), 7.63 (d, J=7.9 Hz, 1H, tagged enzymes was applied. For removal of contaminating CH-11), 7.54 (t, J=7.9 Hz, 1H, CH-12), 7.24 (t, J=8.0 Hz, 1H, CH-5), 13 proteins, the matrix was washed with 5 mL washing buffer I (lysis7.20 (dd, J=8.0, 1.5 Hz, 1H, CH-4); C NMR (175 MHz, MeOD, 300 K): buffer containing 20 mM imidazole) and 5 mL washing buffer II δ=171.1 (C-7), 167.6 (C-14), 152.4 (C-3), 136.8 (C-10), 135.9 (C-8), (lysis buffer containing 40 mM imidazole). Protein elution was 133.4 (C-11), 131.5 (C-12), 129.1 (C-9), 128.0 (C-2), 127.7 (C-5), 127.2 performed by adding 2.5 mL elution buffer (lysis buffer containing (C-13), 125.1 (C-1), 124.3 (C-4), 124.0 (C-6); HRMS (ESI): m/z calcd for 250 mM imidazole). The enzyme solutions were desalted using PD- C14H10ClNO + 4: 292.0371 [M+H] ; found: 292.0372. 10 desalting columns (Cytiva) according to the manufacturer‘s 2-(2,3-Dihydroxyphenyl)benzo[d]oxazole-4-carboxylic acid (2c): 1H specification. For column equilibration, 100 mM Tris·NaCl buffer NMR (700 MHz, MeOD, 300 K): δ=8.04 (dd, J=7.8, 1.0 Hz, 1H, CH- (20 mM Tris, 100 mM NaCl, 100 mL/L glycerol, pH 8) was used. The 6), 7.92 (dd, J=8.0, 1.0 Hz, 1H, CH-4), 7.83 (ddd, J=7.7, 1.8, 0.9 Hz, enzyme solutions were analyzed by SDS PAGE (Figure S8) and the 1H, CH-13), 7.76 (dd, J=2.5, 1.8 Hz, 1H, CH-9), 7.51 (t, J=7.9 Hz, 1H, protein concentration was measured with the UV-Vis spectrometer CH-5), 7.41 (t, J=7.9 Hz, 1H, CH-12), 7.05 (ddd, J=8.1, 2.5, 0.9 Hz, NanoDrop One (Thermo Fisher Scientific) at 280 nm. 1H, CH-11); 13C NMR (175 MHz, MeOD, 300 K): δ=168.0 (C-7), 166.2 In vitro assays: Enzymatic reactions were generally conducted in (C-14), 159.3 (C-10), 152.7 (C-3), 142.7 (C-2), 131.4 (C-12), 128.7 (C-8), 50 mM Tris·HCl buffer (pH 8) with 0.7 μM of each enzyme, 1 mM 128.3 (C-6), 126.0 (C-5), 123.4 (C-1), 120.7 (C-11), 120.4 (C-13), 116.2 ATP and 10 mM MgCl in a total volume of 200 μL. For in vitro (C-4), 115.6 (C-9); HRMS (ESI): m/z calcd for C14H9NO4: 256.0604 [M 2 + synthesis of compounds 1’a and 2a, 1 mM 3-HAA was used as H]+; found: 256.0602. substrate. For synthesis of the derivatives 1’b–f and 2b–f, 0.5 mM 2-Phenylbenzo[d]oxazole-4-carboxylic acid (2e): 1H NMR (700 MHz, 3-HAA and 0.5 mM of the respective benzoic acid-derived building MeOD, 300 K): δ=8.37 (dd, J=7.0, 1.6 Hz, 2H, CH-9, CH-13), 8.04 block were added. Three different combinations of enzymes were (dd, J=7.7, 1.1 Hz, 1H, CH-6), 7.94 (dd, J=8.1, 1.1 Hz, 1H, CH-4), tested: i) MxcE and NatAM, ii) MxcE, NatL2 and NatAM, and iii) 7.63 (m, 1H, CH-11), 7.60 (t, J=7.6, 7.0 Hz, 2H, CH-10, CH-12), 7.52 (t, NatL2 and NatAM. All reactions were incubated for 20 h at 30 °C J=7.9 Hz, 1H, CH-5); 13C NMR (175 MHz, MeOD, 300 K): δ=168.0 (C- and 400 rpm in a thermomixer (Eppendorf). The reactions were 7), 166.1 (C-14), 152.7 (C-3), 142.7 (C-2), 133.5 (C-11), 130.2 (C-10), stopped by addition of 1 V methanol and analyzed by LC–MS. 130.2 (C-12), 129.2 (C-9), 129.2 (C-13), 128.3 (C-6), 127.7 (C-8), 126.0 (C-5), 123.4 (C-1), 116.2 (C-4); HRMS (ESI): m/z calcd for C14H9NO3: 240.0655 [M+H]+; found: 240.0668. Acknowledgements 2-(3-Chlorophenyl)benzo[d]oxazole-4-carboxylic acid (2f): 1H NMR (700 MHz, CDCl3, 300 K): δ=8.27 (s, 1H, CH-9), 8.18 (d, J=7.7 Hz, Financial support from the European Regional Development Fund 1H, CH-13), 8.18 (d, J=7.9 Hz, 1H, CH-6), 7.83 (d, J=7.9 Hz, 1H, CH- (grant EFRE-0300098 to M.N.) is gratefully acknowledged. We 4), 7.60 (d, J=7.7 Hz, 1H, CH-11), 7.54 (t, J=7.9 Hz, 1H, CH-5), 7.52 further thank Professors Stephan Lütz and Oliver Kayser (both TU (t, J=7.7 Hz, 1H, CH-12),; 13C NMR (175 MHz, CDCl3, 300 K): δ=164.3 (C-7), 163.1 (C-14), 150.4 (C-3), 140.6 (C-2), 135.5 (C-10), 133.1 (C-11), Dortmund University) for providing access to their LC/MS facilities. 130.6 (C-12), 128.2 (C-9), 127.8 (C-6), 127.0 (C-8), 126.3 (C-13), 126.1 Open Access funding enabled and organized by Projekt DEAL. (C-5), 120.4 (C-1), 115.6 (C-4); HRMS (ESI): m/z calcd for C14H8ClNO3: 274.0265 [M+H]+; found: 274.0267. Construction of expression plasmids for NatL2, NatAM and MxcE: Conflict of Interest For in vitro analysis, the enzymes NatL2, NatAM and MxcE were produced as His-tagged enzymes in E. coli. For that purpose, the The authors declare no conflict of interest. respective genes were amplified with the primer pairs P12/P13 (natL2), P14/P15 (natAM), and P16/P17 (mxcE) to attach Gibson overhangs. Afterwards, the PCR products were cloned into the Eco53kI restriction site of the plasmid pET28a(+) via Gibson Data Availability Statement assembly. The identity of the plasmids pET28a(+)-natL2, pET28a(+)- natAM, pET28a(+)-mxcE was confirmed via colony PCR (primers The data that support the findings of this study are available in P18/P19) and Sanger sequencing (Figures S5–S7). The validated the supplementary material of this article. plasmids were introduced into E. coli BL21(DE3) by chemical trans- formation. Keywords: benzoxazole · combinatorial biosynthesis · Enzyme production and purification: The expression strains were cultivated in terrific broth (12 gL 1 tryptone/peptone, 24 gL 1 yeast heterologous expression · ligases · Myxococcus xanthus extract, 4 mL/L glycerol, 2.3 gL 1 KH2PO , 12.5 gL 1 4 K2HPO4) at 37 °C until an OD600 nm of 0.6 was reached. Then, the T7 expression system was induced by addition of 1 mM isopropyl-β-d-1-thiogalactopyr- [1] C. Lamberth, J. 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Roberto Villegas-Peñaranda, Accepted manuscript online: December 9, 2022 A. G. Garza, C. N. Boddy, PLoS One 2013, 8, e64858. Version of record online: December 29, 2022 ChemBioChem 2023, 24, e202200635 (8 of 8) © 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH Wiley VCH Donnerstag, 16.02.2023 2305 / 281988 [S. 67/67] 1 14397633, 2023, 5, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cbic.202200635 by Technical University Dortmund, Wiley Online Library on [16/10/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License