Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX
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Expanding the Chemical Space of Nonribosomal Peptide Synthetase-like Enzymes by Domain and Tailoring Enzyme Recombination Johannes W. A. van Dijk† and Clay C. C. Wang*,†,‡ †
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Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, School of Pharmacy, Los Angeles, California 90089, United States ‡ Department of Chemistry, College of Letters, Arts, and Sciences, University of Southern California, Los Angeles, California 90089, United States S Supporting Information *
ABSTRACT: The potential of tailoring enzymes in combination with engineered hybrid nonribosomal peptide synthetase-like enzymes was explored and resulted in methylated and prenylated forms of novel natural products, highlighting the possibilities and limitations of this approach.
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In our previous research, we showed that nonribosomal peptide synthetase (NRPS)-like enzymes from Aspergillus terreus can be easily expressed in our Aspergillus nidulans host and generate their products.11 We also demonstrated that the individual domains can be exchanged to make hybrid enzymes with predictable functionality. Here, we aim to expand the complexity of the products of these enzymes by adding tailoring enzymes. To identify potential modifying enzymes for our small molecules, we looked near the three NRPS-like genes that were previously expressed heterologously. apvA (ATEG_02004.1) produces aspulvinone E (1), which is used in melanin biosynthesis or for further aspulvinone derivatives. Phenguignardic acid (7) is the product of pgnA (ATEG_08899.1), which appears to be a stand-alone enzyme, since the final product is a known secondary metabolite in other species.12 btyA (ATEG_02815.1) produces butrolactone IIa (2), which is a precursor for other butyrolactones found in A. terreus.13 We chose tailoring enzymes from the butyrolactone pathway for recombination with NRPS-like enzyme (hybrids) since there is a neighboring S-adenosyl methionine (SAM)-methyltransferase (ATEG_02816.1/btyB) and the
iosynthetic gene clusters encode a myriad of tailoring enzymes in addition to core backbone-forming enzymes. Among the most frequently found and well-characterized clusters are methyltransferases, prenyltransferases, oxygenases, glycosyltransferase, and aminotransferases.1,2 Several of these types of enzymes are also found in primary metabolism.3 The cytochrome P450 family of monooxygenases plays an important role in drug metabolism,4,5 DNA methyltransferases regulate transcription,6 and prenyltransferases post-translationally modify proteins to confer membrane-binding functionality.7,8 In general, tailoring of small-molecule natural products can be viewed as analogous to post-translational modification of proteins. Elucidating the biosynthetic pathway of natural products often revolves around solving the concerted action of tailoring enzymes, rather than just identifying core enzymes.9 The increasing understanding of all of the different types of modifying enzymes has the potential to increase the diversity and complexity of small molecules that could be generated by metabolic pathway engineering.10 Ideally, a conceptual, novel natural product could be biosynthesized by combining the core enzyme that yields the backbone of interest with the correct set of modifying enzymes. At the same time, a small library of natural products can be generated by expressing a set of enzyme combinations. © XXXX American Chemical Society
Received: May 18, 2018
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DOI: 10.1021/acs.orglett.8b01581 Org. Lett. XXXX, XXX, XXX−XXX
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Organic Letters subsequent prenyltransferase was recently identified as well (ATEG_01730.1/abpB), though not in the vicinity of btyA.14 BtyB methylates butyrolactone IIa at the carboxylic acid to yield butyrolactone II (3). A single subsequent transprenylation by AbpB yields butyrolactone I (4), which can be extracted from wild-type A. terreus. Butyrolactone III, an epoxidated form of butyrolactone I, can also be found in the extract of A. terreus, though the gene responsible for that is not known at this point.13 After reconstituting this pathway in our A. nidulans host, we recombined these tailoring enzymes with functional hybrid NRPS-like enzymes to increase their complexity and gain knowledge on the specificity of these modifying enzymes.
Figure 1. HPLC traces of ethyl acetate extracts of mutant strains with genes from the butyrolactone pathway heterologously expressed at a total spectrum scan by DAD detector. (A) Overexpressed btyA shows butyrolactone IIa (*) production with minor, unidentified, side products. (B) Addition of the methyltransferase results in the production of butyrolactone II (#) as the major product, though * is still present as well. The shift in retention time corresponds to methylation of the carboxylic acid. (C) Further addition of the transprenyltransferase leads to the appearance of mono prenylated butyrolactone I (Δ), though roughly equal amounts of # are observed. Mass spectra, UV absorption, and 1H and 13C NMR of each major compound can be found in the Supporting Information.
butyrolactone II based on retention time and m/z value (Figure 1B). This was confirmed by 2D NMR (Figure S7). The presence of butyrolactone IIa shows that the efficiency of the methyltransferase is not optimal under these conditions, though sufficient methylated product was formed for structural characterization. This successful mutant strain was used for subsequent transformation to add the prenyltransferase ATEG_01730.1. Similar as for the methyltransferase, this gene was amplified from A. terreus genomic DNA and fused to the alcA promoter, flanking regions, and now an AfpyrG marker. Colonies were again analyzed by diagnostic PCR, restreaked, and grown in liquid GMM with MEK induction. LCMS analysis of the extract show the presence of butyrolactone I, based on m/z value and retention time (Figure 1C). This was confirmed by 2-D NMR as well (Figure S8). In addition to butyrolactone I, an equal amount of II can be seen in the LCMS trace, which means the prenyltransferase does not fully convert under these conditions. This could be due to suboptimal relative expression levels, cellular localization, or insufficient culture time. Butyrolactone IIa, however, is completely absent in this strain, suggesting an equilibrium shift toward the final product. It must be noted that ATEG_01730.1 was found to also be responsible for aspulvinone E bisprenylation to aspulvinone H, therefore suggesting a more complex regulatory mechanism. Now that the methyl- and prenyltransferase were shown to be functional in a reconstituted butyrolactone pathway, a previously generated hybrid NRPS-like enzyme was combined with these tailoring enzymes in the same two-step transformation process. CW8504 is a heterologous expression strain with the A domain of pgnA and the T and TE domain of bytA, previously shown to produce a novel compound (phenylbutyrolactone IIa (5)).11 The LCMS traces show the
Engineering of biosynthesis is usually accompanied by a loss of efficiency and thus yield. A robust heterologous host with the capacity to produce gram/liter titers is therefore employed here for pathway engineering.15 As a control and proof of concept, the butyrolactone I pathway from Aspergillus terreus was reconstituted in our Aspergillus nidulans host. The butyrolactone IIa producing mutant strain CW8502 (Figure 1A), that was previously developed in our laboratory, underwent two more cycles of transformation. First, the methyltransferase ATEG_02816.1 was amplified from A. terreus genomic DNA. This gene was merged with an alcA promoter, an Af pyroA marker, and flanking regions to facilitate homologous recombination right after the BtyA gene, thereby recycling the 3′ AfpyrG marker. Colonies were analyzed by diagnostic PCR and for their inability to grow without uridine to confirm that the Af pyrG marker was recycled. A correct colony was restreaked and cultured in liquid glucose minimal media (GMM) as described before, and overexpression of btyA and the methyltransferase btyB was induced by addition of methylethylketone (MEK). Extract analysis by LCMS indicated the production of B
DOI: 10.1021/acs.orglett.8b01581 Org. Lett. XXXX, XXX, XXX−XXX
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Organic Letters appearance of a new peak with an m/z value and retention time that corresponds to methylated product (Figure 2B).
Figure 3. HPLC traces of ethyl acetate extracts of mutant strains with a novel hybrid NRPS-like gene combined the butyrolactone pathway heterologously expressed at a total spectrum scan by DAD detector. (A) TapvA/pgnA hybrid expressed in the A. nidulans host yields hydroxyphenguignardic acid (*) as its major product. (B) Methyltransferase addition did not result in the expected product. (C) Prenyltransferase seems only moderately functional in this context, with only minor prenylated product (Δ) detectable. Mass spectra, UV absorption, and 1H and 13C NMR of each indicated compound can be found in the Supporting Information.
Figure 2. HPLC traces of ethyl acetate extracts of mutant strains with a hybrid NRPS-like gene combined the butyrolactone pathway heterologously expressed at a total spectrum scan by DAD detector. (A) Expression of pgnA/btyA hybrid gene shows phenylbutyrolactone IIa production. (B) Added methyltransferase results in phenylbutyrolactone II production which shows a similar retention shift as butyrolactone II. However, no prenylation was observed on the phenyl side chain. (C) Mass spectra, UV absorption, and 1H and 13C NMR of each major compound can be found in the Supporting Information.
hydroxyphenguignardic acid, no methylation was observed. This can be due to the methyltransferase recognizing a specific molecule, that it has some protein−protein interaction with the C-terminal end of the NRPS-like enzyme, or due to some other form of colocalization that does not occur with a slightly different NRPS-like enzyme or in a heterologous host. The prenyltransferase was already shown to act on multiple targets with hydroxyphenyl side chains, since both butyrolactones and aspulvinones are targets in A. terreus. Hydroxyphenguignardic acid can be added to that group, though the full scope of the targets of this prenyltransferase remains to be explored. Coexpression of ATEG_1730.1 with (part of the) terrequinone A pathway16 could provide some answers. Reciprocally, coexpression of prenyltransferases tdiB and tdiE with (hybrid) enzymes from this study could lead to more novel products and information on the specificity of those prenyltransferases. What this research shows is the capability (and some limitations) of enzyme recombination to expand the chemical space of engineered natural products. Previously, these types of tailoring enzymes have been recombined with native, small NRPS. FtmPS from Neosartorya fischeri produces a cyclic dipeptide brevianamide, which can be prenylated in three different ways by coexpression of three prenyltransferases from A. nidulans.17 In that study, prenylation was also not complete, with the nonprenylated dipeptide still being the main product, similar to what was found in this study. The use of hybrid NRPS-like enzymes in this type of pathway engineering has not been reported before though. A wider, more systematic and automated screening of domain and tailoring enzyme combinations together with structural data of the enzymes should lead to a more diverse library of this type of compound which can be used for bioactivity screening.
Scale up, purification, and NMR analysis of the new compound confirmed methylated phenylbutyrolactone II (6) (Figure S9). The previously reported instability of phenylbutyrolactone IIa is not observed in the methylated molecule. However, addition of the prenyltransferase did not result in any prenylated product (Figure 2C), which was expected since the hydroxyl group on the phenyl side chain is absent and is most likely necessary for aromatic ring prenylation. This recombination approach was applied to a different hybrid NRPS-like enzyme, first reported here. A bishydroxylated form of phenguignardic acid (8) was generated by combing the A domain of apvA, which was shown to be substitutable with the btyA A domain since they activate the same HPPA unit, with the T and TE domain of pgnA (Figure 3A). In this case, the methyltransferase did not methylate the carboxylic acid side chain of the heterocyclic core of the molecule (Figure 3B). This indicates a high specificity of the methyltransferase. Prenyl transfer to this hybrid molecule was achieved, though at a much lower efficiency under these conditions than for the native butyrolactone pathway (Figure 3C), resulting in prenylhydroxyphenguignardic acid (9). Surprisingly, prenylation occurred on the other side of the molecule compared to butyrolactone I, though the conformation of this molecule is likely different. In conclusion, we showed that the butyrolactone pathway can be partially reconstituted in A. nidulans, with the tailoring enzymes working specifically on their target functional groups. The carboxylic acid on the butyrolactone core in phenylbutyrolactone IIa was methylated, despite the absence of the hydroxyl groups on the aromatic side chains. However, when the core was slightly changed but the side chains were intact in C
DOI: 10.1021/acs.orglett.8b01581 Org. Lett. XXXX, XXX, XXX−XXX
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Organic Letters
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b01581. A. nidulans strains and primers used in this study; diagnostic PCR results, sequencing data, compound characterization with spectral data and 2-D NMR (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Johannes W. A. van Dijk: 0000-0001-8558-4655 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Research in the Wang group is funded by the following grants: NIH Grant No. POIGM084077 and NSF Emerging Frontiers in Research and Innovation-MIKS (Grant No. 1136903).
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REFERENCES
(1) Brakhage, A. A. Nat. Rev. Microbiol. 2013, 11, 21−32. (2) Lam, K. S. Trends Microbiol. 2007, 15, 279−289. (3) Green, K. D.; Garneau-Tsodikova, S. Posttranslational Modification of Proteins. In Comprehensive Natural Products II: Chemistry and Biology. Vol 5: Amino Acids, Peptides and Proteins; Mander, L., Liu, H. W., Eds.; Elsevier Science Bv: Amsterdam, 2010; pp 433−468. (4) Meunier, B.; de Visser, S. P.; Shaik, S. Chem. Rev. 2004, 104, 3947−3980. (5) Zanger, U. M.; Schwab, M. Pharmacol. Ther. 2013, 138, 103− 141. (6) Jones, P. A. Nat. Rev. Genet. 2012, 13, 484−492. (7) Maurer-Stroh, S.; Washietl, S.; Eisenhaber, F. Genome Biology 2003, 4, 212. (8) Fierke, C. A.; Zverina, E. A.; Lamphear, C. L.; Wright, E. N.; Danowitz, A. M.; Jennings, B. C. Abstracts of Papers. Proceedings of the 247th National Meeting of the American Chemical Society; Dallas, TX, Mar 16−20, 2014; American Chemical Society: Washington, DC, 2014; Vol. 247, p 1 (9) Keller, N. P.; Turner, G.; Bennett, J. W. Nat. Rev. Microbiol. 2005, 3, 937−947. (10) Meyer, V. Biotechnol. Adv. 2008, 26, 177−185. (11) van Dijk, J. W. A.; Guo, C. J.; Wang, C. C. C. Org. Lett. 2016, 18, 6236−6239. (12) Molitor, D.; Liermann, J. C.; Berkelmann-Lohnertz, B.; Buckel, I.; Opatz, T.; Thines, E. J. Nat. Prod. 2012, 75, 1265−1269. (13) Guo, C.-J.; Knox, B. P.; Sanchez, J. F.; Chiang, Y.-M.; Bruno, K. S.; Wang, C. C. C. Org. Lett. 2013, 15, 3562−3565. (14) Guo, C. J.; Sun, W. W.; Bruno, K. S.; Oakley, B. R.; Keller, N. P.; Wang, C. C. C. Chemical Science 2015, 6, 5913−5921. (15) Chiang, Y. M.; Oakley, C. E.; Ahuja, M.; Entwistle, R.; Schultz, A.; Chang, S. L.; Sung, C. T.; Wang, C. C. C.; Oakley, B. R. J. Am. Chem. Soc. 2013, 135, 7720−7731. (16) Balibar, C. J.; Howard-Jones, A. R.; Walsh, C. T. Nat. Chem. Biol. 2007, 3, 584−592. (17) Wunsch, C.; Mundt, K.; Li, S. M. Appl. Microbiol. Biotechnol. 2015, 99, 4213−4223.
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DOI: 10.1021/acs.orglett.8b01581 Org. Lett. XXXX, XXX, XXX−XXX
