The enormous pool of chemical diversity found in nature serves as an excellent inventory for accessing biologically active compounds. This chemical inventory, primarily found in microorganisms and plants, is generated by a broad range of enzymatic pathways under precise genetic and protein-level control. In vitro pathway reconstruction can be used to characterize individual pathway enzymes, identify pathway intermediates, and gain an increased understanding of how pathways can be manipulated to generate natural product analogs. Moreover, through in vitro approaches, it is possible to achieve a diversification that is not restricted by toxicity, limited availability of intracellular precursors, or preconceived (by nature) regulatory controls. Additionally, combinatorial biosynthesis and high-throughput techniques can be used to generate both known natural products and analogs that would not likely be generated naturally.
We have exploited advances in DNA sequencing technology, genome mining, and microbial metagenomics to identify natural product biosynthetic pathways and open up a vast portion of the unexploited natural product space. In vitro reconstruction1 of natural product biosynthetic pathways (Figure MB-1) can be used to: 1) generate highly pure biosynthetic intermediates and final products; 2) limit competing side reactions and cell metabolites (including some that are potentially toxic) that often occur in vivo, thereby complicating pathway understanding; 3) overcome unknown or poorly controlled regulatory constraints, including posttranslational limitations such as protein–protein interactions; and 4) enable unnatural synthetic building blocks to be used. An example of our approach is in the diversification of type III polyketide synthase (PKS) reactions performed in high throughput2.
Figure MB-1. Schematic of in vitro reconstitution and biosynthetic pathway engineering for generating (unnatural) terpenoids, polyketides and nonribosomally generated peptides. Enzyme notation: terpenoid synthase (TS); halogenase (Hal); ligase (L); thioesterase (TE); aromatase (ARO); cyclase (CYC); methyltransferase (MT); oxygenase/oxidase (Ox); reductase (Re); glycosyltransferase (GT); epimerase (E); peroxidase (PO); prenyltransferase (PT) and carbamoyltransferase (CT).
Polyketide Synthesis on a Chip
The generation of biological diversity by engineering the biosynthetic gene assembly of metabolic pathway enzymes has led to a wide range of “unnatural” variants of natural products. However, current biosynthetic techniques do not allow the rapid manipulation of pathway components and are often fundamentally limited by the compatibility of new pathways, their gene expression, and the resulting biosynthetic products and pathway intermediates with cell growth and function. To overcome these limitations, we have developed an entirely in vitro approach to synthesize analogs of natural products in high throughput1-4. For example, Figure MB-2 highlights the central location of the type III PKS in aromatic metabolism, and in particular the pathway connections among polyphenols, lignin biosynthesis, and oligophenol biosynthesis.
Figure MB-2. Central metabolic pathways involving phenolic compounds, and branch point to type III PKS pathway.
Using several type III polyketide synthases (PKS) together with oxidative post-PKS tailoring enzymes, we performed 192 individual and multienzymatic reactions on a single glass microarray2. Subsequent array-based inhibition screening with a human tyrosine kinase led to the identification of three compounds that acted as modest inhibitors in the low micromolar range. Receptor tyrosine kinases are critical targets for the regulation of cell survival. Cancer patients with abnormal receptor tyrosine kinases (RTK) tend to have more aggressive disease with poorer clinical outcomes. As a result, human epidermal growth factor receptor kinases, such as EGFR (HER1), HER2, and HER3, represent important therapeutic targets (Figure MB-3)4. We have developed an in vitro route to the synthesis and subsequent screening of unnatural polyketide analogues with N-acetylcysteamine (SNAc) starter substrates and malonyl-coenzyme A (CoA) and methylmalonyl-CoA as extender substrates3. The resulting polyketide analogues possessed a similar structural polyketide backbone (aromatic-2-pyrone) with variable side chains. Screening chalcone synthase (CHS) reaction products against BT-474 cells resulted in identification of several trifluoromethylcinnamoyl-based polyketides that showed strong suppression of the HER2-associated PI3K/AKT signaling pathway, yet did not inhibit the growth of non-transformed MCF-10A breast cells. This approach, therefore, enables the rapid construction of analogs of natural products as potential pharmaceutical lead compounds.
Figure MB-3. Type III PKS generation of highly potent anticancer compounds. Upper left is a cartoon depicting the potential interaction of a trifluormethylcinnamoyl-based pyrone against the HER receptor tyrosine kinase isoforms. Right panels depict very low IC50 values (~30 nM) against BT-474 (HER-overexpressing cell line) vs. non-HER overexpressing cell line (MCF-7) and a non-transformed cell line (MCF-10A).
Polyphenol-Based Antimicrobial Compounds
Plant polyphenols are known to have varying antimicrobial potencies, including direct antibacterial activity, synergism with antibiotics and suppression of bacterial virulence. We performed the in vitro oligomerization of resveratrol catalyzed by soybean peroxidase, and the two isomers (resveratrol-trans-dihydrodimer and pallidol) produced were tested for antimicrobial activity5. The resveratrol-trans-dihydrodimer displayed antimicrobial activity against the Gram-positive bacteria Bacillus cereus, Listeria monocytogenes, and Staphylococcus aureus (minimum inhibitory concentration (MIC) = 15.0, 125, and 62.0 μM, respectively) and against Gram-negative E. coli (MIC = 123 μM, upon addition of the efflux pump inhibitor Phe-Arg-β-naphthylamide) (Figure MB-4). In contrast, pallidol had no observable antimicrobial activity against all tested strains. Transcriptomic analysis implied downregulation of ABC transporters, genes involved in cell division and DNA binding proteins. Flow cytometry analysis of treated cells revealed a rapid collapse in membrane potential and a substantial decrease in total DNA content. The active dimer showed >90% inhibition of DNA gyrase activity, in vitro, by blocking the ATP binding site of the enzyme. We thus propose that the resveratrol-trans-dihydrodimer acts to: (1) disrupt membrane potential; and (2) inhibit DNA synthesis. In summary, we introduce the mechanisms of action and the initial evaluation of an active bactericide, and a platform for the development of polyphenolic antimicrobials, which can play a role in expanding the functional range of phytochemicals6.
Figure MB-4. (A) Schematic representation of the mechanisms of defense of bacteria against antibiotics; efflux pumps are dashed yellow square; (B) Scheme of the enzymatic oligomerization of resveratrol mediated by soybean peroxidase (SBP) in aqueous buffer. The major products are resveratrol-trans-dihydrodimer, and pallidol; (C) MIC and IC50 values of the resveratrol-trans-dihydrodimer against Gram-positive B. cereus, L. monocytogenes, S. aureus, and the Gram-negative E. coli (parenthetical values are indicated in µg/mL). Efflux pump inhibitors: Phe-Arg-β-naphthylamide (PABN, 48.1 µM) for E. coli, 1(1-naphthylmethyl)-piperazine (NMP, 441.9 µM) for B. cereus, reserpine (16 µM) for L. monocytogenes, and piperine (350 µM) for S. aureus.
- S.-J. Kwon, M. Mora-Pale, M.-Y. Lee, and J.S. Dordick (2012), "Expanding nature's small molecule diversity via in vitro biosynthetic pathway engineering", Curr. Opin. Chem. Biol. 16, 186-195.
- S.J. Kwon, M.Y. Lee, B. Ku, D.H. Sherman, and J.S. Dordick (2007), “High-throughput, microarray-based synthesis of natural product analogues via in vitro metabolic pathway construction”, ACS Chem. Biol. 2, 419-425.
- M.-I. Kim, S.-J. Kwon, and J.S. Dordick (2009), "In vitro precursor-directed synthesis of polyketide analogues with Coenzyme A regeneration for the development of antiangiogenic agents", Org. Lett. 11, 3806-3809.
- S.J. Kwon, M.-I. Kim, B. Ku, L. Coulombel, J.-H. Kim, J.H. Shawky, R.J. Linhardt, and J.S. Dordick (2009), “Unnatural polyketide analogs selectively target the HER signaling pathway in human breast cancer cells”, ChemBioChem, 11, 573-580.
- M. Mora-Pale, N. Bhan, S. Masuko, P. James, J. Wood, S. McCallum, R.J. Linhardt, J.S. Dordick, and M.A. Koffas, (2015) "Antimicrobial mechanism of resveratrol-trans-dihydrodimer produced from peroxidase-catalyzed oxidation of resveratrol", Biotechnol Bioeng. 112, 2417-2428.
- M. Mora-Pale, S.P. Sanchez-Rodriguez, R.J. Linhardt, J.S. Dordick, and M.A. Koffas, (2013) "Metabolic engineering and in vitro biosynthesis of phytochemicals and non-natural analogues", Plant Sci. 210, 10-24.