Combinatorial engineering of hybrid mevalonate pathways in Escherichia coli for protoilludene production
© Yang et al. 2016
Received: 10 November 2015
Accepted: 4 January 2016
Published: 19 January 2016
Protoilludene is a valuable sesquiterpene and serves as a precursor for several medicinal compounds and antimicrobial chemicals. It can be synthesized by heterologous expression of protoilludene synthase in Escherichia coli with overexpression of mevalonate (MVA) or methylerythritol-phosphate (MEP) pathway, and farnesyl diphosphate (FPP) synthase. Here, we present E. coli as a cell factory for protoilludene production.
Protoilludene was successfully produced in E. coli by overexpression of a hybrid exogenous MVA pathway, endogenous FPP synthase (IspA), and protoilludene synthase (OMP7) of Omphalotus olearius. For improving protoilludene production, the MVA pathway was engineered to increase synthesis of building blocks isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) by sequential order permutation of the lower MVA portion (MvL), the alteration of promoters and copy numbers for the upper MVA portion (MvU), and the coordination of both portions, resulting in an efficient entire MVA pathway. To reduce the accumulation of mevalonate observed in the culture broth due to lower efficiency of the MvL than the MvU, the MvL was further engineered by homolog substitution with the corresponding genes from Staphylococcus aureus. Finally, the highest protoilludene production of 1199 mg/L was obtained from recombinant E. coli harboring the optimized hybrid MVA pathway in a test tube culture.
This is the first report of microbial synthesis of protoilludene by using an engineered E. coli strain. The protoilludene production was increased by approx. Thousandfold from an initial titer of 1.14 mg/L. The strategies of both the sequential order permutation and homolog substitution could provide a new perspective of engineering MVA pathway, and be applied to optimization of other metabolic pathways.
KeywordsProtoilludene Escherichia coli Mevalonate pathway Sequential order permutation Homolog substitution
Protoilludene derivatives, including illudins, marasmanes and melleolides, are known to exert antitumor and antimicrobial activities [1–3]. For example, the most brilliant potential anticancer agent illudin S, which is first isolated from Omphalotus olearius mushroom, has been studied extensively owing to its cytotoxicity to various tumor cell types . These biological properties and medicinal potential have attracted considerable attention since the late 1960s. Illudins, marasmanes and melleolides can be synthesized from protoilludene by different oxygenation reactions. For example, P450 monooxygenases for the biosynthesis of illudin have been identified from O. olearius . However, protoilludene is naturally produced in a small quantity and its purification from biological material suffers from low yields. Hence, metabolic engineering of microorganisms, such as Escherichia coli, is an alternative and attractive route for the production of protoilludene.
Protoilludene biosynthesis begins with the formation of the universal precursors, isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which can be generated via the methylerythritol-phosphate (MEP) pathway and the mevalonate (MVA) pathway . Isopentenyl pyrophosphate and dimethylallyl pyrophosphate are condensed to form farnesyl diphosphate (FPP) by FPP synthase. Linear FPP undergoes multiple electrophilic cyclizations and rearrangements to generate tricyclic protoilludene with an action of protoilludene synthase, which has been isolated from various species including O. olearius, Armillaria gallica, and Stereum hirsutum [7–9]. O. olearius protoilludene synthase (OMP7) exhibits a superior catalytic efficiency (Kcat/Km) of (13.0 ± 2.0) × 104 M−1 s−1 among those protoilludene synthases (Additional file 1: Table S1) .
Strains used in this study
E. coli AO
E. coli DH5α harboring pTAO
E. coli AO/NA
E. coli DH5α harboring pTAO and pSNA
E. coli AO/MvL1–6
E. coli DH5α harboring pTAO and pSMvL1–6
E. coli AO/L1–L6
E. coli DH5α harboring pTAO, pSMvL1–6 and pBMvUL
E. coli AO/M1–M13
E. coli DH5α harboring pTAO and pSMvL1–13-MvUM
E. coli AO/H1–H13
E. coli DH5α harboring pTAOMvUH and pSMvL1–13
Results and discussion
Establishment of a protoilludene biosynthesis pathway in E. coli
Thus, the protoilludene synthesis plasmid pTAO was co-transformed with plasmid pSNA , which encodes a hybrid exogenous MVA pathway for sufficient supply of IPP and DMAPP, into E. coli DH5α, resulting in the strain E. coli AO/NA. Gas chromatographic analysis showed a specific peak with retention time of 5.7 min, which was subsequently confirmed as protoilludene by GC–MS (Fig. 2b). For 48 h of culture, the strain E. coli AO/NA produced 517 mg/L of protoilludene with an undesired accumulation of mevalonate as much as 571 mg/L (Fig. 2c), indicating the suboptimal performance of MVA pathway encoded by pSNA. It is thus required to redesign the MVA pathway, especially the lower portion of the MVA pathway for protoilludene production.
Optimization of the MvL portion of the MVA pathway by sequential order permutation
Coordination of MvU and MvL portions of MVA pathway for protoilludene production
Both MvUs and MvLs were then expressed in all combinations in E. coli to find an optimal combination of the two portions for protoilludene production. As the MvUL plasmid (pBMvUL) is compatible with the lower MVA portion plasmids (pSMvL1–6) and the protoilludene plasmid (pTAO), E. coli can be transformed with the three plasmids for the combination of MvUs and MvLs in protoilludene production. However, the MvUM plasmid (pSMvUM) is not compatible with pSMvL1–6 derived from the same cloning vector (pSTV28) and MvUM and MvL1–6 are combined in pSMvL1–6-MvUM (Additional file 1: Fig. S4). The MvUH portion was cloned into pTAO plasmid, resulting in pTAO-MvUH, because the MvUH plasmid (pTMvUH) is not compatible with the same vector originated pTAO plasmid (Additional file 1: Fig. S4). Escherichia coli AO/H1–H6 strains harboring pTAO-MvUH and pSMvL1–6 produced a little amount of protoilludene (<35 mg/L; Fig. 4c), accompanying with accumulation of a large amount of mevalonate (>1300 mg/L). It indicated the MvUH produced too much mevalonate beyond the capacity of MvLs and the metabolic unbalance between MvUH and MvLs caused even a significant decrease of cell growth (Additional file 1: Table S2). In contrast, there was no significant accumulation of mevalonate in the strains of E. coli AO/L1-L6 (pTAO/pSMvL1–6/pBMvUL) and E. coli AO/M1–M6 (pTAO/pSMvL1-6-MvUM), which suggested the lower capacity of the upper portions MvUL and MvUM than the lower portion MvLs (Fig. 4d). In contrast, strains E. coli AO/L1–L6 and E. coli AO/M1–M6 did not exhibit significant mevalonate accumulation (Fig. 4d). However, the poor mevalonate supply from MvUL compared to MvUM seems to restrict the protoilludene production. The highest protoilludene production of 721 mg/L was observed in E. coli AO/M2, which represented a 1.4-fold increase to the production from E. coli AO/NA.
Homolog substitution of the lower MVA portion genes
It is demonstrated the feasibility of producing protoilludene in engineered E. coli. Heterologous expression of the MVA pathway encoded by pSNA enabled the strain E. coli AO/NA to produce 517 mg/L of protoilludene, but mevalonate was accumulated in a significant amount as 571 mg/L due to the unbalanced upper and lower portions of the MVA pathway. To create a balanced efficient MVA pathway, we sequentially permuted the order of genes in the lower portion of the MVA pathway (MvL) and coordinated their expression with the upper portion of the MVA pathway (MvU) by alternations of copy-number and promoter of plasmids. Through this approach, 721 mg/L of protoilludene was produced with reduced accumulation of mevalonate in the strain E. coli AO/M2. The substitution of mevalonate kinase from S. pneumoniae with the homolog from S. aureus further increased protoilludene production to 1199 mg/L. These results suggest that the optimized MVA pathway is efficient to supply IPP and DMAPP for protoilludene production and also can serve as a platform IPP/DMAPP synthesis pathway for production of other valuable terpenes.
Bacterial strains and growth conditions
Escherichia coli DH5α were grown in 2YT medium (16 g tryptone, 10 g yeast extract, and 5 g sodium chloride per 1L) at 37 °C for plasmid construction, and at 30 °C for protoilludene production. The seed culture grown overnight at 37 °C was inoculated with an optical density at 600 nm (OD600) of 0.1 into 2YT medium containing 2 % (v/v) glycerol. Escherichia coli strains (Table 1) harboring the lower portion of the MVA pathway were cultured with addition of 4 mM mevalonate. Ampicillin (100 μg/mL), chloramphenicol (50 μg/mL), kanamycin (50 μg/mL) and 0.2 mM IPTG were added as required. To harvest protoilludene produced during culture, 1 mL of decane was initially overlaid on 4 mL of culture broth. Cell growth was determined by measuring the OD600. All experiments were carried out in duplicate.
Construction of plasmids
Basic molecular biology procedures, including restriction enzyme digestion and bacterial transformation, were carried out as described in the literature . DNA fragments were amplified by PCR using Pfu DNA polymerase (SolGent, Daejeon, Korea) according to the manufacturer’s instructions. BglBricks assembly  was applied for construction of various plasmids. The schematic diagram of the constructs is shown in figures and the detailed construction process was depicted in Additional file 1. All plasmids and primers used in this study are described in Additional file 1: Table S4.
Identification and quantification of protoilludene
The decane phase of the two-phase culture was collected and centrifuged for 10 min at 12,000 rpm to remove cell debris, and subsequently subjected to gas chromatography (GC) and gas chromatography-mass spectrometry (GC–MS). The production of protoilludene was quantified using an Agilent Technologies 7890A gas chromatograph equipped with a flame ionization detector (FID). One μL of sample was injected at a split ratio of 1:10, and separated on a 19091 N-133 HP-INNOWAX column (length, 30 m; internal diameter, 0.25 mm; film thickness, 250 μm). The oven temperature was initially held at 80 °C for 1 min and was increased at a rate of 10 °C/min to 250 °C, where it was held for 1 min. Nitrogen was used as the carrier gas with an inlet pressure of 39 psi. The detector temperature was maintained at 260 °C. GC–MS analysis was run on a GCMS-2010 ultra mass spectrometer (Shimadzu, Tokyo, Japan). Purified protoilludene was used as the standard compound to construct the standard curve (R2 > 0.99) for the estimation of protoilludene production (Additional file 1: Fig. S1).
Quantification of mevalonate
Mevalonate concentration was determined by GC analysis. Culture filtrate was adjusted to pH 2 with 3 M HCl, incubated at 45 °C for 1 h, saturated with anhydrous Na2SO4, and extracted with ethyl acetate. The resulting samples were analyzed for mevalonate concentration using an Agilent Technologies 7890A gas chromatograph. The analytical temperature of the GC was controlled at an initial temperature of 180 °C for 1 min, then ramped to 200 °C gradually at 2.5 °C/min and held for 2 min. The detector temperature was maintained at 260 °C.
SWK and CW conceived the idea and designed the experiments. LY carried out the experiments. LY, CW and JZ analyzed the data. LY, SWK and CW drafted the manuscript. All authors read and approved the final manuscript.
This work was supported by a Grant (NRF-2013R1A1A2008289) and a Grant (NRF-2012M1A2A2671831) from the National Research Foundation, MSIP and a Grant from the Next-Generation BioGreen 21 Program (SSAC, Grant#: PJ01106201), Rural Development Administration, Korea.
The authors declare that they have no competing interests.
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- Reina M, Orihuela JC, Gonzalez-Coloma A, de Ines C, de la Cruz M, del Val GA, Torno JR, Fraga BM. Four illudane sesquiterpenes from coprinopsis episcopalis. Phytochemistry. 2004;65:381–5.View ArticleGoogle Scholar
- Wang XN, Shen JH, Du JC, Liu JK. Marasmane sesquiterpenes isolated from Russula foetens. J Antibiot. 2006;59:669–72 (Tokyo).View ArticleGoogle Scholar
- Bohnert M, Nutzmann HW, Schroeckh V, Horn F, Dahse HM, Brakhage AA, Hoffmeister D. Cytotoxic and antifungal activities of melleolide antibiotics follow dissimilar structure-activity relationships. Phytochemistry. 2014;105:101–8.View ArticleGoogle Scholar
- Schobert R, Knauer S, Seibt S, Biersack B. Anticancer active illudins: recent developments of a potent alkylating compound class. Curr Med Chem. 2011;18:790–807.View ArticleGoogle Scholar
- Wawrzyn GT. Discovery and characterization of sesquiterpenoid biosynthetic pathways from Basidiomycota. Retrieved from the University of Minnesota Digital Conservancy. http://hdl.handle.net/11299/162701.
- Martin VJ, Pitera DJ, Withers ST, Newman JD, Keasling JD. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Nat Biotechnol. 2003;21:796–802.View ArticleGoogle Scholar
- Engels B, Heinig U, Grothe T, Stadler M, Jennewein S. Cloning and characterization of an Armillaria gallica cDNA encoding protoilludene synthase, which catalyzes the first committed step in the synthesis of antimicrobial melleolides. J Biol Chem. 2011;286:6871–8.View ArticleGoogle Scholar
- Wawrzyn GT, Quin MB, Choudhary S, Lopez-Gallego F, Schmidt-Dannert C. Draft genome of Omphalotus olearius provides a predictive framework for sesquiterpenoid natural product biosynthesis in basidiomycota. Chem Biol. 2012;19:772–83.View ArticleGoogle Scholar
- Quin MB, Flynn CM, Wawrzyn GT, Choudhary S, Schmidt-Dannert C. Mushroom hunting by using bioinformatics: application of a predictive framework facilitates the selective identification of sesquiterpene synthases in basidiomycota. Chembiochem. 2013;14:2480–91.View ArticleGoogle Scholar
- Nowroozi FF, Baidoo EE, Ermakov S, Redding-Johanson AM, Batth TS, Petzold CJ, Keasling JD. Metabolic pathway optimization using ribosome binding site variants and combinatorial gene assembly. Appl Microbiol Biotechnol. 2014;98:1567–81.View ArticleGoogle Scholar
- Wang C, Yoon SH, Jang HJ, Chung YR, Kim JY, Choi ES, Kim SW. Metabolic engineering of Escherichia coli for alpha-farnesene production. Metab Eng. 2011;13:648–55.View ArticleGoogle Scholar
- Anthony JR, Anthony LC, Nowroozi F, Kwon G, Newman JD, Keasling JD. Optimization of the mevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene. Metab Eng. 2009;11:13–9.View ArticleGoogle Scholar
- Lim HN, Lee Y, Hussein R. Fundamental relationship between operon organization and gene expression. Proc Natl Acad Sci USA. 2011;108:10626–31.View ArticleGoogle Scholar
- Yoon SH, Lee SH, Das A, Ryu HK, Jang HJ, Kim JY, Oh DK, Keasling JD, Kim SW. Combinatorial expression of bacterial whole mevalonate pathway for the production of beta-carotene in E. coli. J Biotechnol. 2009;140:218–26.View ArticleGoogle Scholar
- Nishizaki T, Tsuge K, Itaya M, Doi N, Yanagawa H. Metabolic engineering of carotenoid biosynthesis in Escherichia coli by ordered gene assembly in Bacillus subtilis. Appl Environ Microbiol. 2007;73:1355–61.View ArticleGoogle Scholar
- Anderson JC, Dueber JE, Leguia M, Wu GC, Goler JA, Arkin AP, Keasling JD. BglBricks: a flexible standard for biological part assembly. J Biol Eng. 2010;4:1.View ArticleGoogle Scholar
- Andreassi JL 2nd, Dabovic K, Leyh TS. Streptococcus pneumoniae isoprenoid biosynthesis is downregulated by diphosphomevalonate: an antimicrobial target. Biochemistry. 2004;43:16461–6.View ArticleGoogle Scholar
- Voynova NE, Rios SE, Miziorko HM. Staphylococcus aureus mevalonate kinase: isolation and characterization of an enzyme of the isoprenoid biosynthetic pathway. J Bacteriol. 2004;186:61–7.View ArticleGoogle Scholar
- Sambrook J, Russell DW. Molecular cloning: a laboratory manual. New York: Cold Spring Harbor Laboratory Press; 2001.Google Scholar