6943 ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 300, No.1, January, pp. 416-422, 1993 Overproduction of Soluble Trichodiene Synthase from Fusarium sporotrichioides in Escherichia colr David E. Cane,*,2 Zhen Wu,* John S. Oliver,* and Thomas M. Hohnt *Department 01 Chemistry, Box H, Brown University, Providence, Rhode Island 02912; and tMycotoxin Research Unit, USDA/ARS, National Center lor Agricultural Utilization Research, Peoria, Illinois 61604 Received August 10, 1992, and in revised form September 18, 1992 1) to any of 200 known cyclic sesquiterpenes. Although Trichodiene synthase is a sesquiterpene cyclase iso­ only a relative handful of these enzymes have been iso­ latedfrom variousfungal specieswhichcatalyzes thecy­ lated and subjected to mechanistic study, they are all op­ clization of farnesyl diphosphate (FPP) to trichodiene. erationally soluble proteins, either monomers or homo­ The trichodiene synthase gene (To.x5) ofFusarium spo­ dimers of subunit M r 40,000-60,000, and require no rotrichioides has previously heen cloned and expressed cofactor other than a divalent metal cation, Mg2+ usually as 0.05-0.1% oftotal cell protein in Escherichia coli. We being preferred (1-3). By far the best studied of these have used polymerase chain reaction to amplify the tri­ cyclases has been trichodiene synthase, a fungal protein chodiene codingsequencecarriedon the plasmidpTS56­ responsible for the conversion ofFPP to trichodiene (3), 1. The resulting DNA, carrying a Bamm restriction site the parent hydrocarbon of the trichothecane family of and the T7 gene 10 ribosome binding site and transla­ mycotoxins (4, 5). Extensive mechanistic studies have tional spacer element immediately upstream ofthe ATG supported a cyclization mechanism in which FPP is ini­ start codon as well as a HindIll siteadjacent to thetrans­ lational stop codon, was inserted into the corresponding tially rearranged to its tertiary allylic isomer, nerolidyl sites of the expression vector pLM1. The latter vector diphosphate (NPP, 2), which in turn undergoes further carried the promoter and translational leader sequence ionization, cyclization, and rearrangement to give tricho­ diene (6-9) (Scheme I). All the various electrophilic re­ from T7 gene 10 and the E. coli rmBT1 T 2 tandem tran­ scription terminator. This construct was cloned into E. actions and rearrangements are believed to take place at coli BL21(DE3). The resulting transformants, when in­ a single active site. According to this picture, the folding duced with isopropyl ,B-D-thiogalactoside, produced tri­ of the acyclic substrate at the active site is a major de­ chodiene synthase as 20-30% of total soluble protein. terminant of the structure and stereochemistry of the The recombinant synthase, which could be purified five­ eventually formed sesquiterpene product (10). fold to homogeneity by ammonium sulfate precipitation, Trichodiene synthase, which was first isolated from ion-exchange chromatography on Q Sepharose, and gel apple mold fungus Trichothecium roseum (11), has been filtration on Superose 12, was identical to nativeprotein purified to homogeneity from the T-2 toxin producer, Fu­ in steady-state kinetic parameters and mobility on so­ sarium sporotrichioides and has been shown to be a homo­ dium dodecyl sulfate-polyacrylamide gel electrophoresis dimer of 45-kDa subunits (12). The closely related tri­ and had the expected MENFP N-terminal sequence. chodiene synthase of Gibberella pulicaris (anamorph F. © 1993 Academic Press, Inc. sambucinum) has been partially purified as well (13). Screening of a Agtll genomic library ofF. sporotrichioides DNA with antibodies to the cyclase resulted in isolation of the structural gene for trichodiene synthase (Tox5), Sesquiterpene synthases catalyze the cyclization ofthe 3 encoded in a 1182-bp open reading frame containing a universal acyclic precursor farnesyl diphosphate (FPP , 60-nt in-frame intron (14). In vitro excision ofthe intron andsubcloning ofthe coding sequence intothe Escherichia I This investigation was supported by a grant from the National In· stitutes of Health, GM30301, to D.E.C. 2 To whom correspondence should be addressed. 3 Abbreviations used: FPP, farnesyl diphosphate; NPP, nerolidyl di· thiogalactopyranoside; SDS-PAGE, sodium dodecylsulfate-polyacryl· phosphate; PCR, polymerase chain reaction; EDTA, ethylenediamine· amide gel electrophoresis; PVDF, polyvinylidene difluoride; roo, ribosome tetracetic acid disodium salt; LB, Luria-Bertani; IPTG, isopropyl poD· binding site. 416 0003-9861/93 $5.00 Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved. OVERPRODUCTION OF TRICHODIENE SYNTHASE IN Escherichia coli 41"1 _~_~Opp ~ .' '. "':::: I "':::: I :..... "':::: I Ii . ~ : or ·opp II OPP . 1 FPP 2 NPP H 1 0.... -~\~..t_ •._•. - ~~~:~r ~ Y -OPP 2NPP 3 Trichodiene SCHEME 1. Cyclization of farnesyl diphosphate (FPP) to trichodiene through intermediacy of nerolidyl diphosphate (NPP). coli expression vector pDR540 resulted in bacterial synthase. We therefore sought to obtain sufficient quan­ expression oftrichodiene synthase, albeit at relatively low tities ofenzyme for further mechanistic andenzymological levels corresponding to 0.05-0.1% oftotal cellular protein studies. We now report the high-level overexpression of (15). Interestingly, transformed cells produced trichodiene trichodiene synthase and the purification and preliminary itself at levels of up to 60 ,ug/liter, presumably due to characterization of the recombinant cyclase. utilization ofendogenous FPP by the recombinant cyclase. The F. sporotrichioides Tox5 gene has also been cloned EXPERIMENTAL PROCEDURES into tobacco (Nicotiana tabacum), resulting in heterolo­ Materials. Plasmid pLMl and E. coli strain BL21(DE3) were gifts gous expression of trichodiene synthase and the produc­ from Professor Gregory L. Verdine of Harvard University. E. coli strain tion of detectable quantities of trichodiene in the plant XLI-Blue was from Stratagene (San Diego, CAl and strain JMI09(DE3) host (16). was from Promega (Madison, WI). Restriction enzymes Seal andBamHI were from Stratagene and HindIII was from Promega. T4 DNA ligase The corresponding trichodiene synthase gene of G. pul­ was purchased from Promega. Pfu DNA polymerase was obtained from icaris has also been isolated by using the F. sporotrichioides Stratagene. Oligonucleotides were synthesized by the phosphoramidite gene as a hybridization probe to screen a library of G. method on a BioSearch 8700 by Charles Sutterland. Bulk matrix Q pulicaris DNA (17). The two sequences showed an 89% Sepharose and Superose 12 were obtained from Sigma (St. Louis, MO) homology at the nucleotide level, including the 60-nt in­ and Bio-Gel P-6DG was purchased from Bio-Rad (Richmond, CAl. [1­ 3HJFPP (16.7 I'Ci/l'mol) was synthesized as previously described (7). tron, and a 96% identity at the amino acid level, the pri­ All other materials used for recombinant DNA manipulations, enzyme mary difference lying in the presence of an additional assay, and protein purification were analytical grade or higher. All buffers nine amino acids at the C-terminus of the G. pulicaris and nutrient broths were prepared with doubly deionized nanopure grade synthase. Although trichodiene synthase had no overall water. homology to any other known gene, significantly, both Methods. Restriction endonuclease digestions, DNA ligations, synthases were found to contain an aspartate rich motif preparation and transformation of competent cells, plasmid minipreps, and other standard recombinant DNA manipulations were carried out previously noted in several isoprenoid chain elongation according to published procedures (20), except that after restriction en­ enzymes, including FPP synthase (18). Thus the tricho­ zyme digestion, a Millipore Ultrafree-Probind filter unit was used to diene synthase of F. sporotrichioides had the sequence remove protein in place ofphenol-chloroform extraction. Maxipreps of VLDDSKD starting at amino acid 98, while the G. puli­ plasmid DNA by the alkaline lysis method (20) followed by precipitation caris cyclase had the sequence VLDDSSD at the same with 13% polyethyleneglycol were used to prepare double-stranded DNA for sequencing. DNA sequencing on plasmid products was carried out position. Furthermore, both enzymes contained a short by the Sanger dideoxy chain termination method using the Sequenase sequence rich in basic amino acids (DRRYR-F. sporo­ 2.0 kit (U.S. Biochemical) according to the standard protocols and with trichioides, DHRYR-G. pulicaris) closely analogous to a primers developed earlier for the sequencing ofthe trichodiene synthase presumptive active site peptide previously implicated in gene (14). PCR was carried out in a Coy thermal cycler (Ann Arbor, MIl. SDS-PAGE was performed in 14 cm X 14 cm X 1 mm 12% gels pyrophosphate binding by avian FPP synthase (19). by the method of Laemmli (21) and proteins were visualized by staining Nothing more is known currently about the active site of with Coomassie brilliant blue R-250. Densitometric analysis of dried trichodiene synthase, or indeed any other sesquiterpene SDS-PAGE gels was performed on a LKB Ultroscan XL laser densi- 418 CANE ET AL. tometer. lmmunoblot detection oftrichodiene synthase was carried out Expression of trichodiene synthase by E. coli BL21(DE3J/ as previously described using lmmobilon-P PVDF transfer membranes pZW03. Growth and production conditions were optimized to give the (Millipore) and rabbit antiserum against F. sporotrichioides