Expression of the trichodiene synthase gene of Fusarium sporotrichioides in Escherichia coli results in sesquiterpene production.
Trichodiene synthase is a sesquiterpene cyclase involved in the biosynthesis of trichothecene mycotoxins. We report that insertion of the unaltered trichodiene synthase gene of Fusarium sporotrichioides into the Escherichia coli expression vector pDR540 produced an inactive polypeptide with a molecular weight approximately 2000 greater than that of trichodiene synthase. This result is consistent with the presence of an intron in the trichodiene synthase gene, and prompted us to specifically delete a putative 60-nucleotide intron sequence. Insertion of the intron-deleted open reading frame into pDR540 resulted in the production of active enzyme. Trichodiene synthase activity in crude extracts from induced cultures was 0.07 nmol/min/mg of protein and represented 0.05-0.10% of the total cell protein. A cross-reactive protein was present with the same apparent molecular weight as the subunit of native trichodiene synthase. The recombinant enzyme was partially purified and shown to have properties closely resembling those of the native enzyme. Trichodiene was detected in ethyl acetate extracts from induced cultures at a concentration of 60 micrograms/liter after 4.5 h. These findings support the primary structure recently reported for trichodiene synthase and demonstrate that the expression of a sesquiterpene cyclase in E. coli results in sesquiterpene production.
Hohn
T M
TM
Agricultural Research Service, U.S. Department of Agriculture, Peoria, Illinois 61604.
Plattner
R D
RD
D001483:Base Sequence; D019755:Carbon-Carbon Lyases; D002872:Chromosome Deletion; D005670:Fusarium / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D005800:Genes, Fungal; D007438:Introns; D008190:Lyases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D010957:Plasmids; D015183:Restriction Mapping; D012717:Sesquiterpenes / Q000302:isolation & purification*
D012717:Sesquiterpenes; D008190:Lyases; D019755:Carbon-Carbon Lyases; C051130:trichodiene synthetase