OVERPRODUCTION, IN ESCHERICHIA-COLI, OF SOLUBLE TAXADIENE SYNTHASE, AKEY ENZYME IN THE TAXOL BIOSYNTHETIC-PATHWAY

Taxadiene synthase catalyzes the conversion of the universal precursor of diterpenoids, geranylgeranyl diphosphate, to taxadiene, a key inte rmediate in Taxol (paclitaxel) biosynthesis. The gene encoding taxadie ne synthase was cloned recently. Here we report a method for the heter ologous overexpression of cDNA encoding taxadiene synthase in Escheric hia coli using a thioredoxin fusion expression system, which increase the solubility of expressed protein. Taxadiene synthase cDNA was ampli fied by polymerase chain reaction and then subcloned into pET3d and pE T32a(+) to form pET3dTX and pET32TX, respectively. The expressed taxad iene synthase from E. coli BL21(DE3)/pET3dTX was present completely as inclusion bodies. The transformant E. coli BL21(DE3)/pET32TX produced a thioredoxin fusion taxadiene synthase (15-20% of total soluble prot ein) where induced with isopropyl beta-D-thiogalactopyranoside at low temperature (20 degrees C). The recombinant enzyme was purified by a s ingle step with a His-binding metal affinity column. The maximal produ ction attained was 13 mg of purified, active fusion protein per 500 ml culture of E. coli BL21(DE3)/pET32TX. The purified recombinant taxadi ene synthase fusion protein was similar to native protein in steady-st ate kinetic parameters and mobility on sodium sulfate-polyacrylamide g el electrophoresis. The protein purified from E. coli BL21(DE3)/pET3dT X had the expected N-terminal (AQLSFNA) sequence. (C) 1988 Academic Pr ess.