Heterologous Expression And Preliminary Immobilization Study On The Enzymes For The Biosynthesis Of Terrequinone A

Terrequinone A, a kind of the antitumorigenic nature product, is a second metabolite isolated from Aspergillus nidulans, which have great broad application prospects and market potential. However, the natural product has a low solubility and poor stability in both polar and nonpolar media, which strongly restricts their incorporation in many formulations. In addition, its derivatives share a complex dihydroxybenzoquinone core and vary mainly in the pattern of prenylation on the indole substituents. Synthesis of this natural product using chemical methods was limited due to its adverse environmental impact, safety and waste output. As the development of synthetic biology, biosynthsis method can be used on the production of the natural product without the tedious blocking and deblocking steps that are common in enantio-and region-selective organic synthesis. Such high selectivity also affords efficient reactions with few by-products.In our work, we performed the heterologous expression of the gene cluster in the Terrequinone A biosynthesis routine from the Aspergillus nidulans in the E.coil by genetic engineering technology. The synthetic gene cluster was cloned from Aspergillus nidulan by nested PCR techniques and the gene was amplified and inserted into the expression vector pET24a(+). The recombinant plasmid was transfected into expression host E. coli BL21(DE3) or Rosetta (DE3). Five recombinant enzyme protein (TdiA. TdiB. TdiC. TdiD. TdiE) was produced after IPTG induction, respectively. we also examined the expression efficiency of eukaryotic proteins that contain rare codons in prokaryotichost E. coli. The recombinant proteins were purified using immobilization metal affinity chromatography. IPA was biosynthesized using TdiD as enzyme and L-tryptophan as substrate in vitro and TdiA was used to catalyze IPA into Didemethylasterriquinone D, intermediate in the production rutine of Terrequinone A. Moreover, TdiB, TdiC and TdiE was combined with TdiD, TdiA and cofactor to biocatalyze the preparation of final product, Terrequinone A. Based on the catalytic properties of the free enzymes, we introduced the immobilized enzyme technology into the Terrequinone A biosynthesis to improve the biosynthetic efficiency. A preliminary research results showed that IPA could be biosynthiszed using the amino-immobilized TdiD as biocatalyst and L-tryptophan as substrate, and could be used to prepare Didemethylasterriquinone D using the amino-immobilized TdiA, respectively.The aim of this research is to improve the enzyme biosynthetic efficiency for natural products in vitro. It was perspected to contribute to the solution for the drawback in innovative natural products drug production, such as less content in plant extract, low recovery in chemical methods synthesis and reduced the costs of natural medicine production.