Identification And Application Research On The Post-modification Genes Of Helvolic Acid Gene Cluster

Microbial conversion is playing an irreplaceable role in the synthesis of steroid medicines, among which hydroxylation and C1,2 dehydrogenation are the most important reactions. Aspergillus fumigatus and Metarhizium anisopliae, fungi which produce steroid antibiotics like helvolic acid, have a great capability for steroid hydroxylation and C1,2 dehydrogenation. Steroid antibiotics shares the structural similarity with steroid drugs and has unsaturated double bond between C1 and C2 positions and several hydroxyl groups. It's convinced that research on the mechanism of steroid antibiotics synthesis in the molecular level is necessary for the understanding of the role fungi play in the microbial conversion of steroid drugs. This study aims to identify the functions of post-modification genes of helvolic acid gene cluster and investigate their value in steroid drugs industry.1. Cloning of helvolic acid gene cluster of M. anisopliae and analysis of its gene functions.M. anisopliae, commonly used strain in the hydroxylation reaction of steroid drugs, has a high level of helvolic acid production. A fosmid library was constructed for the helvolic acid gene cluster of this strain and 4 pairs of degenerate primers designed on the basis of information from genome of A. fumigatus and EST library of M. anisopliae.3 positive clones were obtained and were sequenced. Finally we discovered a 23-kb sequence of the gene cluster, consisting of one cyclase, one 3-keto-△1-dehydrogenase, one short chain dehydrogenase, two acyl tansferase and 4 P450 genes. Additionally, the gene composition of this cluster shows a similarity with that of A. fumigatus.2. Functional analysis of P450 in the cluster from A. fumigatusThe specific catalytical functions of 4 P450 genes in the cluster from A. fumigatus are unknown. In this study, one of the P450 genes was analyzed by means of Agrobacterium tumeficiens mediated gene inactivation tool. The wide type and P450-knock-out mutant were cultured synchronously and was compared by HPLC with their fermentation broth. Testing results showed no helvolic acid and any of its detectable derivatives existing in the fermentative culture of the mutant strain in contrast to those in the wide type, which demonstrated the vital role of P450 during the helvolic acid synthesis.3. Functional determination and enzymatic characterization of 3-keto-△1-dehydrogenase in the cluster from A. fumigatus in the field of steroid drugs conversion3-keto-△1-dehydrogenase (KsdDF) in the cluster is believed to catalyze the formation of the double bond between C1 and C2 positions. However, whether it could catalyze the dehydrogenation of C1 and C2 hydrogen atoms of 3-keto-4-en-steroid drugs is still unknown. Furthermore, different from bacterial membrane protein KsdD, fungal KsdDF was found as a soluble protein without any obvious membrane embedded sequence by amino acid sequence analysis. After expression of ksdDF in E. coli BL21(λDE3) and localization of active KsdDF enzyme, almost all the KsdD activity was found in the intercellular supernatant, which implied that this enzyme was active as a soluble protein, rather than a membrane-bound one. Km value of this KsdDF with the substrate AD is 2×10-4mol/l. When expressed in P. pastoris KM71 and GS115, KsdDF was also found to exsist in the intercellular supernatant, which further confirmed this fungal enzyme to be a soluble protein. Kinetics parameters of KsdDF expressed in P. pastoris GS115 intracelluarly are as follows:Km 5×10-5mol/l, Vm 51.68U/mg. However, KsdDF from intracellular expression with pPIC3.5k showed high boiactivity, while KsdDF from extracellular expression with pPIC9k did not show any detectable activity. As previous experiments have shown that the bacterial KsdDs are membrane-bound proteins, we speculated the KsdDF from A. fumigatus is an outer membrane-associated fraction and membrane fractions are needed for its configuration formation.4. Construction of ksdDFtransformed Pichia pastoris engineering strain.As an inner membrane-bound protein with two groups of membrane embedded sequences, bacterial KsdDs are mostly heterologously expressed as inclusion body and are hard to be successfully constructed into engineering strain with high KsdD activity. By contrast, ksdDF could be expressed as a highly active soluble protein in E. coli and P. pastoris. P. pastoris KM71 and GS115 was transformed with pPIC3.5K containing ksdDF, and the recombinant strains could transform 1.0g/l AD into ADD and 1.5g/l AD into ADD and boldenone(BD) respectively at 100% conversion rate at the shake flask fermentation level. ADD and BD, the products of recombinant GS115, could transform mutually by the 17-hydroxysteroid dehydrogenase of P. pastoris, which is determined by the reducing power. According to this, BD accounted for more than 80% in BD/ADD mixture by addition of 3% glucose at the end of fermentation. The recombinant P. pastoris KM71 and GS115 have shown tremendous producing capability of ADD and BD, which provides follow-up for magnifying research of process engineering.To sum up, this paper has revealed the finding of helvolic acid gene cluster from M. anisopliae. Compared with the cluster from A. fumigatus, the gene similarity is low, but it can be concluded that synthesis route of helvolic acid and gene components are semblable between the two clusters. Moreover, our work has identified the significant position P450 plays in the helvolic acid synthesis and laid a method foundation for the identification of other genes in the hecvolic acid cluster. Most importantly, via research on ksdDfF from A. fumigatus, a soluble KsdD has been indentified and successfully constructed into two engineering strains which efficiently transform AD into ADD and BD respectively.