Gene for biosynthesis of core structure of ophiobolin
A gene for biosynthesis of core structure of ophiobolin, the gene being the AuOS gene of Aspergillus sp. 094102, deposited with the accession number CCTCC No: M208153, the gene sequence thereof being shown as SEQ ID NO. 1. Also provided is a method of preparation of ophiobolin using the gene.
1. A cDNA, wherein the cDNA comprises the nucleic acid sequence of SEQ ID NO: 2.
2. A method for preparing an ophiobolin precursor, the method comprising:
1) extracting RNA from mycelia of Aspergillus sp. 094102 which has been deposited in China Center for Type Culture Collection (CCTCC) with an accession number: CCTCC NO. M208153;
2) reverse-transcribing the RNA of 1) into a cDNA;
3) conducting PCR amplification of the cDNA of 2) with a pair of primers to obtain a PCR product mixture;
4) recovering a target DNA fragment from the PCR product mixture of 3), wherein the target DNA fragment comprises the nucleic acid sequence of SEQ ID NO: 2 ligated to recognition sites of restriction enzymes Nde I and Hind III;
5) ligating the target DNA fragment into a first vector to obtain a first recombinant plasmid;
6) digesting the target DNA fragment from the first recombinant plasmid of 5) and inserting the target DNA fragment into a second vector to obtain a second recombinant plasmid;
7) transforming the second recombinant plasmid of 6) into Escherichia coli BL21 to obtain an engineered Escherichia coli strain;
8) cultivating the engineered Escherichia coli strain of 7) to obtain a target protein encoded by the target DNA fragment;
9) purifying the target protein; and
10) in vitro synthesizing an ophiobolin precursor from a first substrate and a second substrate using the target protein as a catalyst, wherein the first substrate is selected from dimethylallyl diphosphate, geranyl diphosphate, farnesyl diphosphate, and geranylgeranyl diphosphate, the second substrate is isopentenyl diphosphate, and the ophiobolin precursor has a molecular structure as follows:
3. The method of claim 2 , wherein the pair of primers of 3) comprise the nucleic acid sequence of each of SEQ ID NO: 4 and SEQ ID NO: 5, respectively.
4. A recombinant plasmid, wherein:
the engineered plasmid is a pET28a vector comprising a target DNA sequence; and
the target DNA sequence comprises the nucleic acid sequence of SEQ ID NO: 2.
5. An engineered Escherichia coli strain, the engineered Escherichia coli strain comprising the recombinant plasmid of claim 4 .