Preliminary biotransformation of hiosciamine in Saccharomyces cerevisiae. Sequencing of cDNA encoding H6H in Brugmansia candida anthers

MARCONI, PATRICIA; CARDILLO, ALEJANDRA BEATRIZ; GIULIETTI, ANA MARÍA

Santiago de Chile

Congreso; XII International Biotechnology Symposium and Exhibition. 2004; 2004

Brugmansia candida is a native South-American plant which produces tropane alkaloids mainly scopolamine. This alkaloid is synthesized from hyoscyamine by action of Hyoscyamine 6[b]-hydroxylase (H6H). Recently we reported the isolation of h6h cDNA from B. candida h6hmRNA by RT-PCR. Total RNA was isolated from anthers of mature plants with RNeasy Plant Kit (Qiagen). After cDNA synthesis using Superscript II reverse Transcriptase (Life Technologies), PCR reaction was done on bases of Hyoscyamus niger h6h gene specific primer design due to high homology founded for the transcript among Solanaceae family. Two isolated fragments (aprox. 1.1 kb) was cloned in Escherichia coli using pCR2.1 (Invitrogen). The automated sequence analysis was done using M13 forward and reverse primers. The average of insert size of 1.1 Kb allow to obtain 502 and 509 bp to each fragment in forward sense and 510 and 517 in reverse sense. Bioinformatic tools were used in order to search homologies between each fragment isolated and h6h from Hyosciamus muticus. The DNA sequence of the insert has a high homology (97/98%) with the DNA sequence of mRNA h6h from H. muticus. The sequence of the insert revealed that the ATG and STOP codon are present in each sequence cloned. Current experimental work is being performed in order to develop h6h cDNA expression in Saccharomices cerevisiae for biotransformation purposes.