Identification of promoter elements in the Dolichospermum circinale AWQC131C saxitoxin gene cluster and the experimental analysis of their use for heterologous expression

BMC Microbiology

article

D’Agostino, Paul M.¹  Al-Sinawi, Bakir²  Mazmouz, Rabia²  Muenchhoff, Julia²  Neilan, Brett A.²  Moffitt, Michelle C.¹ 

[1] School of Science, Western Sydney University;School of Biotechnology and Biomolecular Sciences, University of New South Wales;Department of Chemistry, Technische Universität München;Faculty of Chemistry and Food Chemistry, Technische Universität Dresden;Centre for Healthy Brain Ageing, School of Psychiatry, University of New South Wales;School of Environmental and Life Sciences, University of Newcastle

关键词: Cyanobacteria;    Dolichospermum circinale;    Anabaena circinalis;    Saxitoxin;    Paralytic shellfish toxins;    Regulation;    Promoter;    Transcription;    Heterologous expression;   

DOI  :  10.1186/s12866-020-1720-3

学科分类：放射科、核医学、医学影像

来源: BioMed Central

【 摘 要 】

Dolichospermum circinale is a filamentous bloom-forming cyanobacterium responsible for biosynthesis of the paralytic shellfish toxins (PST), including saxitoxin. PSTs are neurotoxins and in their purified form are important analytical standards for monitoring the quality of water and seafood and biomedical research tools for studying neuronal sodium channels. More recently, PSTs have been recognised for their utility as local anaesthetics. Characterisation of the transcriptional elements within the saxitoxin (sxt) biosynthetic gene cluster (BGC) is a first step towards accessing these molecules for biotechnology. In D. circinale AWQC131C the sxt BGC is transcribed from two bidirectional promoter regions encoding five individual promoters. These promoters were identified experimentally using 5′ RACE and their activity assessed via coupling to a lux reporter system in E. coli and Synechocystis sp. PCC 6803. Transcription of the predicted drug/metabolite transporter (DMT) encoded by sxtPER was found to initiate from two promoters, PsxtPER1 and PsxtPER2. In E. coli, strong expression of lux from PsxtP, PsxtD and PsxtPER1 was observed while expression from Porf24 and PsxtPER2 was remarkably weaker. In contrast, heterologous expression in Synechocystis sp. PCC 6803 showed that expression of lux from PsxtP, PsxtPER1, and Porf24 promoters was statistically higher compared to the non-promoter control, while PsxtD showed poor activity under the described conditions. Both of the heterologous hosts investigated in this study exhibited high expression levels from three of the five sxt promoters. These results indicate that the majority of the native sxt promoters appear active in different heterologous hosts, simplifying initial cloning efforts. Therefore, heterologous expression of the sxt BGC in either E. coli or Synechocystis could be a viable first option for producing PSTs for industrial or biomedical purposes.

【 授权许可】

CC BY|CC0