Analysis of promoter regions in the saxitoxin gene cluster identifies potential bottlenecks in heterologous biosynthesis

Background: 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. Results: In D. circinale AWQC131C the sxt BGC is transcribed from two bidirectional promoter regions encoding five individual promoters. These promoters were identified experimentally using 5ʹ 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, P sxtPER and P sxtPER2 . In E. coli , strong expression of lux from P sxtP , P sxtD and P sxtPER was observed while expression from P orf24 and P sxtPER2 was remarkably weaker. In contrast, heterologous expression in Synechocystis sp. PCC 6803 showed that expression of lux from P sxtP , P sxtPER , and P orf24 promoters was statistically higher compared to the non-promoter control, while P sxtD showed poor activity under the described conditions. Conclusions: Both of the heterologous hosts investigated in this study exhibited high expression levels from three of the five sxt promoters. These results indicate that future experiments to clone the complete sxt BGC into either heterologous host should result in the transcription of the complete pathway following engineering of the least active promoters. Therefore, heterologous expression of the sxt BGC in either E. coli or Synechocystis could be a viable option for producing PSTs for industrial or biomedical purposes.