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.

中文翻译：

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圆孢子虫AWQC131C毒素基因簇中启动子元件的鉴定及其异源表达的实验分析。

圆孢子虫是一种形成丝状花开的蓝细菌，负责生物合成包括麻毒素在内的麻痹性贝类毒素（PST）。PST是神经毒素，其纯化形式是监测水和海鲜质量的重要分析标准，以及研究神经元钠通道的生物医学研究工具。最近，PST因其作为局麻药的效用而得到认可。毒素（sxt）生物合成基因簇（BGC）中的转录元件的表征是将这些分子用于生物技术的第一步。在圆环衣原体AWQC131C中，sxt BGC从编码五个单独启动子的两个双向启动子区域转录而来。这些启动子使用5'RACE进行了实验鉴定，并通过与大肠杆菌和集胞藻（Synechochocystis sp。）中的lux报告系统偶联来评估了它们的活性。PCC6803。发现由sxtPER编码的预测药物/代谢物转运蛋白（DMT）的转录起始于两个启动子PsxtPER1和PsxtPER2。在大肠杆菌中，观察到PsxtP，PsxtD和PsxtPER1中lux的强表达，而Porf24和PsxtPER2中的lux显着弱。相反，在异胞藻属中异源表达。PCC 6803显示，与非启动子对照相比，PsxtP，PsxtPER1和Porf24启动子的lux表达在统计学上更高，而PsxtD在上述条件下显示出较弱的活性。在这项研究中研究的两个异源宿主都从五个sxt启动子中的三个显示了高表达水平。这些结果表明，大多数天然sxt启动子在不同的异源宿主中均具有活性，从而简化了初始克隆工作。因此，在工业或生物医学目的生产PST时，sxt BGC在大肠杆菌或集胞囊藻中的异源表达可能是可行的首选。