Genetically encoded biosensors for the non-invasive monitoring of the production of recombinant neurotoxins

肉毒神经毒素（BoNT）是由厌氧菌肉毒梭菌天然产生的，目前已知有七种不同的毒素血清型。已知BoNT引起肉毒中毒，肉毒中毒是人类的肌肉麻痹疾病;肉毒杆菌神经毒素是已知的毒性最大的物质之一，因为低至1 ng/kg的浓度被证明对人类是致命的（Sorouri等人，2017年）。其中，已知BoNT血清型A对人类最危险，由于其极强的效力，加上易于通过简单的生化合成生产。因此，BoNT/A被疾病、控制和预防中心分类为A型生物恐怖剂（Arnon等人，2001年）。尽管其毒性，BoNT/A最近已被用于医学，用于治疗以不正确的肌肉收缩为特征的病症，例如肌张力障碍、中风、多发性硬化症、半面痉挛、局灶性痉挛、多汗症（Laing，Laing & O 'Sullivan，2008）和美容医学，如应用注射来减少皮肤皱纹（Liu et al.，2012).用于医疗应用的BoNT基于重组毒素生产;一个关键的例子是Ipsen Pharma生产的生物药物DYSPORT，用于治疗神经系统疾病患者的肌肉骨骼或平滑肌疾病（Pharma，2019）。DYSPORT基于BoNT血清型A神经毒素的天然活性，其导致神经冲动向肌肉的传递减少，从而有助于缓解肌肉过度活动。在体内，肉毒杆菌神经毒素充当锌金属蛋白酶，其靶向并水解SNARE复合物中的不同蛋白质。SNARE蛋白的切割导致神经递质活性的阻断，这导致外周神经末梢的麻痹（肉毒中毒）;这进而导致呼吸困难或死亡（Sikorra等人，2016).存在由重组产生的毒素（例如BoNT）的生物加工产生的若干挑战。由于高毒性，生产需要在生物安全条件下进行，这限制了可用于检测最终产品的质量和数量控制测量（Pharma，2019）。即使实现了这样的测量，也要使用离线采样方法，这意味着测试速度较慢且吞吐量较低。目前，科学家们正试图优化重组神经毒素的生产过程，以提高产量和质量。其他研究也试图通过延长基于神经毒素的药物的功效并改善其定位来改善BoNT的药物应用，以减少脱靶活性（Rossetto，Pirazzini & Montecucco，2015）。这些研究需要生物工艺优化，包括测试不同参数如何影响所有生物加工步骤。因此，需要对样品进行体内高通量测试，以支持生物加工和制造的更快开发和优化。这可以通过一系列基因编码的生物传感器来实现，这将加快检测过程，因为生物传感器允许对关键参数进行更简单、非侵入性的测量。基因生物传感器可以用作体内分析设备，检测生物反应并将其耦合到荧光输出。存在各种类型的生物传感器，其可用于以稳健且具有成本效益的方式监测由重组BoNT表达引起的当前挑战。（Eivazzadeh-Keihan等人，2018年）。作为长期计划，其他生物传感器可用于监测重组BoNT的产生，例如基于转录的生物传感器作为生产率的指示，或基于FRET的生物传感器，用于通过跟踪SNAP-25结构域的结合和切割来监测神经毒素活性（Rossetto，Pirazzini & Montecucco，2015）; BoNT/A的天然靶标之一。

Botulinum neurotoxins (BoNTs) are naturally produced by the anaerobic bacteria Clostridium botulinum and currently, there are seven different known serotypes of the toxin. BoNTs are known to cause botulism, a muscle paralysis illness in humans; botulinum neurotoxins are amongst of the most toxic substances known, as concentrations as low as 1ng/kg are proven to be fatal to humans (Sorouri et al., 2017). Amongst all, the BoNT serotype A is known to present the most danger to mankind, due to its extreme potency, combined with ease of production via simple biochemical synthesis. Hence, BoNT/A is classified as a type A bioterrorism agent by the Centre for Disease, Control and Prevention (Arnon et al., 2001). Despite its toxicity, BoNT/A has recently been exploited in medicine, for the treatment of disorders characterised by incorrect muscle contraction, such as dystonia, stroke, multiple sclerosis, hemifacial spasms, focal spasticity, hyperhidrosis (Laing, Laing & O'Sullivan, 2008) and aesthetic medicine, like the application of injections to reducing skin wrinkles (Liu et al., 2012).BoNTs used in medical applications are based on recombinant toxin production; a key example is the biological drug DYSPORT, manufactured by Ipsen Pharma for the treatment of musculoskeletal or smooth muscle disorders in patients with neurological disease (Pharma, 2019). DYSPORT is based on the native activity of BoNT serotype A neurotoxins, which cause reduced transmission of nerve impulses to the muscle and in turn, this helps to relieve muscle hyperactivity. In-vivo, botulinum neurotoxins act as zinc metalloproteases which target and hydrolyse different proteins in the SNARE-complex. Cleavage of the SNARE proteins leads to blockage of neurotransmitter activity, which causes paralysis of the peripheral nerve terminals (botulism); in turn, this leads to breathing difficulties or death (Sikorra et al., 2016).There are several challenges arising from bioprocessing of recombinantly produced toxins, such as BoNTs. Due to the high toxicity, production needs to be done under biosafety conditions, which limits the quality and quantity control measurements that can be done to test the final product (Pharma, 2019). Even when such measurements are achieved, off-line sampling methods are used, which means that testing is slower and low throughput. Currently, scientists are attempting to optimise production processes of recombinant neurotoxins, in order to improve yield and quality. Other studies are also trying to improve the pharmaceutical applications of BoNTs by extending the efficacy of neurotoxin-based drugs and improving their localisation, in order to reduce off-target activity (Rossetto, Pirazzini & Montecucco, 2015). Such studies require bioprocess optimisation which involves testing how different parameters affect all bioprocessing steps. Therefore, there is a demand for in-vivo, high throughput testing of samples to support a faster development and optimisation of the bioprocessing and manufacture. This can be achieved through a series of genetically encoded biosensors which will speed up the detection process, as biosensors allow for a simpler, non-invasive measurements of key parameters.Genetic biosensors can be used as in-vivo analytical devices that detect a biological response and couple it to a fluorescence output. Various types of biosensors exist, which can be used to monitor of the current challenges arising from recombinant BoNTs expression, in a robust and cost-effective way. (Eivazzadeh-Keihan et al., 2018). As a long-term plan, other biosensors can be used to monitor production of recombinant BoNTs, such as transcription-based biosensors as an indication of productivity rates, or FRET-based biosensor, used to monitor neurotoxin activity through tracking the binding and cleavage of SNAP-25 domains (Rossetto, Pirazzini & Montecucco, 2015); one of the native targets of BoNT/A.