A novel biosynthetic strategy for the production of a key cryptophycin precursor

说明(申请人提供)：隐膜蛋白是一类结构多样的聚酮/非核糖体多肽天然产物，具有强大的抗癌活性。事实上，一种隐霉素合成类似物在治疗对铂耐药的卵巢癌方面表现出了希望。尽管有这种令人印象深刻的活性，但由于与治疗相关的临床上重大的周围神经病变，隐孔蛋白成为一种有益的癌症化疗药物的过程受到了影响。然而，这些天然产物充满希望的治疗谱促使Alluvium Bioscions继续发现和开发一种可以进入临床评估的隐霉素化合物。为此，Alluvium目前正在开发一种高通量、固相化学酶法生产技术，以使人们能够获得结构多样化的隐霉素化合物，这些化合物可以很容易地筛选出所需的药理特性。这一努力的关键是获得四个化学碎片或单元，它们在固相上组装，然后生物催化转化为成熟的隐霉素化合物。所需的三个单元要么可以从市场上获得，要么可以通过简单的合成方法很容易地获得，而第四个单元4-羟基苯丙烯酸目前必须通过定制的合成策略产生，这导致了低的总收率和高的每种化合物的成本。为了增加其隐霉素化学酶法生产技术的价值，Alluvium Biosciences致力于开发一种低成本、高产量的解决方案来生产4-羟基苯丙氨酸。因此，在这一阶段的SBIR方案中，Alluvium Biosciences将开发一种新的生物合成技术，利用细菌发酵来生产这种关键的隐霉素聚酮中间体。具体地说，该策略旨在设计和设计一种生物合成途径，由已知的隐霉素生物合成酶组成，该途径将指导4-羟基苯基洛替酸在大肠杆菌宿主内的生物合成。在构建工程生物合成途径并将其转移到细菌宿主中后，将建立生产所需隐霉素中间体的发酵条件。一旦概念验证得到证明，第二阶段的研究工作将集中在进一步提炼基因工程细菌菌株，并将发酵技术转移到大规模生产系统，以低成本生产4-羟基苯丙稀酸。第二阶段的工作还将制定生物合成生产化学上不同的4-羟基苯洛替酸类似物的战略。由该发酵工艺生产的纯化的4-羟基苯丙烯酸和结构类似物将作为通过Alluvium的固相化学酶技术规模化生产理想的隐霉素化合物的起始材料。 公共卫生相关性：癌症是一个重要的全球人类健康问题，值得为发现和开发新的治疗方法而进行大量研究投资。隐孢子素是一种已知的、有效的抗癌化合物，由于无法忍受的副作用，已被临床试验淘汰。这项拟议的研究旨在帮助开发一种新的技术，以快速产生可能显示出较少副作用的隐霉素类似物，从而使医生能够利用隐孢子素来对抗这种经常致命的疾病。

DESCRIPTION (provided by applicant): The cryptophycins are a structurally diverse class of polyketide/non-ribosomal peptide natural products that possess potent anticancer activity. In fact, a cryptophycin synthetic analogue has demonstrated promise in treating platinum resistant ovarian cancer. Despite this impressive activity, the development of the cryptophycins into a beneficial cancer chemotherapeutic agent has suffered due to clinically significant peripheral neuropathy that correlates with treatment. However, the promising therapeutic spectrum of these natural products has motivated Alluvium Biosciences to pursue the continued discovery and development of a cryptophycin compound that that can enter clinical evaluation. To this end, Alluvium is currently developing a high throughput, solid-phase chemoenzymatic production technology to enable access to structurally diverse cryptophycin compounds that can be readily screened for desirable pharmacological properties. Key to this effort is the availability of four chemical fragments, or units, which are assembled on solid-phase and subsequently biocatalytically transformed into mature cryptophycin compounds. Three of the required units are either commercially available or can be readily obtained by simple synthetic methods, while the fourth unit, 4- hydroxy phenyloctenoic acid, must currently be generated by custom synthetic strategies that result in low overall yields and high cost per compound. To add value to its cryptophycin chemoenzymatic production technology, Alluvium Biosciences is motivated to develop a low-cost, high yielding solution for the production of 4-hydroxy phenyloctenoic acid. Accordingly, in this Phase I SBIR proposal, Alluvium Biosciences will develop a novel biosynthetic technology that employs bacterial fermentation to produce this critical cryptophycin polyketide intermediate. Specifically, the proposed strategy aims to design and engineer a biosynthetic pathway, comprised of known cryptophycin biosynthetic enzymes, which will direct the biosynthesis of 4- hydroxy phenyloctenoic acid within an E. coli bacterial host. Subsequent to the construction and transfer of the engineered biosynthetic pathway into the bacterial host, fermentation conditions will be established for the production of the desired cryptophycin intermediate. Once proof-of-concept is demonstrated, Phase II research efforts will focus on further refinement of the genetically engineered bacterial strain and transfer of the fermentation technology toward a large-scale production system for the low-cost generation of 4-hydroxy phenyloctenoic acid. Efforts in Phase II will also develop strategies for the biosynthetic production of chemically diverse 4-hydroxy phenyloctenoic acid analogues. Purified 4-hydroxy phenyloctenoic acid, and structural analogues, produced by this fermentation technology will serve as starting material for the scalable production of desirable cryptophycin compounds via Alluvium's solid-phase chemoenzymatic technology. PUBLIC HEALTH RELEVANCE: Cancer represents a significant global human health concern that justifies substantial research investments for the discovery and development of novel treatments. Cryptophycin is a known, potent anti-cancer compound that has been dropped from clinical testing due to intolerable side-effects. This proposed research seeks to aid in the development of a novel technology for the rapid generation of cryptophycin analogues that may display fewer side effects, thereby enabling cryptophycin to be utilized by physicians in the battle against this oft-deadly disease.