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

描述（由申请人提供）：隐菌素是一种结构多样的聚酮类/非核糖体肽天然产物，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铂耐药卵巢癌方面已经显示出前景。尽管具有这种令人印象深刻的活性，但由于与治疗相关的临床显着的周围神经病变，隐菌素发展成为有益的癌症化疗药物受到了影响。然而，这些天然产物有希望的治疗谱促使冲积生物科学公司继续发现和开发一种可以进入临床评估的隐藻素化合物。为此，冲积公司目前正在开发一种高通量、固相化学酶生产技术，以获得结构多样的隐藻素化合物，这些隐藻素化合物可以很容易地筛选出理想的药理特性。这项工作的关键是四种化学片段或单元的可用性，它们在固相上组装，随后生物催化转化为成熟的隐藻素化合物。其中三个所需的单位要么是商业上可用的，要么可以通过简单的合成方法轻松获得，而第四个单位，4-羟基苯乙烯酸，目前必须通过定制合成策略生成，导致总收率低，每个化合物的成本高。为了增加其隐藻素化学酶生产技术的价值，冲积生物科学公司积极开发一种低成本、高产的4-羟基苯乙烯酸生产方案。因此，在这个I期SBIR提案中，冲积生物科学公司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藻素聚酮中间体。具体来说，提出的策略旨在设计和设计一个生物合成途径，由已知的隐藻素生物合成酶组成，这将指导大肠杆菌宿主体内4-羟基苯乙烯酸的生物合成。随着工程生物合成途径的构建和转移到细菌宿主中，将建立发酵条件以生产所需的隐藻素中间体。一旦概念验证得到证实，第二阶段的研究工作将集中在进一步改进基因工程菌株，并将发酵技术转移到大规模生产系统中，以低成本生产4-羟基苯乙烯酸。第二阶段的工作还将制定化学上多样化的4-羟基苯乙烯酸类似物的生物合成生产策略。通过这种发酵技术生产的纯化的4-羟基苯乙烯酸及其结构类似物将作为冲积物公司的固相化学酶技术大规模生产所需的隐藻素化合物的起始材料。

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.