21ENGBIO: Polyphosphate-based synthetic pathway (PSP) for bioconversion of isopentenols to isoprenoid precursors using recombinant cell lysates

类异戊二烯（也称为萜类）是一大类天然产物，存在于所有生物王国中，有超过80,000种独特的化学结构，高度适应宿主生物的需求。自古以来，类异戊二烯从一些草药或动物肝脏中提取，用于制药、香料、香料、着色剂、维生素和商品化学品。根据MarketWatch的数据，2020年全球萜烯市场价值为5.594亿美元，预计到2026年底将达到7.743亿美元。由于对与工业有关的类异戊二烯的需求不断增长，增加萜类化合物的供应已变得至关重要。然而，天然来源的类异戊二烯产量低，限制了其大规模生产用于工业应用。另一方面，化学合成类异戊二烯通常是不切实际的，因为化学复杂性高，产量低，和环境问题。因此，生物催化生产类异戊二烯是一种有吸引力的替代方法，可以从自然资源中化学提取和化学合成。在所有已知的生物中，类异戊二烯来源于两种五碳（C5）二磷酸盐，二磷酸二甲基烯丙基（DMAPP）和二磷酸异戊烯基（IPP）。接下来，这些前体结合生成较长的二磷酸戊烯基（C10、C15和C20），经其他酶转化为具有高度结构和化学多样性的萜类骨架和复杂的类异戊二烯。类异戊二烯前体DMAPP和IPP是通过MVA或MEP途径自然产生的，这两种途径是具有复杂调控的长生物合成途径。尽管这些途径已被设计用于在大肠杆菌和酿酒酵母中生产类异戊二烯，但它们的复杂性和固有的低效率给类异戊二烯的工业生产带来了重大挑战。此外，在体内生产类异戊二烯的传统生物催化平台受到中间体和产物的细胞毒性、细胞膜屏障和副细胞活性的阻碍。为了克服天然类异戊二烯途径和体内生物转化所带来的关键限制，我们建议开发一种基于廉价起始底物的短合成途径，并利用在大肠杆菌中重组表达并用作细胞裂解物的新型酶。具体来说，我们将开发一种人工途径来生产类异戊二烯前体（DMAPP和IPP），该途径基于多磷酸依赖的三激酶级联反应，使用异戊二烯醇（prenol和异戊二醇）作为起始底物。首先，提出的途径将使用纯化重组酶的一锅转化进行优化。之后，选择的蛋白将在大肠杆菌细胞中共表达，全细胞裂解物将用于多磷酸盐驱动的异戊烯醇和相关醇向类异戊烯前体的生物转化。提出的突破性想法包括开发一种短合成途径，以廉价的底物和重组细胞裂解物作为低成本的酶源来生产昂贵的类异戊二烯前体。这种方法有几个主要优点，包括：(1)低成本的强效酶来源；(2)、廉价的起始底物（戊二醇、异戊二醇、聚磷酸盐）；(3)、消除细胞毒性问题（可以使用高浓度底物）；(4)消除底物/产物运输的膜屏障；(5)，可以直接控制反应参数（底物、辅因子或酶的负载）。这个原理验证项目将建立一个灵活的平台，从廉价的底物中生产天然和非天然类异戊二烯前体。接下来，可以通过添加其他酶来扩展这一途径，以生物催化生产更复杂的类异戊二烯，这些类异戊二烯在医药、香料、化妆品、香料、营养和农用化学品中有着广泛的应用。

Isoprenoids (also known as terpenoids) comprise a large class of natural products present in all kingdoms of life with over 80,000 unique chemical structures highly tailored to the requirements of host organisms. Since ancient times, isoprenoids were extracted from some herbs or animal liver for applications as pharmaceuticals, flavours, fragrances, colorants, vitamins, and commodity chemicals. According to MarketWatch, the global terpenes market was valued at 559.4 M USD in 2020, and it is expected to reach 774.3 M USD by the end of 2026. As the demand for industrially relevant isoprenoids is continuously growing, increasing availability of terpenoids has become critical. However, low yields of isoprenoids from natural sources are limiting their mass production for industrial applications. On the other hand, chemical synthesis of isoprenoids is generally impractical due to high chemical complexity, low yields, and environmental concerns. Therefore, biocatalytic production of isoprenoids represents an attractive alternative to chemical extraction from natural sources and chemical synthesis. In all known organisms, isoprenoids are derived from the two five-carbon (C5) diphosphates, dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP). Next, these precursors are combined to generate longer prenyl diphosphates (C10, C15, and C20), which are converted by other enzymes to terpenoid backbones and complex isoprenoids with high structural and chemical diversity. The isoprenoid precursors DMAPP and IPP are naturally produced through either the MVA or the MEP pathway, which are long biosynthetic pathways with complex regulation. Although these pathways have been engineered for isoprenoid production in Escherichia coli and Saccharomyces cerevisiae, their complexity and inherent inefficiency present significant challenges for industrial production of isoprenoids. Furthermore, traditional biocatalytic platforms for in vivo production of isoprenoids are hampered by cell toxicity of intermediates and products, cell membrane barriers, and side cellular activities. To overcome the critical limitations imposed by natural isoprenoid pathways and in vivo biotransformations, we propose to develop a short synthetic pathway based on inexpensive starting substrates and exploiting novel enzymes recombinantly expressed in E. coli and used as cell lysates. Specifically, we will develop an artificial pathway for the production of isoprenoid precursors (DMAPP and IPP) based on a polyphosphate-dependent three-kinase cascade using isopentenols (prenol and isoprenol) as starting substrates. First, the proposed pathway will be optimised using one-pot transformations with purified recombinant enzymes. Afterwards, the selected proteins will be co-expressed in E. coli cells, and whole cell lysates will be used for polyphosphate-driven biotransformation of isopentenols and related alcohols to isoprenoid precursors. The proposed breakthrough idea involves the development of a short synthetic pathway to produce expensive isoprenoid precursors using cheap substrates and recombinant cell lysates as a low-cost enzyme source. This approach offers several major advantages including: (1), low-cost source of robust enzymes; (2), cheap starting substrates (prenol, isoprenol, polyphosphate); (3), abrogation of cell toxicity problems (high substrate concentrations can be used); (4), elimination of membrane barriers for substrate/product transport; (5), enabling direct control over reaction parameters (loadings of substrates, cofactors, or enzymes). This proof of principle project will establish a flexible platform for producing both natural and non-natural isoprenoid precursors from inexpensive substrates. Next, this pathway can be extended by adding other enzymes for biocatalytic production of more complex isoprenoids with numerous applications in medicine, fragrances, cosmetics, flavours, nutrition, and agrochemicals.