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

异戊二烯类化合物(也称为萜类化合物)包括一大类天然产物，存在于所有生命王国中，有80,000多种独特的化学结构，非常适合宿主生物体的要求。自古以来，从一些草药或动物肝脏中提取异戊二烯类化合物，用于制药、香料、香料、着色剂、维生素和商品化学品。根据市场观察，2020年全球萜烯市场价值为559.4美元，预计到2026年底将达到774.3美元。随着工业上对异戊二烯类化合物的需求不断增长，增加萜类化合物的可获得性变得至关重要。然而，天然来源的异戊二烯产量较低，限制了它们在工业应用中的大规模生产。另一方面，由于化学复杂性高、产率低和环境问题，化学合成类异戊二烯通常是不切实际的。因此，生物催化生产异戊二烯类化合物是一种有吸引力的替代天然来源的化学提取和化学合成的方法。在所有已知的生物中，异戊二烯类化合物来自两个五碳(C5)二磷酸盐，二甲基烯丙基二磷酸(DMAPP)和异戊烯基二磷酸(IPP)。接下来，这些前体结合在一起生成较长的戊烯基二磷酸盐(C10、C15和C20)，这些二磷酸盐被其他酶转化为具有高度结构和化学多样性的萜类主链和复杂的异戊二烯。类异戊二烯前体DMAPP和IPP是通过MVA或MEP途径自然产生的，这两条途径都是具有复杂调控的长时间生物合成途径。虽然这些途径已经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给工业化生产类异戊二烯带来了巨大的挑战。此外，用于体内生产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带来的严重限制，我们建议开发一种基于廉价起始底物和开发在大肠杆菌中重组表达的用作细胞裂解物的新型酶的短合成途径。具体地说，我们将开发一种人工途径来生产类异戊二烯前体(DMAPP和IPP)，该途径基于以异戊烯醇(戊烯醇和异戊二烯醇)为起始底物的依赖于多聚磷酸的三激酶级联。首先，将使用纯化的重组酶进行一锅转化来优化所提出的途径。然后，选定的蛋白质将在大肠杆菌细胞中共表达，整个细胞裂解产物将用于聚磷酸盐驱动的异戊烯醇和相关醇的生物转化为异戊二烯前体。提出的突破性想法包括开发一条短的合成路线，使用廉价的底物和重组细胞裂解物作为低成本的酶来源来生产昂贵的类异戊二烯前体。这种方法有几个主要的优点，包括：(1)低成本的健壮的酶来源；(2)廉价的起始底物(戊烯醇、异丙二酚、聚磷酸盐)；(3)消除了细胞毒性问题(可以使用高底物浓度)；(4)消除了底物/产物运输的膜障碍；(5)能够直接控制反应参数(底物、辅因子或酶的负荷)。这一原则证明项目将建立一个从廉价底物生产天然和非天然异戊二烯前体的灵活平台。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异戊二烯类化合物，在医药、香料、化妆品、香料、营养和农用化学品中有许多应用。

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.