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

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.

All natural isoprenoids (or terpenoids) are derived from two interconvertible five-carbon hemiterpene diphosphates, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), produced via either the mevalonate or non-mevalonate pathways. To overcome numerous limitations associated with natural isoprenoid pathways and in vivo production of terpenoids, we propose to develop a synthetic pathway for the bioconversion of cheap isopentenols (prenol and isoprenol) to expensive isoprenoid precursors (DMAPP and IPP) using a polyphosphate-based three-kinase cascade and recombinant E. coli cell lysates as a low-cost enzyme source.
Recently, we have demonstrated the presence of prenol kinase activity in the E. coli kinase ThiM producing dimethylallyl phosphate (DMAP) as product. Furthermore, we have established a polyphosphate-dependent ATP regeneration system, which increased prenol phosphorylation activity of purified ThiM several times. In the proposed project, this kinase cascade will be extended by adding an isopentenyl phosphate kinase for the phosphorylation of DMAP and isopentenyl phosphate (IP) to DMAPP and IPP. This pathway will be optimised using purified enzymes followed by their co-expression in E. coli cells and in vitro bioconversion of isopentenols to isoprenoid precursors using recombinant E. coli cell lysates.
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 an 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).