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

Lead Research Organisation: Bangor University
Department Name: Sch of Natural Sciences

Abstract

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.

Technical Summary

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).

Publications

10 25 50
 
Description - identified at least four new bacterial enzymes with phenol kinase activities which are 2-5 times higher than that of the benchmark kinase ThiM from E. coli;
- identified nine proteins showing high kinase activities against phenol-like substrates, which are promising biocatalysts for the biosynthesis of non-natural terpenoids;
- a new research idea has been developed focused on screening known pyrophosphokinases to perform double phosphorylation of isopentenols to isoprenoid precursors in one enzyme reaction;
- selected 20 potential pyrophosphokinases from different phylogenetic clusters, and these clones are currently in the protein purification pipeline.
Exploitation Route After finishing biochemical characterization and product analysis of novel kinases, the results of this work will be published in a peer-review journal and will be available to a broad academic community, as well as for industrial researchers developing various natural and non-natural terpenoid product.
Sectors Chemicals

Healthcare

Manufacturing

including Industrial Biotechology

Pharmaceuticals and Medical Biotechnology

 
Description Economic impact: this project has already identified several promising biocatalysts for the biotransformation of cheap substrates to non-natural terpenoid products, which are of high interest for industry resulting in new partnerships with the UK companies.
First Year Of Impact 2023
Sector Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Title Collection of protein expression plasmids and purified enzymes 
Description Established a collection of 65 protein expression plasmids encoding selected enzymes, which produce 42 soluble proteins under standard expression conditions in E. coli cells. 
Type Of Material Technology assay or reagent 
Year Produced 2023 
Provided To Others? No  
Impact This enzyme collection will be used in our lab and shared with our collaborators in the UK for developing novel enzyme cascades and synthetic pathways for the biotransformation of isopentenols to valuable chemicals. 
 
Title Enzymatic screens for isopentenol phosphorylation activity 
Description Currently, we are optimizing novel enzymatic screens for isopentenol phosphorylation activity, which is based on the formation of prenylated FMN by FMN prenyl transferases and spectrophotometric detection of prenylated FMN. 
Type Of Material Technology assay or reagent 
Year Produced 2022 
Provided To Others? No  
Impact The optimized protocol for detection of isopentenol kinase activity will be published in a peer-review journal and will be made available for a broad research community. 
 
Title Novel assay for isopentenol kinase cascade 
Description Developed a novel biochemical assay for validating the proposed kinase cascades using purified isoprene synthases as reporter enzymes. 
Type Of Material Technology assay or reagent 
Year Produced 2023 
Provided To Others? No  
Impact The proposed assay will facilitate the identification of promising isopentenol kinases for applications in biotechnology. 
 
Description Short pathways to isoprenoid precursors 
Organisation University of Manchester
Department Manchester Institute of Biotechnology MIB
Country United Kingdom 
Sector Academic/University 
PI Contribution Proposed enzyme cascades for the bioconversion of isopentenols to isoprenoid precursors.
Collaborator Contribution Proposed biosynthetic pathways for the bioconversion of isoprenoid precursors to monoterpenoids
Impact Submitted a joint grant application to BBSRC (Responsive mode, 2022) for a collaborative project, which was not funded, so we are looking for other funding opportunities.
Start Year 2022
 
Description Bangor University Community Day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Bangor University Community Day (14th October 2023): our postdoc Anna Khusnutdinova and two PhD students were involved in organising and presenting the Biotechnology Science Stall including three demonstrations based on our laboratory research for local schools and parents.
Year(s) Of Engagement Activity 2023
 
Description International Congress Thermophiles-2023 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Our postdoc Anna Khusnutdinova was involved in the preparation and running the 16th International Congress "Thermophiles-2023, which took place at Bangor University (Bangor, Wales, UK) from 29th August to 2nd September 2023, which involved up to 100 scientists and PhD students from UK, Europe, and America.
At this conference, Anna also presented a poster entitled "Computational and experimental approaches for discovering thermostable enzymes".
Year(s) Of Engagement Activity 2023
 
Description Open Days at Bangor University 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Approximately 40 pupils visited our lab during Open Days at Bangor University and asked questions about our research projects.
Year(s) Of Engagement Activity 2022