13 ERA IB TERPENOSOME: Engineered compartments for monoterpenoid production using synthetic biology

Lead Research Organisation: University of Manchester
Department Name: Chemistry

Abstract


Technical Summary

Terpenoids are commercially valuable natural fine chemicals with a variety of uses as drugs (taxol, artemisinin), nutraceuticals (carotenoids), cosmetics (astaxanthin), and agriculturals (gibberellins). Monoterpenoids are mainly produced in plants and are used as flavours (e.g. limonene), and fragrances (e.g. carveol), as well as precursors for pharmaceutical molecules. However, the natural production of commercially relevant monoterpenes is limited, since their extraction from plants is costly and inefficient, and the alternative chemical synthesis involves long and complex, multi-step protocols. Monoterpenes produced from bacteria using heterologous biosynthesis pathways would be ideal for sustainability and compound diversity; however, so far it has produced low yields, while sesquiterpenes have been successfully overproduced by, e.g., Martin and coworkers (2003, Nature Biotech 21:796-802). TERPENOSOME will develop yeast and bacterial monoterpenoid producing strains using synthetic biology approaches, which bypass many of the production constraints associated with chemical synthesis and isolation from natural resources.

Metabolosomes are recently discovered bacterial organelles, which are large structures composed of an exterior protein shell with multiple enzymes compartmentalised on the inside. There are currently only two examples reported in the literature for the localisation of green fluorescent protein inside ethanolamine utilization metabolosomes (Choudhary et al., 2012). These serve as excellent proof-of-concept examples to show: (1) that non-native proteins can be targeted inside metabolosomes, and (2) that metabolosomes can be used as closed scaffolds inside host organisms.

TERPENOSOME will be the first to apply the metabolosome to synthetically designed monoterpene biosynthesis for production improvement, side product reduction through improved metabolic control and higher local flux, and to avoid toxicity limiting production levels.

Planned Impact

N/A

Publications

10 25 50
 
Description The engineered bacteria with the monoterpenoid biosynthesis pathways are producing limonene, geraniol and linalool which are all monoterpenoids and are the source of smells for lemon, rose and lavendar. This is a consortium with 5 partners (Takano, Scrutton - Universirty of Manchester, Braus - University of Goettingen, Bruheim-NTNU, Trefzer- ThermoFischer GeneArt, Finkelmeier-Aroma Chemicals Services International) who equally contributed to the findings.
Below are some of our significant achievements.
1) We constructed artificial compartments into E. coli, including a variant using the eutLK genes of Salmonella typhimurium and we analysed for the formation of bacterial microcompartments (BMCs) using a fluorescent tagging. We also encapsulated the limonene monoterpene production pathway. The expression of shell proteins detrimentally effects limonene production, likely due to increased metabolic loading of E. coli and showed slower growth. To overcome metabolic load, a minimal protein scaffold was constructed by using two (eutSM) and a single (eutM) shell protein and to attach the GPPS and LimS to these protein scaffold for enhanced limonen production. Initial results show some promise, however this element of the project remains only 50% complete, further analysis are required to determine limonene production in absence of minimal shell proteins.
2)Yeast host and peroxisome engineering. Deletion library for the production of Saccharomyces cerevisiae strains with constantly high numbers of peroxisomes were constructed resulting in four single deletions, six double deletions, four triple deletions, and one quadruple deletion mutant. mCherry-protein tagged with a SKL-tag for transport into peroxisomes was integrated into the chromosome of each strain and the number of peroxisomes per cell was quantified via fluorescence microscopy. The strain with the highest number had 433% increase in peroxisomes per cell compared to the control (100%) after one day cultivation in complex medium. Two strains with highest numbers of peroxisomes and no growth deficiencies which gave 327% and 341% increase, were chosen for the further experiments. Two enzymes were deleted which are involved in the turnover of geraniol to geranyl acetate and citronellol.This resulted in the reduction of citronellol to 12-fold. Several addition/mutations to increase terpene production was created and the integrated strains were analysed for geraniol production. The ratio of peroxisomal to cytosolic geraniol yield was shifted from 0.63 to 1.15 in the mutated strains, showing that the enrichment of peroxisomes and the synthesis of geraniol inside the peroxisomes produces higher yields of geraniol.
3) Fermentation, monoterpene production analysis, and metabolites in the engineered hosts. Metabolomics analysis for E. coli and yeast were developed which includes a headspace GC-MS/MS method, capillary ion chromatography (capIC) -MS/MS method for the phosphometabolome and improved the chromatographic resolution of sugar phosphates which is necessary for detailed monitoring of upper glycolytic pathway. 80 metabolites have been upgraded with 13C internal standards . A large experimental study with almost 40 batch and chemostat cultivations were conducted and tested was carbon, phosphate and nitrogen limitation at four different growth rates (0.06, 0.12, 0.18 and 0.24 h-1), while in batch four different carbon sources were used (glucose, galactose, fructose and sucrose). This generated a large data set that is currently under investigation.
4) Identification of biosynthesis enzymes for monoterpene biosynthesis and Redesign, DNA synthesis and assembly of the monoterpenoid biosynthesis pathway. Gene cluster of a heterologous monoterpenoid biosynthesis pathway (MBP) in E. coli has been refactored, synthesised and assembled. Additional monoterpenoid synthase genes have been synthesised for modular modification of the MBP. Limonene synthase has been purified and activity confirmed with/without N-terminal metabolosome tag in in vitro biotransformation assays. MBP for limonene synthesis has been introduced to E. coli and production of limonene initial studies showed a yield of >100 mg/L/OD. By culture and host optimisation, the production was increased to 530mg/L which is 10x increase compared to the non-optimised construct. Geraniol was also tested which showed 160mg/L which is 5x increase production and by addition of an acetyltransferase, a 25% conversion to geranyl acetate was achieved. Linalool was also tested and achieved a high amount. Second generation of the MVA pathway, and a genome integrated version of the MBP was also created and the comparison of the production is being tested before the end of the project.
5) Redesign, DNA synthesis of the monoterpenoid biosynthesis pathway. Sequence design and optimization (including elimination of unwanted motifs and secondary structures) which resulted in 11kb differentially regulated construct and 49 monoterpene synthase were synthesized. In addition codon-optimized and refactored E.coli EUT microcompartment genes as well as a hybrid of the bacteria E.coli and S. typhimurium, with and without his tags, for protein purification. Further 5 genes were synthesised for yeast. For improving optimization and producibility of constructs, data mining and data analysis were conducted. Based on several thousand data items taken from our database (sequences versus complexity of production), multiple data mining approaches were used to identify criteria which relatively precisely reflect the sequence complexity and lead to new optimized designs. The analysis showed that one can mainly limit to the measurement of similarities between windows of size 20 and some easy to check GC content conditions. Furthermore we have now introduced a simple three-level hierarchy (traffic-light system) that captures the complexities sufficiently and can easily visualised and is now integrated into our online system.

Some problems were encountered during the project:
1)There was a problem with personnel in Partner 5 (Scrutton) as the PDRA appointed left the position. A new PDRA was appointed only a few months later. Therefore, a no cost extension until the 31st January 2018 was requested to BBSRC and was approved.
2) Partner 3 (NTNU) had major construction work and change of laboratory space twice throughout the project period, the lab was not up running for several months twice, but this had no consequences for the progress of the overall project.
3) Partner 6 (GeneArt) At the beginning of the project, there was some delay, as the position for a bioinformatician for the project could only be filled in April 2015. Nevertheless, various planned objectives have been achieved
These problems were not a major obstacle and the objectives were met.

Some publications are still pending and are being taken forward by the 5 leads.
Exploitation Route The end compounds generated may have use to industry who requires new or modified monoterpenes for fragrances. The technology developed can also be taken forward by the same industries but also other academics and industry who have interest in generating chemical diversity using synthetic biology approaches.
Scrutton has further commercialised some of the compounds produced in the project and talking to industry for further possible collaborations.
Sectors Agriculture, Food and Drink,Chemicals,Energy,Environment,Healthcare,Pharmaceuticals and Medical Biotechnology

URL http://www.mib.ac.uk/research/portfolio/terpenosome/
 
Description External advisor for European Commission Scientific Committee on Emerging and Newly Identified Health Risks on synthetic biology
Geographic Reach Europe 
Policy Influence Type Citation in other policy documents
Impact Made huge impact on the policy for defining Synthetic Biology, identifying the risks for Synthetic Biology and future recommendations in Synthetic Biology risk assessment at a European level.
 
Description Cold Spring Harbour Laboratory course in Synthetic Biology
Amount £500 (GBP)
Organisation Society for General Microbiology (SGM) 
Sector Academic/University
Country United Kingdom
Start 06/2014 
End 08/2014
 
Title MIBiG 
Description Bacteria, fungi and plants produce an enormous variety of small functional molecules with manifold biological activities, e.g., as antibiotics, immunosuppressants, and signaling molecules. The biosynthesis of such molecules is encoded by compact genomic units (biosynthetic gene clusters). Over the past decades, hundreds of biosynthetic gene clusters encoding the biosynthesis of secondary metabolites have been characterized. Although dozens of biosynthetic gene clusters are published and thousands are sequenced annually (with or without their surrounding genome sequence), very little effort has been put into structuring this information. Hence, it is currently very difficult to prioritize gene clusters for experimental characterization, to identify the fundamental architectural principles of biosynthetic gene clusters, to understand which ecological parameters drive their evolution, and to obtain an informative 'parts registry' of building blocks for the synthetic biology of secondary metabolite biosynthesis. Therefore, developing a genomic standard for experimentally characterized biosynthetic gene clusters (e.g., Minimum Information about a BIosynthetic Gene cluster, MIBiG) would be of great value to the field of microbial secondary metabolism. Building on the MIxS standards for ecological and environmental contextualization, information on, e.g., enzyme function, substrate specificities, functional subclusters, regulatory and transport systems, operon structure, chemical moieties of the end compound and its intermediates, biosynthetic precursor compounds, compound bioactivity and molecular targets and compound toxicity could be added to allow cross-linking the information to biochemistry, pharmaceutical properties, genomic structure and ecology. Using the already developed computational pipeline for analysis of biosynthetic gene clusters antiSMASH (http://antismash.secondarymetabolites.org/), which has quickly become a standard in the field, information on characterized biosynthetic gene clusters will be linked to the untapped wealth of thousands of unknown gene clusters that have recently been unearthed by massive genome sequencing efforts. Taken together, this has the potential to guide the characterization of new metabolites by allowing to optimize the sampling of diversity at different levels and to identify the biochemical, genomic and ecological parameters that are key predictors of pharmaceutically relevant biological activities. Moreover, it can transform the unordered pile of literature on secondary metabolites into a structured and annotated catalogue of parts that can be used as building blocks to design new biochemical pathways with synthetic biology. 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? Yes  
Impact MIBiG will the better description and contextualization of sequence data, of important sequence elements, with concrete biotechnological applications that will increase the visibility and applicability of the GSC and its mission in the fields of applied microbiology, synthetic biology, natural products chemistry and enzymology. 
URL http://gensc.org/projects/mibig/
 
Description Fermentation and Metabolomics 
Organisation Norwegian University of Science and Technology (NTNU)
Country Norway 
Sector Academic/University 
PI Contribution This collaboration is part of the consortium from 13 ERA IB TERPENOSOME: Engineered compartments for monoterpenoid production using synthetic biology project. Takano group shared optimal cultivation settings for wild type and engineered monoterpene production hosts and strains producing high yields of monoterpenes which they can use for their metabolomics studies.
Collaborator Contribution This collaboration is part of the consortium from 13 ERA IB TERPENOSOME: Engineered compartments for monoterpenoid production using synthetic biology project. Prof Per Bruheim (Partner 3) led WP5 Fermentation, monoterpene production analysis, and metabolites in the engineered hosts. Metabolomics analysis for E. coli and yeast were developed which includes a headspace GC-MS/MS method, capillary ion chromatography (capIC) -MS/MS method for the phosphometabolome and improved the chromatographic resolution of sugar phosphates which is necessary for detailed monitoring of upper glycolytic pathway. 80 metabolites have been upgraded with 13C internal standards for improved quantitative accuracy and precision which was crucial for the quantitative metabolomics. A large experimental study with almost 40 batch and chemostat cultivations were conducted and tested was carbon, phosphate and nitrogen limitation at four different growth rates (0.06, 0.12, 0.18 and 0.24 h-1), while in batch four different carbon sources were used (glucose, galactose, fructose and sucrose). This generated a large data set that is currently under investigation.
Impact A large experimental study with almost 40 batch and chemostat cultivations were conducted and tested was carbon, phosphate and nitrogen limitation at four different growth rates (0.06, 0.12, 0.18 and 0.24 h-1), while in batch four different carbon sources were used (glucose, galactose, fructose and sucrose). This generated a large data set that is currently under investigation. Takano joins the work with the interpretation of these results. This work will be completed with writing of a publication. There is no similar study being published by any research group, and we expect quite high interest in these data and findings.
Start Year 2014
 
Description Redesign, DNA synthesis of the monoterpenoid biosynthesis pathway 
Organisation Thermo Fisher Scientific
Country United States 
Sector Private 
PI Contribution This collaboration is part of the consortium from 13 ERA IB TERPENOSOME: Engineered compartments for monoterpenoid production using synthetic biology project. Takano group fed back on the DNA synthesis quality and the use of the new web interface and prediction tools to Thermo Fischer GeneArt.
Collaborator Contribution This collaboration is part of the consortium from 13 ERA IB TERPENOSOME: Engineered compartments for monoterpenoid production using synthetic biology project. Axel Trefzer was the contact for Thermo Fischer GeneArt and led WP2: Redesign, DNA synthesis and assembly of the monoterpenoid biosynthesis pathway. Sequence design and optimization (including elimination of unwanted motifs and secondary structures) which resulted in 11kb differentially regulated construct and 49 monoterpene synthase were synthesized for Partner 5 (Scrutton). In addition codon-optimized and refactored E.coli EUT microcompartment genes as well as a hybrid of the bacteria E.coli and S. typhimurium, with and without his tags, for protein purification for Partner 1 (Takano). Further 5 genes were synthesised for Partner 2 (Braus). For improving optimization and producibility of constructs, data mining and data analysis were conducted. Based on several thousand data items taken from our database (sequences versus complexity of production), multiple data mining approaches were used to identify criteria which relatively precisely reflect the sequence complexity and lead to new optimized designs. The analysis showed that one can mainly limit to the measurement of similarities between windows of size 20 and some easy to check GC content conditions. Furthermore a simple three-level hierarchy (traffic-light system) that captures the complexities sufficiently and can easily be visualised is now integrated into our online system
Impact Meetings and visits - In Manchester in Sep. 2014 and April 2015 - Project partner MIB visiting our Geneart production site in July 2015 - In Trondheim in June 2016 - Skype call in December 2016 - Meeting in Göttingen in May 2017 Publication in prep: Engineered peroxisomes as a new platform for the production of monoterpenoids in yeast Joint publication together with Partner 2 (Braus) Partner 6(Geneart) This collaboration was multi-disciplinary
Start Year 2014
 
Description Untargetted metabolomics 
Organisation Norwegian University of Science and Technology (NTNU)
Country Norway 
Sector Academic/University 
PI Contribution We will be analysing the data from Per Bruhiem's group on untargetted metabolomics and use the data for metabolic modelling of E. coli
Collaborator Contribution They will provide us with data on untargetted metabolomics
Impact This collaboration is multi-disciplinary with my team contributing the computational analysis and NTNU providing experimental data.
Start Year 2014
 
Description Yeast peroxisome production 
Organisation University of Göttingen
Country Germany 
Sector Academic/University 
PI Contribution This collaboration is part of the consortium from 13 ERA IB TERPENOSOME: Engineered compartments for monoterpenoid production using synthetic biology project. Prof Braus (Partner2) led WP4 and also was part of the steering committee. His group worked on the yeast host and peroxisome engineering. We provided Prof Braus with optimal cultivation settings for wild type and engineered monoterpene production hosts (WP 3 and 4) - E. coli and yeast strains that were constructed in the WPs (a set of eight engineered LW2591Y yeast strains which produce geraniol by Partner 2.
Collaborator Contribution This collaboration is part of the consortium from 13 ERA IB TERPENOSOME: Engineered compartments for monoterpenoid production using synthetic biology project. Prof Braus (Partner2) led WP4 and also was part of the steering committee. His group worked on the yeast host and peroxisome engineering. PEX/ATG deletion library for the production of Saccharomyces cerevisiae strains with constantly high numbers of peroxisomes were constructed resulting in four single deletions, six double deletions, four triple deletions, and one quadruple deletion mutant. mCherry-protein tagged with a SKL-tag for transport into peroxisomes was integrated into the chromosome and the number of peroxisomes per cell was quantified via fluorescence microscopy. The strain with the highest number (PEX30/PEX31 deletion) had 433% peroxisomes per cell compared to the control (100%) after one day cultivation in complex medium. Two strains with highest numbers of peroxisomes and no growth deficiencies (PEX30/32 deletion (327%) and PEX30/31/ATG36 deletion (341%)) were chosen to delete ATF1 and OYE2, which are involved in the turnover of geraniol to geranyl acetate and citronellol. This resulted in the reduction of citronellol to 12-fold. Several addition/mutations to increase terpene production was created. Two strains produced higher amounts of geraniol amounts than the control strain. The ratio of peroxisomal to cytosolic geraniol yield was shifted from 0.63 in the control to 1.10-1.15 (PEX30/31/ATG36 deletion), showing that the enrichment of peroxisomes and the synthesis of geraniol inside the peroxisomes produces higher yields of geraniol.
Impact Meetings and visits - In Manchester in Sep. 2014 and April 2015 - Project partner MIB visiting Geneart production site in July 2015 - In Trondheim in June 2016 - Skype call in December 2016 - Meeting in Göttingen in May 2017 Two-weeks stay of postdoc at department of Takano/Scrutton: cultivation of yeast strains and extraction and GCMS analysis of geraniol for screening of different promoters, gene origins and integration methods. Possible publications in preparation: 1)Metabolite profiling of Saccharomyces cerevisiae grown on different carbon sources and growth rates 2)Engineered peroxisomes as a new platform for the production of monoterpenoids in yeast This collaboration was multi-disciplinary
Start Year 2014
 
Title PROCESS OF PRODUCING MONOTERPENES 
Description The present invention relates to a process of producing a monoterpene and/or derivatives thereof. The process comprises the steps of: a) providing a host microorganism genetically engineered to express a bacterial monoterpene synthase (mTS); and b) contacting geranyl pyrophosphate (GPP) with said bacterial mTS to produce said monoterpene and/or derivatives thereof. The present invention also relates to a microorganism for use in producing a monoterpene and/ or derivatives thereof and a recombinant microorganism adapted to conduct the step of converting geranyl pyrophosphate (GPP) into a monoterpene and/or derivatives thereof by expression of a bacterial mTS. It was shown to produce 1,8 cineole using 1,8 cineole synthase and to produce linalool using linalool synthase, both from Streptomyces clavuligerus. 
IP Reference WO2018142109 
Protection Patent granted
Year Protection Granted 2018
Licensed No
Impact n/a
 
Description MIB Open Day Stands/Tours 
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 At Institute Open Day members of research group presented exhibits on topics of enzyme catalysis, synthetic biology, light activated biology and 'proteins' in general. Also demonstrated use of laboratory equipment on lab-tours run for attending students. Event was well received by both students and their teachers and seemed to inspire interest in the subject.

No defined impacts realised to date
Year(s) Of Engagement Activity 2012,2013,2014,2015,2016
 
Description iGEM participation 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Undergraduate students
Results and Impact Takano has organised the Manchester iGEM team since 2012 with Prof Rainer Breitling. The iGEM competition (International Genetically Engineered Machine competition: https://igem.org/Main_Page) is a prestigious international synthetic biology event, with more than 300 participating teams from international universities (form all over the world), who present their summer research at a Giant Jamboree in Boston. iGEM is a major opportunity for undergraduate students to acquire interdisciplinary and transferable skills and to show their achievements in an international setting. The total number of participants is well over 500 with each team having more that 10 members.

Our Manchester teams have been very successful and have achieved a gold medal for four years; in 2016 they won not only the gold medal, but also scooped the special award for 'Best Computational Model' - and were also shortlisted for the 'Best Education and Public Engagement' award. We had very interesting topics from Palm oil production in E. coli to alcohol patch on skin to detect and make aware alcohol consumption. Many discussions involving NGOs(e.g. friends of the earth, green peace, alcohol anonymous) and public institutions (e.g. police, NHS hospitals, FBI) and industry (cheese makers, brewery, confectionery). All of the teams achievements have increased awareness in synthetic biology and sparked many discussion afterwards. We still receive interest for a topic which was done in 2012.

More information of the Manchester teams can be found at
http://2018.igem.org/Team:Manchester
http://2017.igem.org/Team:Manchester
http://2016.igem.org/Team:Manchester;
http://2015.igem.org/Team:Manchester-Graz;
http://2013.igem.org/Team:Manchester
Year(s) Of Engagement Activity 2013,2014,2015,2016,2017
URL http://2018.igem.org/Team:Manchester