Reverse engineering the soil microbiome: detecting, modeling, and optimizing signal impacts on microbiome metabolic functions

Lead Research Organisation: University of Manchester
Department Name: Chemistry

Abstract

Overview: Our project will provide fundamental insights into the roles of signals in mediating the ecology of soil microbes and suppression of plant diseases. This work establishes a foundation for engineering functional soil microbiomes for precision agriculture. Our team consists of experts in soil ecology, genetic engineering, metabolomics, and community modeling from the UK and USA.

Objectives: 1) Develop and test genetic recorder (GR) strains to 'listen and report' on signals in the soil that regulate primary and secondary metabolic pathways in Streptomyces spp. isolated from disease suppressive soils; 2) Model and test how species-species interactions that rely on primary and secondary metabolic induction impact multi-species communities; and 3) Discover effects of potential signals on Streptomyces metabolism and harness signals to optimize microbial functional
capacities in soil.

Methods: 1) We will create GRs to detect the activation of genes/pathways of interest in soil microbes using serine integrase-mediated recombination. The GRs will be triggered by yet unknown chemical, physical, and biological signals to produce a state change that can be easily quantified using Next-Generation Sequencing technology. Using these GRs, we will be able to simultaneously record the activation of hundreds of metabolic activities, across diverse microbial species, in a single high-throughput experiment. 2) We will utilize genome-scale metabolic models,
transcriptomics, and metabolomics to connect signals to functions. We will extend existing metabolic modeling platforms to incorporate novel functionality to understand how signals will influence the physiology of individual bacteria and alter emergent ecosystem dynamics. Transcriptomic and metabolomic data will be used to validate and extend current knowledge of exo-metabolite roles in system behavior and advance systems-level understanding of soil microbiomes. 3) We will screen potential signals for their direct effects on Streptomyces antibiotic inhibitory and nutrient use phenotypes in vitro. We will use the GRs to screen presumptive signals for their role in mediating Streptomyces primary and secondary metabolic activities, providing both a signal discovery platform and a direct comparison with phenotypic data. We will create signal-optimized isolate combinations or isolate/signal combinations and test their capacities to reduce plant diseases on wheat and radish seedlings in soil. Finally, we will test the effectiveness of the GRs in detecting signals in complex soil communities.

Intellectual Merit: The proposed research will advance fundamental understanding of the roles of signals in mediating the assembly, dynamics, and functional behaviors of complex soil microbiomes; provide novel tools for studying signaling dynamics in vivo, with potential to serve as sensors of microbial activities in soil; enhance systems-level understanding and modeling of primary and secondary metabolic activities within microbiomes; and test the capacity of signal-optimized inoculants to enhance plant health and productivity in soil systems.
sustainable cropping systems worldwide.

Planned Impact

Education, training, and participation of underrepresented groups: We will recruit and train five post- doctoral research associates (PDRA), while actively recruiting additional graduate students and undergraduate researchers to our project. At both institutions, the PDRA and graduate students will work within highly-collaborative interdisciplinary teams, and gain experiences in biotechnology, agriculture, and synthetic biology. We have budgeted funds to support each PDRA for a cross-institution short-term research exchange to enhance scientific training. To encourage participation of nderrepresented groups, the UMN team will work with the McNair Scholars Program, which provides fellowships for undergraduates from underrepresented groups. The U-M team will participate in the Life Sciences Teachers Summer School to engage local school teachers with the research undertaken on this project, and will be involved in the U-M Work Experience program which mentors 14-15 year-old students in the opportunity for hands-on research. The U-M team will participate in outreach events such as `Discover Day' (targeting students planning to go to University) and `Science Star Days' (to inspire younger children to pursue science). Finally, all PIs will incorporate our research findings into undergraduate courses and offer opportunities for undergraduate research experiences throughout the term of the project.

Outreach and stakeholder interactions: Relevant findings from this project will be shared in twice-annual meetings with Minnesota crop producers. PI Kinkel is the co-founder of a small business (Jord Bioscience) that could potentially license IP generated within this proposal to commercialize and facilitate its adaptation in precision agriculture domestically and internationally.

Dissemination of research to enhance understanding: The PIs will disseminate results to the scientific community through diverse, high-impact scientific journals. PIs, postdocs, and graduate students will present research findings at major national and international conferences. We will communicate our work to the general public via UMN and U-M press offices to highlight project findings. We will propose a UMN Bell Museum of Natural History Café Scientifique (CS) session, which provides a unique happy hour program at for adults to learn about cutting-edge research. Finally, the project partners are committed to the use of social media, including Twitter, to disseminate project findings.

Sponsored Research Symposia and events: In conjunction with the visit of the U-M team to the UMN, we will sponsor a full-day research symposium for students, staff, PDRA, and faculty, focusing on "Convergent Research to Advance Understanding and Applications in Microbiome Science". In addition, all PDRA will present departmental or program seminars on their cross-institutional visit, and seminars will be required to be in an academic discipline outside of the student's home institution. This will challenge students to take a truly interdisciplinary approach to the presentation of their research project. Finally, post-doctoral scientists at UMN will jointly develop and teach a one-semester Special Topics course on the soil microbiome, with an emphasis on the biotic signals of plant/microbial origin.

Publications

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Description The project has just started and has severly been effeceted by the Covid pandemic and travel restricitions. The project progress is now delayed by at least 6 months (due to the first lockdown from March-July 2020 and subsquent 50% occupancy). We have been able to make substantial progress. However, the progress is still slow compared to normal times, as we do not have access to the lab 100%. We also have further negative impact as this is a joint project with US partners at the University of Minnesota. The planned kickoff meeting was cancel and hope to meet in person later in 2021 or early in 2022..
There have been delays in establishing a collaboration agreement between the University of Manchester and Minnesota that will allow material transfer, perhaps due to the effect of pandemic-related staffing shortage in both universities' contract offices. We anticipate we will have a future-proof agreement in place soon. However, so far the transfer of key strains and plasmids has been delayed.

Key Achievements:
i) Identification and testing of CRISPR plasmids for GR integration
We have identified a series of CRISPR plasmids with varied regulation of Cas9 expression via different promoters and a theophylline responsive riboswitch. We have successfully tested these in model Streptomyces strains, prior to their use to install the GRs into our model microbiome.
ii) Generation of genome-scale metabolic models
We have built 10 genome-scale metabolic models for the 10 Streptomyces isolates that make up our model microbiome.
iii) Integration of proteome allocation theory to the COMETS framework
We have tested the compatibility between proteome allocation principles and the modelling COMETS framework. We have successfully used COMETS to simulate the growth of a E. coli/Salmonella enterica co-culture with acetate-methionine cross-feeding interactions based on proteome-constrained models.
iv) All UK and US team members attended the Signals in the Soil Virtual Workshop, 15- Sept 2020. We have uploaded a project video and the US PI answered the questions in Project Lighting Talk Question session. Two PDRAs (one UK and one US) presented a poster each and had some interesting discussions.

Overall progress
Our project aims to understand signalling among 10 Streptomyces isolates using multiple technologies and datasets. Genomic and meta-transcriptomic data have already been collected by our US collaborators, and we are now positioned to collect untargeted metabolomics in the coming period.
While our novel genetic recorders are being prototyped in model systems, we are establishing the use of CRISPR to install the GRs into the chromosomes of our isolates, through the tuning of Cas9 expression in a series of plasmids generated in the Takano lab. We have successfully disrupted glycerol uptake using this tool in other Streptomyces strains, and now look to apply it to our 10 isolates and other Streptomyces strains.
Our US collaborators have been curating the 10 original metabolic models by including secondary metabolism and signal and toxin matrices, while we have been incorporating constraints based on resource or proteome allocation theory. This new approach will play an important role in determining metabolic strategies to improve predictions of metabolic fluxes and community-level interactions in the COMETs frameworks. We have also investigated the role of ensemble modelling in studying the uncertainties of model topology and proteomic cost parameters. We now will integrate and iterate our models with our wetlab datasets.
Exploitation Route N/A
Sectors Agriculture, Food and Drink,Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description SitS collaboration 
Organisation University of Minnesota
Country United States 
Sector Academic/University 
PI Contribution The collaboration with University of Minnesota is throught the NSF - UKRI/NERC Signals in the Soil (SitS) program. We have a project entitled "Reverse engineering the soil microbiome: detecting, modeling, and optimizing signal impacts on microbiome metabolic functions" with our partners in University of Minnesota: Prof Linda Kinkel, Dr Michael Smanski, Dr William Harcombe; and from the University of Manchester: Prof Eriko Takano and Prof Rainer Breitling. In addition, we have two PDRAs (an expert in Streptomyces molecular biology - Jack Connolly, an expert in microbial community modelling - Hong Zeng who was appointed June 2020 - Feb 2021) and one technician - Kamila Schmidt with expertise in untargeted metabolomics recruited at University of Manchester from this funding. We are contributing in all of the Objectives of the project. Objective 1: Develop and test Genetic Recorder strains for regulated primary and secondary metabolic pathways in ten sympatric Streptomyces isolates from a model soil community - where Prof Takano contributes her expertise on Streptomyces small signalling molecules, high throughput pathway assembly and Streptomyces CRISPR/Cas9 technology. Objective 2: Predict and test the mechanisms of primary and secondary metabolic induction in multi-species (2-10 isolates) communities - where Prof Breitling contributes his expertise on modelling and computational genome and untargeted metabolite analysis. Objective 3: Discover effects of potential signals on Streptomyces metabolism and harness signals to optimize microbial functional capacities in soil - where we provide our signal molecules that we have already isolated.
Collaborator Contribution Our partners from University of Minnesota: Prof Linda Kinkel, Dr Michael Smanski, Dr William Harcombe are also all involved in the three objectives. Dr Michael Smanski in Objective 1 for his expertise in Streptomyces genetics, Dr William Harcombe in metabolite modelling, and Prof Linda Kinkel in soil ecology and the link with Streptomyces in disease suppression.
Impact The project requires a very close collaboration with all the 5 groups involved (mentioned above) which is a multi-disciplinary, ranging from computational modelling and metabolite analysis, untargeted metabolomics, Streptomyces genetics, synthetic biology, and soil ecology. We have setup a Google Drive for the project where all presentation, documents, data are shared. We also have a scheduled monthly zoom meeting with all the PIs and PDRA and PhD students involved in the project. During this meeting each group presents an update as well as one group giving a more indepth presentation. There was a physical annual meeting planned by NSF-UKRI at Washington DC in 2020, however due to the Covid pandemic and travel restrictions, this was changed to a virtual meeting. The virtual meeting was held 15-17- September 2020 and all the group members attended this meeting. We presented a joint video and PI slides which summarises our project and presented in the lighting talk question session. In addition, one of the University of Manchester PDRA (Jack Connolly) and University of Minnesota PDRA (Jeremy, Chacón) gave a poster presentation. COVID-19 has been extremely disruptive to the beginning of our project and the effect is still continuing. Particularly in the wet lab, as there was no lab access during lockdown for 4 months. We now have lab access 50% of the time since July 2020. This access restriction is planned to go on as long as the 2M physical distancing rules from the government does not change. We have been able to make some progress. However, the progress is still slow compared to normal times, as we do not have access to the lab 100%. There have been delays in establishing a collaboration agreement between the University of Manchester and Minnesota that will allow material transfer, perhaps due to the effect of pandemic-related staffing shortage in both universities' contract offices. We anticipate we will have a future-proof agreement in place soon. However, so far the transfer of key strains and plasmids has been delayed.
Start Year 2019
 
Description iGEM 2020 
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 Organised the Manchester iGEM team for 2020. iGEM is an annual international student competition in synthetic biology, providing a great interdisciplinary learning opportunity for our students from all faculties. This year's Manchester iGEM team worked throughout the summer in very unusual circumstances. The team project was Hipposol, aimed at producing an eco-friendly sunscreen, derived from hippopotamus sweat, in genetically engineered bacteria.
They were probably the first iGEM team to successfully use computational retrosynthesis for their project, and with the help of the Manchester Business School they comprehensively explored the social and economic implications of their ideas using a Responsible Research and Innovation approach.
The Manchester 2020 iGEM team won a Gold medal and was nominated for the "Best Supporting Entrepreneurship" award in this year's iGEM (Virtual) Giant Jamboree
Year(s) Of Engagement Activity 2020
URL https://2020.igem.org/Team:Manchester