title: Exometabolomics Assisted Design and Validation of Synthetic Obligate Mutualism.
abstract: Synthetic microbial ecology has the potential to enhance the productivity and resiliency of biotechnology processes compared to approaches using single isolates. Engineering microbial consortia is challenging; however, one approach that has attracted significant attention is the creation of synthetic obligate mutualism using auxotrophic mutants that depend on each other for exchange or cross-feeding of metabolites. Here, we describe the integration of mutant library fitness profiling with mass spectrometry based exometabolomics as a method for constructing synthetic mutualism based on cross-feeding. Two industrially important species lacking known ecological interactions, Zymomonas mobilis and Escherichia coli, were selected as the test species. Amino acid exometabolites identified in the spent medium of Z. mobilis were used to select three corresponding E. coli auxotrophs (proA, pheA and IlvA), as potential E. coli counterparts for the coculture. A pooled mutant fitness assay with a Z. mobilis transposon mutant library was used to identify mutants with improved growth in the presence of E. coli. An auxotroph mutant in a gene (ZMO0748) with sequence similarity to cysteine synthase A (cysK), was selected as the Z. mobilis counterpart for the coculture. Exometabolomic analysis of spent E. coli medium identified glutathione related metabolites as potentially available for rescue of the Z. mobilis cysteine synthase mutant. Three sets of cocultures between the Z. mobilis auxotroph and each of the three E. coli auxotrophs were monitored by optical density for growth and analyzed by flow cytometry to confirm high cell counts for each species. Taken together, our methods provide a technological framework for creating synthetic mutualisms combining existing screening based methods and exometabolomics for both the selection of obligate mutualism partners and elucidation of metabolites involved in auxotroph rescue.
authors.affiliation: Lawrence Berkeley National Laboratory , Berkeley, California 94720, United States.
authors.forename: Suzanne M
authors.initials: SM
authors.lastname: Kosina
authors.affiliation: Lawrence Berkeley National Laboratory , Berkeley, California 94720, United States.
authors.forename: Megan A
authors.initials: MA
authors.lastname: Danielewicz
authors.affiliation: Lawrence Berkeley National Laboratory , Berkeley, California 94720, United States.
authors.forename: Mujahid
authors.initials: M
authors.lastname: Mohammed
authors.affiliation: Lawrence Berkeley National Laboratory , Berkeley, California 94720, United States.
authors.forename: Jayashree
authors.initials: J
authors.lastname: Ray
authors.affiliation: Lawrence Berkeley National Laboratory , Berkeley, California 94720, United States.
authors.forename: Yumi
authors.initials: Y
authors.lastname: Suh
authors.affiliation: Sandia National Laboratory , Livermore, California 94550, United States.
authors.forename: Suzan
authors.initials: S
authors.lastname: Yilmaz
authors.affiliation: Sandia National Laboratory , Livermore, California 94550, United States.
authors.forename: Anup K
authors.initials: AK
authors.lastname: Singh
authors.affiliation: Lawrence Berkeley National Laboratory , Berkeley, California 94720, United States.|University of California Berkeley , Berkeley, California 94720, United States.
authors.forename: Adam P
authors.initials: AP
authors.lastname: Arkin
authors.affiliation: Lawrence Berkeley National Laboratory , Berkeley, California 94720, United States.
authors.forename: Adam M
authors.initials: AM
authors.lastname: Deutschbauer
authors.affiliation: Lawrence Berkeley National Laboratory , Berkeley, California 94720, United States.
authors.forename: Trent R
authors.initials: TR
authors.lastname: Northen
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D018920:Coculture Techniques; D004926:Escherichia coli / Q000235:genetics / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D055432:Metabolomics / Q000379:methods*; D059013:Microbial Consortia / Q000502:physiology; D009154:Mutation; D015203:Reproducibility of Results; D013559:Symbiosis; D058615:Synthetic Biology / Q000379:methods*; D017355:Zymomonas / Q000378:metabolism / Q000502:physiology*
chemical_list: D000596:Amino Acids; D029968:Escherichia coli Proteins
keywords: cross-feeding; exometabolomics; mass spectrometry; microbial synthetic biology; mutant fitness profiling; synthetic obligate mutualism