Exometabolomics Assisted Design and Validation of Synthetic Obligate Mutualism

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.