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. In this paper, 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 rescuemore » 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.« less

Authors:

Kosina, Suzanne M. ^[1]; Danielewicz, Megan A. ^[1]; Mohammed, Mujahid ^[1]; Ray, Jayashree ^[1]; Suh, Yumi ^[1]; Yilmaz, Suzan ^[2]; Singh, Anup K. ^[2]; Arkin, Adam P. ^[3]; Deutschbauer, Adam M. ^[1]; Northen, Trent R. ^[1]

---

+ Show Author Affiliations

1. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
2. Sandia National Lab. (SNL-CA), Livermore, CA (United States)
3. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States)

Publication Date:

Wed Feb 17 00:00:00 EST 2016

Research Org.:

Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Org.:

USDOE Office of Science (SC), Biological and Environmental Research (BER)

OSTI Identifier:

1429352

Grant/Contract Number:  

AC02-05CH11231

Resource Type:

Accepted Manuscript

Journal Name:

ACS Synthetic Biology

Additional Journal Information:

Journal Volume: 5; Journal Issue: 7; Related Information: © 2016 American Chemical Society.; Journal ID: ISSN 2161-5063

Publisher:

American Chemical Society (ACS)

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; cross-feeding; exometabolomics; mass spectrometry; microbial synthetic biology; mutant fitness profiling; synthetic obligate mutualism