Elucidating Physiology of Complex Microbial Systems through Co-Culture 13c-Metabolic Flux Analysis

Microbial
communities carry out efficient biological transformations that are the result
of multiple complementary metabolic systems working together. In bioprocess
applications, co-culture systems have unique advantages over mono-culture
systems in optimizing substrate utilization and product yield, i.e. co-culture systems
allow for the independent optimization and assembly of various complementary
metabolic capabilities into functional systems, where the diversity of
metabolic pathways and ability of microorganisms to exchange metabolites
dramatically increases the space of possible metabolic conversions.

Co-culture
systems are now increasingly applied in metabolic engineering to optimize utilization
of mixed substrates, for production of mixed products, and distribution of
complex metabolic pathways among species. Among the many omics tools used to
gain insight into the physiology of these systems, fluxomics
is the most direct and relevant method to study the in vivo metabolic state of co-cultures. In the past, ¹³C-metabolic
flux analysis (¹³C-MFA) of co-culture systems required physical
separation of proteins or cells to resolve individual population fluxes. In
this work, we demonstrate for the first time that it is possible to determine
metabolic flux distributions in multiple species simultaneously without the
need for physical separation of cells or proteins, or overexpression of
species-specific products. Instead, metabolic fluxes for each species in a
co-culture are estimated directly from isotopic labeling of total biomass
obtained using conventional mass spectrometry approaches such as GC-MS. In
addition to determining metabolic fluxes, the approach quantifies the relative
population size of each species in a mixed culture and interspecies metabolic
exchange, thus enabling detailed studies of species dynamics and interactions
in co-cultures.

We
will demonstrate application of our novel co-culture ¹³C-MFA approach
on the following three diverse and industrially relevant co-culture systems:

i.      
A
co-culture system in which each species consumes the same substrate but
metabolizes the substrate through different pathways, specifically a co-culture
of E. coli knockouts Δpgi and Δzwf.

ii.    
A
co-culture system in which each species consumes the same substrate but
produces a different product, specifically a co-culture of wild-type E. coli and S. cerevisiae.

iii.  
A
co-culture system in which one species consumes a particular substrate and produces
a by-product that serves as the substrate for the second species, specifically a
glucose consuming E. coli and a
glucose non-consuming E. coli strain
(ΔptsI) that uses acetate produced by the first
strain as its carbon source.

Through the use of co-culture ¹³C-MFA,
we are able to elucidate the metabolic state of the various co-culture systems
as well as investigate species dynamics and interspecies metabolite exchange. Moreover,
an improved metabolic network model of S.
cerevisiae for ¹³C-MFA was developed through parallel tracer
experiments. In summary, our work provides a powerful novel approach to study
co-culture systems that will greatly facilitate rational design of co-culture systems
in metabolic engineering.