Multi-Modular Platform for Engineering
Self-Sustained Scalable Microbial
Consortia with Programmable Output
Alex S. Beliaev
Pacific Northwest National Laboratory,
Richland WA
12-th Annual World Congress on Industrial Biotechnology
July 19-22, 2015
Montréal, Canada
August 7, 2015
1
Moving Beyond Single Species
Modern biotechnology needs to harness the metabolic
potential of diverse microbes
Limitation of single species
engineering:
improvements come at the
expense of robustness and
stability
well-controlled
environments are required
In nature, robustness and stability are addressed through
functional redundancy and compartmentalization
1
Functional Compartmentalization of
Natural Ecosystems
CO2
Autotroph
Natural communities are are built
upon photoautotroph-heterotroph
Interactions
Carbohydrates
Photosynthesis
Other
anabolism
Heterotroph
O2
Carbohydrate
polymers
Corg CO2
Carbohydrate
polymers
Micro/macr
o-nutrients
(N, P, Fe)
C3-4 intermediates
NADH
Biomass, other
respiration/fermentation
products
Autotroph-heterotroph interactions
are based on the exchange of
commodities and currencies
Interactions evolve into more
complex interrelationships such as
synergism and complementation
of function
Breadth and diversity of
interactions offer ways to control
composition and output
2
Autotroph-Heterotroph Consortium as
Engineering Framework
Design based on
compartmentalization:
- driver
- processor
- controller
CO2
Flexibility via
interchangeable
modules with various
functional outputs (plugand-play)
Interactions can be programmed via built-in regulatory circuits and
feedback loops for self-control and adaptability to environmental
fluctuations
Generalizable Approach for Development
of Riboswitch Based Circuits
PT7
Coding sequence
Riboswitch
Absence of ligand
ligand
repression
induction
Presence of ligand
In Vivo Selection of Riboswitch Controlled
Devices
TetA confers resistance only at an individual cell-level
Bacteriostatic mechanism of action exerts fitness effect upon the organism
TetA expression is a function of aptamer-ligand Kd,
In Vivo Riboswitch Enrichment and
Identification
High-throughput
sequencing of the
switch sequences
2.1 – 4.0 million reads/experiment
~ 1700 THP switch sequences (sequenced >5 times)
~ 2800 pAF switch sequences (sequenced >5 times)
THP responsive sequences
pAF responsive sequences
Activator sequences 103
Activator sequences 105
Repressor sequences 92
Repressor sequences 146
In Vivo Riboswitch Enrichment and
Identification
Switch sequences clustered into several groups
THP activator seq
M-fold predictions
THP repressor seq
FUTURE DIRECTIONS
Model binding kinetics to understand assembly rules that
confer functionality
Examine different in vivo enrichment strategies (batch vs.
continuous)
Apply methodology to
identify novel riboswitch
devices
Proof-of-principle
demonstration for building
riboswitch controlled
synthetic microbial
consortia
9
ACKNOWLEDGEMENTS
PNNL Team:
Dr. Ryan McClure
Dr. Hans Bernstein
Dr. Hyun Seob Song
Dr. Stephen Lindemann
Dr. Moiz Charania
Eric Hill
Collaborators:
Carothers Lab (UW)
Gallivan Lab (Emory)
Funding:
DOE BER Genomic Science
Program
PNNL Lab Directed Research
and Development Program