JBEI Highlights November 2014

1. Improving microbial bio-gasoline production
in Escherichia coli using tolerance engineering
1Linshiz, et al., “PaR-PaR: Laboratory Automation System.” ACS Synth. Biol. 2:216-222 (2013).
2Linshiz, et al., “PR-PR: Cross-Platform Laboratory System.” ACS Synth. Biol. Article ASAP (2014).
Background
• Isopentenol, a valuable bio-gasoline target has recently been
engineered for production in E. coli (George et al 2014) but is
also toxic to the microbial host.
Approach
• To engineer robust hosts we took a systems biology approach
towards host optimization.
• Whole genome transcriptomics was conducted to identify genes
that were upregulated in response to isopentenol exposure.
Upregulated genes were tested for their potential in improving
tolerance and also increasing production.
Significance
• MdlB is the first E. coli native transporter shown to improve tolerance to and production of a short
chain alcohol and is an important discovery in the area of solvent tolerance engineering.
Foo JL#, Jensen HM#, Dahl RH, George K, Keasling JD, Lee TS,Leong SSJ, Mukhopadhyay A*. Improving
microbial bio‐gasoline production in Escherichia coli using tolerance engineering. mBio 2014
Outcomes
• Several candidates were found that, when
overexpressed, conferred improved isopentenol
tolerance.
• MetR, a regulator provided the greatest
improvement in production when integrated with a
production strain.
• MdlB, an ABC transport protein, also improved
tolerance and production.
MdlB, an ABC transport
pump improves isopentenol
tolerance and improves
production by 12-60 %

2. Structure of the OsSERK2 leucine-rich repeat
extracellular domain
1Linshiz, et al., “PaR-PaR: Laboratory Automation System.” ACS Synth. Biol. 2:216-222 (2013).
2Linshiz, et al., “PR-PR: Cross-Platform Laboratory System.” ACS Synth. Biol. Article ASAP (2014).
Background
• Somatic embryogenesis receptor kinases (SERKs)
are leucine-rich repeat (LRR)-containing integral
membrane receptors that are involved in the
regulation of development and immune responses in
plants.
• It has recently been shown that rice SERK2
(OsSERK2) is essential for XA21-mediated resistance
to the pathogen Xanthomonas oryzae pv. oryzae.
Approach and Outcomes
• The crystal structures of the LRR domains of
OsSERK2 and a D128N OsSERK2 mutant,
expressed as hagfish variable lymphocyte receptor
(VLR) fusions have been solved.
• In BAK1, the D122N mutation causes a phenotypic
variation that results in increased growth at the
expense of defense against pathogens. The
analogous mutation was made in OsSERK2 (D128N).
The result is a shift in the hydrogen bonding to a
residue (R152) that important for binding the co-
receptor.
Significance
• Understanding SERK structure and function will help optimize the growth-defense balance to
maximize crop yields for the growing biofuel demand.
McAndrew et al. (2014). "Structure of the OsSERK2 leucine‐rich repeat extracellular domain". Acta
Crystallogr D Biol Crystallogr, 70(Pt 11), 3080‐3086.
N
C

3. Theory, Practice and Prospects of X-ray
and Neutron Scattering for Lignocellulosic
Biomass Characterization: Towards Understanding Biomass Pretreatment
1Linshiz, et al., “PaR-PaR: Laboratory Automation System.” ACS Synth. Biol. 2:216-222 (2013).
2Linshiz, et al., “PR-PR: Cross-Platform Laboratory System.” ACS Synth. Biol. Article ASAP (2014).
Background
• In this publication, we provide an overview of the theory, current practice and
future prospects of WAS and SAS scattering techniques for probing lignocellulosic
biomass structures with an emphasis on understanding biomass pretreatment and
recalcitrance.
• Our review focused on hierarchical structures of plant cell wall and their
correlations with biomass recalcitrance on the nano/micrometer scale as well as
cell wall chemistry.
• As nano-composites, the integrity of plant cell walls is maintained by interactive
interactions among the three major biomacromolecules and alterations of a
specific structure such as a particular inter-linkage triggers a series of events via
either chemical or physical interactions, therefore, structural characterizations on
the length scale of cell walls are necessary to describe the effects of biomass
pretreatment on the recalcitrant structures in plant cell walls.
Significance/perspective
• First review on the importance of WAS and SAS techniques for
fundamental studies of lignocellulosic biomass processing for
biofuels applications.
• The techniques bridge the knowledge gap at multi-length scales.
• In-situ measurements of structural changes during biomass
pretreatment using WAS and SAS provide rich dynamic information.
• Analysis of SAS data from lignocellulosic biomass samples is not
trivial and SAS data in the higher q region (around 0.1A-1) need to
be analyzed with caution.
• Collaborative discussions with instrument scientist are encouraged.
Gang Cheng, Xin Zhang , Blake A. Simmons and Seema Singh, Energy and Environmental Science,
accepted November 2014
SANS study showed that the lignin aggregates consist
of nanometer‐sized subunits and are released during
IL pretreatment
The scattered intensity is integrated over the
scattering vector q and plotted versus the
azimuth angle  to measure microfibril angle
using SAS