JBEI Highlights May 2015

1. Understanding limonene toxicity in E. coli
Outcomes
• A mutation in alkyl hydroperoxidase
allowed significantly improved
growth in the presence of limonene
• This led to the hypothesis that
limonene forms a toxic
hydroperoxide, which was verified
by several methods
Background
• Limonene, a promising biofuel
candidate, was previously shown to
be highly toxic to E. coli
Approach
• We allowed E. coli to evolve
tolerance towards limonene and
sequenced the evolved strain,
which was highly limonene-resistant
Significance
• Laboratory evolution is a powerful method to uncover tolerance phenotypes
• We obtained insight into the mechanism of limonene toxicity and have a
strain that is highly tolerant to the toxic hydroperoxide, which forms
spontaneously in aerobic conditions.
Spontaneous oxidation of limonene to limonene-
hydroperoxide and detoxification by AhpCL177Q.
Chubukov, V., Mingardon, F., Schackwitz, W., Baidoo, E. E., Alonso‐Gutierrez, J., Hu, Q., Lee, T. S., Keasling, J. D., &
Mukhopadhyay, A. (2015). "Acute limonene toxicity in Escherichia coli is caused by limonene‐hydroperoxide and
alleviated by a point mutation in alkyl hydroperoxidase (AhpC)". Appl Environ Microbiol, 81(14), 4690‐4696. doi,
10.1128/AEM.01102‐15
Strains expressing AhpCL177Q are
highly tolerant to limonene.
Limonene-hydroperoxide is
the major toxic compound to
E. coli. Non-oxidized
limonene is relatively non-
toxic to wild type E. coli.
The spontenous
E. coli mutant
was resequenced
at the DOE Joint
Genome Institute.

2. Metabolic engineering for the high-yield
production of isoprenoid-based C5 alcohols in
E. coli
Outcomes
• 60% increase in the yield of 3-methyl-3-buten-1-ol by engineering the Shine-Dalgarno sequence of nudB2
• Achieved final titers of 2.23 g/L of 3-methyl-3-buten-1-ol (~70% of pathway-dependent theoretical yield)2
1George, et al., “Correlation Analysis of Targeted Proteins and Metabolites to Assess and Engineer Microbial Isopentenol
Production.” Biotechnology and Bioengineering. 111(8):1648-1658 (2014).
2George, et al., “Metabolic engineering for the high-yield production of isoprenoid-based C5 alcohols in E. coli.” Scientific
Reports. doi:10.1038/srep11128 (2015).
Background
• Branched five carbon (C5) alcohols
are attractive targets for microbial
production due to their desirable fuel
properties and importance as platform
chemicals
• Optimization of Initially engineered
two-plasmid system for 3-methyl-3-
buten-1-ol has been performed1
Approach
• NudB, a promiscuous phosphatase,
was identified as a likely pathway
bottleneck by metabolite profiling, and
RBS engineering on NudB was
attempted.
• Further engineering on mevalonate
kinase expression and C5 alcohol
recovery was also attempted.
Significance
• Engineered a heterologous isoprenoid pathway in E. coli for the high-yield
production of 3-methyl-3-buten-1-ol, 3-methyl-2-buten-1-ol, and 3-methyl-1-butanol
RBS engineering to improve 3-methyl-3-buten-1-ol titer and reduce IPP
accumulation.
~70% theoretical
yield and 2.2 g/L
titer in shake flask
w/ overlay

3. Engineering of plant cell walls for enhanced
biofuel production
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 current advances in
engineering of plants with improved properties as feedstocks for biofuels
production.
• The review focused on the following targets:
• Reducing lignin
• Decreasing inhibitors such as acetate
• Increasing hexose/pentose ratio
• Increasing cell wall sugar content
Significance/perspective
• Up to date review on the advances in cell wall
engineering.
• Summary of many recent studies including several
from the BRCs.
• Annotated reference list with 87 references to
mostly recent literature
• Discussion of remaining challenges, especially trait
stacking and predicting plant performance under a
range of environmental conditions.
Loque D, Scheller HV, Pauly M (2015) Engineering of plant cell walls for enhanced biofuel production.
Curr Opin Plant Biol 25: 151‐161.
An example of
engineering to obtain
high density and low
lignin. In these plants,
normal lignin was
maintained in the
vessels while low
lignin was restricted to
fiber cells.
Biomass is
composed of
different
polysaccharides
and lignin. The
detailed
composition
differs between
different types of
feedstock.

4. An unusual xylan in Arabidopsis primary cell
walls is synthesised by GUX3, IRX9L, IRX10L
and IRX14
Background
• The polysaccharide xylan is a major component of
biomass (2nd only to cellulose).
• It is a hindrance to cellulose deconstruction (blocks
cellulase access); difficult to depolymerise.
Approach and Outcomes
• We identified and characterized a novel xylan structure in
Arabidopsis. This xylan had glycosidic linkages resistant
to standard xylan degrading hydrolases.
• This xylan also has an novel pattern of side-chain
spacing, pointing to exquisite molecular control over
glycan synthesis. This pattern has unknown function.
• Using a multi ‘omics approach, we identified candidate
glycosyltransferase genes for its biosynthesis.
• We used reverse genetics to test and confirm these
predictions in planta.
• We developed a high-throughput, non-radioactive
methods for characterizing glycosyltransferase activity in
vitro.
Significance
• We characterized a novel xylan structure and identified the majority of the
genes responsible for its synthesis.
Mortimer, JC et al. (2015). An unusual xylan in Arabidopsis primary cell walls is
synthesised by GUX3, IRX9L, IRX10L and IRX14. Plant Journal. doi, 10.1111/tpj.12898
Cellulose Xylan
Work led by Prof. Paul
Dupree, University of
Cambridge, UK.

5. Trends In Microbiology: engineering solvent
tolerant microbes
The review
I reviewed strain engineering, primarily as it pertains to
bacterial solvent tolerance, and on the benefits and
challenges associated with expression of membrane‐
localized transporters in improving solvent tolerance and
production.
Background
During microbial production of solvent‐like compounds,
such as advanced biofuels and bulk‐chemicals,
accumulation of the final product can negatively impact
the cultivation of the host microbe and limit the
production levels. Consequently, improving solvent‐
tolerance is becoming an essential aspect of engineering
microbial production strains. Transporters specifically
have emerged as a powerful category of proteins that
bestow tolerance and often improve production but are
difficult targets for cellular expression.
Mukhopadhyay, A. (2015). "Tolerance engineering in bacteria for the production of advanced biofuels and chemicals". Trends
Microbiol. doi, 10.1016/j.tim.2015.04.008

6. SRM-based approach to assess
organelle profiles in plant samples
Outcomes
• It is indeed possible to use the SRM approach (targeted mass spectrometry) to profile organelle abundance in a plant
sample
• We also demonstrated that the approach can be used to assess organelle profiles in plant tissues e.g. the abundance of
plastid markers was significantly higher in photosynthetic tissue.
Parsons and Heazlewood (2015). Beyond the Western front: targeted proteomics and organelle abundance
profiling. Front. Plant Sci. 5, 301. doi: 10.3389/fpls.2015.00301
Background
• The characterization of subcellular
organelles involved in cell wall
biosynthesis requires high purity
preparations for proper analyses.
• Although western blotting enables
profiling of organelle purity and
contamination, there is a limited
availability of antibodies in plant
science
Approach
• Use targeted mass spectrometry
approaches to profile protein extracts
to determine suitability of this method
for organelle profiling.
Significance
• We have developed a collection of SRM transitions or peptide targets that can be now be used
to easily and quickly assess plant organelle profiles in fractions associated with cell wall
biosynthesis e.g. plasma membrane and Golgi apparatus.
Non‐photosynthetic tissue Photosynthetic tissue
SRM signal for plastid markers from mass spectrometry
1 2 3
1 2 3

7. Complex regulation of prolyl-4-hydroxylases
impacts root hair expansion
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
• Prolyl-4-hyroxylase is an enzyme responsible for converting
proline to hydroxyproline. The Hydroxyproline residues are
subsequently glycosylated. Thus, this step is essential for
formation of many, possibly all, glycans and polysaccharies
in the plant cell wall.
• Plants have several P4H isoforms and their individual roles
have not been understood.
Approach and Outcomes
• Mutants in three major P4H isoforms expressed in
Arabidopsis roots were studied.
• P4H5 has an essential function different from P4H2 and
P4H13, which are partly redundant.
• Protein-protein interactions were investigated by co-
localization, bimolecular fluorescence complementation
(BiFC) and FRET (Förster resonance energy transfer).
• The P4H proteins form homodimers, and additionally forms
heterodimes with P4H5
• P4H5 is localized in the Er when P4H5 is absent. Only in the
presence of P4H5 is the P4H2 protein targeted to Golgi.
Significance
• Understanding P4H function is important since these enzymes are necessary for glycan formation
in plant cell walls. The conditional targeting of P4H2 is an important illustration of the role of protein-
protein interactions in the localization of function fo Golgi resident proteins involved in cell wall
formation. This is a vastly understudied area of research.
Velasquez al. (2015). "Complex regulation of prolyl‐4‐hydroxylases impacts root hair expansion". Mol
Plant 8(5), 734‐746.
Left: In roots, deficiency in
P4H is seen in the absence
or deficiency in root hairs.
Below: Protein‐protein
interactions were
investigated with a range of
methods. Results obtained
with BiFC are shown.