JBEI Highlights July 2015

1. Comprehensive structural analysis of the
terminal Myxalamid reductase domain for the
engineered production of primary alcohols
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
• The terminal reductase (R) domain from the non-
ribosomal peptide synthetase (NRPS) module MxaA
in Stigmatella aurantiaca Sga15 catalyzes a non-
processive four-electron reduction to produce the
myxalamide family of secondary metabolites
• Despite widespread use in nature, a lack of structural
and mechanistic information concerning reductive
release from polyketide synthase (PKS) and NRPS
assembly lines principally limits our ability to redesign
R domains with altered or improved activity.
Approach and Outcomes
• The crystal structures of the MxaA reductase in the
presence and absence of the cofactor NADPH have
been solved.
• In addition, computational and structural findings
provided a basis for mechanistic investigations and, in
the process, delivered a rationally altered variant with
improved activity towards highly reduced substrate.
Significance
• The results presented here provide a comprehensive understanding of these unique termination
domains and, in the process, set a strong foundation for future efforts to generate new PKS- or
NRPS-based routes to diverse terminal alcohol containing compounds.
J.F. Barajas et al. (2015). ”Comprehensive structural analysis of the terminal myxalamid reductase domain for the
engineered production of primary alcohols". Chem Biol, DOI: http://dx.doi.org/10.1016/j.chembiol.2015.06.022
PCPPCP
NADPH NADPH
RR

2. Natural products as biofuels and bio-based
chemicals: fatty acids and isoprenoids
Background
• Natural products are best known for their uses in medicine and
agriculture. To date, more than 20,000 natural products have been
identified from bacteria, fungi, plants, and marine sources. Although
these compounds are not typically considered as candidates for liquid
transportation fuels, a number of fatty acid-derived and isoprenoid
natural products are being developed for use as renewable biofuels and
bio-based chemicals.
Significance
• This comprehensive review summarizes recent work (2005-2015) on
fatty acid-derived compounds (fatty acid alkyl esters, fatty alcohols,
medium- and short-chain methyl ketones, alkanes, -olefins, and long-
chain internal alkenes) and isoprenoids, including hemiterpenes (e.g.,
isoprene and isopentanol), monoterpenes (e.g., limonene), and
sesquiterpenes (e.g., farnesene and bisabolene).
• Accelerated R&D activity in biofuels over the past decade has facilitated
the discovery of a variety of enzymes that enable biochemical
conversion of fatty acids (and intermediates of fatty acid biosynthesis)
to a range of industrially relevant compound classes.
• To date, gram-per-liter titers (in batch culture) and moderate yields have
been reported for the fatty acid-derived methyl ketones and fatty acid
ethyl esters and for the isoprenoids isopentanol, bisabolene, and
farnesene. Given the extremely rapid pace of developments in
synthetic biology and systems biology, dramatic improvements in
production are within reach.
H. Beller et al. (2015). "Natural products as biofuels and bio‐based chemicals: fatty acids and
isoprenoids". Natural Product Reports, doi: 10.1039/c5np00068h .

3. Biocomposite adhesion without added resin:
understanding the chemistry of the direct
conversion of wood into adhesives
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
• Wood is a ubiquitous natural resource that is a
cornerstone material for industrialized society.
• Composite materials based on wood require
adhesives, most of which are currently derived from
non-renewable sources.
• The realization of an effective bio-renewable adhesive
for the production of composite materials is highly
desired.
Approach and Outcomes
• Laser modification of wood surfaces created a unique
wood surface that could undergo bonding by hot
pressing.
• Compositional analysis of the material at the surface
revealed it was predominately composed of cellulose
and lignin that had been partially depolymerized.
• This produces a very strong bonded interface suitable
for composite materials based on wood.
Significance
• As cellulose and lignin are two of the most abundant polymers found in the world, the manipulation
of them into a structural heat setting resin provides a new potential route for the direct utilization of
biomass.
J.A. Dolan et al. (2015). "Biocomposite adhesion without added resin: understanding the chemistry of the
direct conversion of wood into adhesives". RSC Adv., 5(82), 67267‐67276.
Illustrative model of laser induced changes of a wood surface
at the nanoscale level and hot pressed bondline illustrating
potential adhesion mechanism.

4. Tight regulation of plant immune responses by
combining promoter and suicide exon elements
Outcomes
• Developed a very tight expression system by stacking the suicide exon technology on top of the DEX inducible promoter system
• Regenerated viable and healthy plants harboring the resistance gene and the dual regulated effector that can be induced on
demand to initiate the HR phenotype
• Demonstrate that this dual regulation strategy is generalizable to control the expression of various toxic proteins or biosynthetic
enzymes
Use of a dual transcription and splicing regulation to eliminate
leaky hypersensitive response
Gonzalez, et al. (2015) “Tight regulation of plant immune responses by combining promoter and
suicide exon elements.” NAR. DOI: 10.1093/nar/gkv655.
Background
• Effector genes regulated by inducible promoters
cause background cell death due to leaky protein
expression.
• Leaky protein expression from inducible
promoters is a common limitation in transgenic
plant generation
Approach
• Utilization of a suicide exon to tightly regulate the
expression of effector proteins.
• Stacking of the DEX inducible promoter system
on top of the suicide exon technology to further
control the expression of effector proteins
Significance
• This dual regulation system is generally applicable to genes that initiate severe or undesired
phenotypes upon leaky expression and provides a straightforward and promising way to generate
many previously unattainable transgenic crops.

5. Phylogenomics databases for facilitating
functional genomics in rice
Outcomes
• Functional genomics studies of rice genes belonging to a single family can be facilitated by phylogenomic analysis
that integrates diverse types of biological information.
• Future prospects to advance analysis of gene function include coupling phylogenomic analyses with computational
predictions of gene function.
Background
Among the 42,653 predicted (non-
transposon) genetic elements in the rice
genome, 21,998 encode multiple members
that make up 3,865 protein families. Thus,
each gene within the rice genome has a
51.6% possibility of being functionally
redundant (21,998 / 42,653).
Approach
Phylogenomics databases for six large
gene families, i.e., those for kinase,
glycosyltransferases (GTs), glycoside
hydrolases (GHs), transcription factors
(TFs), transporters, and cytochrome p450
monooxygenases (P450s), have been
constructed.
Significance
• In this review key features and applications of these databases has been introduced to
serve as a very useful guide in the post-genomics era of the research.
K.H. Jung et al. (2015). "Phylogenomics databases for facilitating functional genomics in rice". Rice (N Y), 8(1), 60.

6. Recent innovations in analytical methods for
the qualitative and quantitative assessment
of lignin
Background
• Lignin has frequently been considered a waste‐product from
the deconstruction of plant cell walls, in attempts to isolate
polysaccharides that can be hydrolyzed and fermented into
fuel or other valuable commodities.
• In order to develop useful applications for lignin, accurate
analytical instrumentation and methodologies are required
to qualitatively and quantitatively assess.
• This review seeks to provide a comprehensive overview of
many of the advancements achieved in evaluating key lignin
attributes.
J.S. Lupoi et al., (2015) “Recent innovations in analytical methods for the qualitative and
quantitative assessment of lignin”. Renewable and Sustainable Energy Reviews, 49, 871–906.
Schematic representation
of lignin including syringyl
(S, blue), guaiacyl (G,
green), and p-
hydroxyphenol (H, red)
phenylpropanoid moieties,
and lignin–lignin linkages
Common
fragments of
lignin measured
after pyrolysis.
Significance/perspective
• High‐throughput, multivariate analysis modeling is an
effective technique to efficiently screen biomass for key
biofuel traits.
• Advances in NMR, imaging, mass spectrometry,
computational approaches, and vibrational spectroscopy
have facilitated the construction of toolbox.
• Techniques developed for probing lignin structure, linkages
to carbohydrates, and quantifying specific functionalities
and linkages present in lignin and pyMBMS have provided
another innovative technique for probing lignin monomer
content.
• Further advances in these analytical tools can only build
upon the foundation laid by the research described in this
review.

7. Localization of polyhydroxybutyrate in
sugarcane using Fourier-transform infrared
microspectroscopy and multivariate imaging
Background
• The production of bio-based, biodegradable plastics
from or in plants can assist in supplanting those
manufactured using fossil fuels.
• Polyhydroxybutyrate (PHB) is a biodegradable
polyester that has been evaluated as a possible
candidate for relinquishing the use of conventional
plastics.
Approach and Outcomes
• PHB, possessing similar properties to polyesters
produced from non-renewable sources, has been
previously engineered to be expressed in
sugarcane.
• This manuscript illustrates the coupling of a Fourier-
transform infrared microspectrometer, equipped
with a focal plane array (FPA) detector, with
multivariate imaging to successfully identify and
localize PHB aggregates in planta.
Significance
• This study demonstrates the power of IR microspectroscopy to rapidly image plant sections to
provide a snapshot of the chemical composition of the cell.
• While PHB was localized in sugarcane, this method is readily transferable to other value-added co-
products expressed in different plant tissues.
Images of polyhydroxybutyrate‐containing sugarcane (a) without and (b) with spectral points
selected. The images were collected using a 128 × 128 focal plane array. c Re‐constructed
image using the second principal component; (d) Loadings plot for the second principal
component used in re‐constructing the image in (c).
J.S. Lupoi et al. (2015). "Localization of polyhydroxybutyrate in sugarcane using Fourier‐transform infrared
microspectroscopy and multivariate imaging". Biotechnol Biofuels, 8, 98.

8. Standards for plant synthetic biology:
a common syntax for exchange of DNA parts
Outcomes
• Developed standards for Type IIS restriction endonuclease-mediated assembly
• Defined a common syntax of 12 fusion sites to enable the facile assembly of eukaryotic transcriptional units
Twelve fusion sites have been defined allowing a multitude
of standard parts to be generated to build synthetic genes.
Patron et al. (2015) “Standards for plant synthetic biology: a common syntax for
exchange of DNA parts.” New Phytol. DOI: 10.1111/nph.13532 (2015).
Background
• Synthetic biology requires the development of
biological parts for the engineering of plants.
• In the plant field, synthetic biology tools are
rather limited, none compatible with each others,
and are poorly exchangeable between labs
Approach
• Standardization of DNA parts will accelerate
plant bioengineering.
• An international consortium including inventors,
developers and adopters of Golden Gate cloning
methods agreed to use a Type IIS genetic
grammar for plants that is also extendible to all
eukaryotes
Significance
• Enables sharing characterized DNA parts between laboratory across the world, speedup
fundamental research discoveries, leverage crop engineering, reduce experimental costs.

9. Engineering secondary cell wall deposition
enhances monomeric sugar release after
low temperature ionic liquid pretreatment
Outcomes
• The reduced lignin Arabidopsis engineered lines resulted in high levels of monomeric sugar release at lower pretreatment
temperatures as compared to the wild type.
• Ionic liquid pretreatment of the engineered Arabidopsis at 70 °C for 5 hours resulted in improved saccharification
efficiency and increased hemicellulose recovery for the pretreated biomass and produced similar total sugar yields as
compared to those obtained after pretreatment at 140 °C for 3 hours.
Background
Increasing the accumulation of polysaccharides
in biomass and improving biomass digestibility
could have significant beneficial impacts on the
cost of lignocellulosic biofuel production both by
increasing fermentable sugar yield per acre and
reducing the severity of pretreatment Approach
Approach
We engineered Arabidopsis strains with
reduced lignin levels that carry mutations of
C4H and NST1, which, respectively, restrict
lignin biosynthesis to vessels, and enhance
polysaccharide levels with little to no apparent
negative impact on growth phenotype. These
strains were tested using IL pretreatment for
reducing severity of the pretreatment.
Significance
Similar sugar recovery at the lower temperature pretreatment supports the hypothesis that reducing
lignin can reduce the necessary severity of pretreatment needed, and increased polysaccharide
deposition can increase glucose recovery on a mass basis.
0
5
10
15
20
25
30
35
ut 70 140 ut 70 140 ut 70 140 ut 70 140
wt mut1 mut2 mut3
SugarRecovery%ofBiomass
xylose
glucose
**
+
--
**
++
--
**
++
--
*
--
**
--
**
--
*
+ +
C. Scullin et al. (2015) "Restricting lignin and enhancing sugar deposition in secondary cell walls enhances
monomeric sugar release after low temperature ionic liquid pretreatment". Biotechnol Biofuels, 8, 95.

10. Polarized Raman micro-spectroscopy:
diagnosing cell wall microfibril organization in
engineered plants
Outcomes
• Protocols can be translated for liquid-handling robots, microfluidic devices, automated microscopes, and people.
• DNA assembly and mutatagenesis protocols were successfully performed on a robot, a microfluidic device, and by hand.
L. Sun et al. (2015). "Non‐invasive imaging of cellulose microfibril orientation within plant cell walls by
polarized Raman microspectroscopy". Biotechnol Bioeng. doi, 10.1002/bit.25690
Background
• Reliable screening methods
are needed to assess the
consequence of genetic
mutation on the cell wall
properties, ideally with
predictive value on the
mechanical stability of the
feedstock plant.
Approach
• Develop an optical approach
(including automated
analysis) that combines the
orientation sensitivity of
polarized light with the
macromolecular specificity of
Raman Microspectroscopy
Significance
• Enables quick assessment of mutation on the order of plant cell wall microfibril
organization for a predictive understanding of mechanical plant properties.

11. MUCI10 produces galactoglucomannan that
maintains pectin and cellulose architecture in
Arabidopsis seed mucilage
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
• Mannans are abundant hemicelluloses in plants
• The biosynthesis is not fully understood.
Approach and Outcomes
• muci10 is a mutants with altered seed mucilage
• muci10 is mutated in a glycosyltransferase in the GT34 family.
• Arabidopsis seed mucilage was shown to contain abundant
galactoglucomannan. This is different from seed
galactomannans described in other species and from mannan in
vegetative tissue.
• MUCI10 is an alpha-1,6-galactosyltransferase that adds
galactose to the glucomannan backbone.
• Branched mannans are important for cellulose deposition.
Significance
• Most glycosyltransferases still have unknown function. This study
increases our basic understanding of cell wall biosynthesis and
provides new tools for cell wall engineering. Understanding
galactoglucomannan biosynthesis is important since it is the
most abundant hemicellulose in gymnosperm wood. Knowing the
enzymes required for glucomannan biosynthesis enables
engineering of bioenergy crops plants with increased
hexose/pentose ratio, which would be advantageous for
fermentation into fuels and bioproducts.
Voiniciuc et al. (2015). "MUCI10 Produces Galactoglucomannan That Maintains Pectin and Cellulose
Architecture in Arabidopsis Seed Mucilage”. Plant Physiol, doi 10.1104/pp.15.00851.
muci10 has decreased seed mucilage. The csla2
mutant which cannot synthesize the glucomannan
backbone is shown for comparison
MUCI10 adds
alpha‐1,6‐linked
galactose
residues to the
glucomannan
backbone

12. Interlaboratory study to evaluate the
robustness of capillary electrophoresis with
mass spectrometry for peptide mapping
Background
• Due to the different separation mechanism, peptides
missed in reversed-phase LC–MS can be detected by
CE–MS, rendering this technology an attractive
complementary or alternative tool for peptide
mapping.
• In an effort to illustrate the robustness and portability
of CE–MS for peptide mapping, an international team
was formed, which includes 13 independent
laboratories from companies and universities in the
United States, Canada, Australia, and Europe.
Approach and Outcomes
• Trypsin digested BSA was chosen as a model system.
• All laboratories analyzed the sample on two
consecutive days.
• Migration time and peak intensity of a representative
set of ten target peptides were evaluated and a
statistical analysis was performed based on the
principles of the ISO 5725-2 guideline
Significance
• Repeatability levels of 9–12% RSD obtained by these laboratories agree with results reported by
former studies employing CE–MS- or LC–MS-based methods.
• This study emphasizes the importance of tightly controlled system suitability tests for a successful
method transfer across multiple sites.
C. Wenz et al. (2015). "Interlaboratory study to evaluate the robustness of capillary electrophoresis with mass
spectrometry for peptide mapping". J Sep Sci. doi, 10.1002/jssc.201500551
Repeatability and
reproducibility (given
as % RSD) of tm
(laboratories 1–11)
(A) and %A
(laboratories 1–9) (B)
were plotted against
their general mean
for all target peptides.
Linear regression
trendlines are
indicated as dashed
lines. Correlation
coefficient (R2) values
are given as
insets.