JBEI Research Highlights - September 2018

1. Three members of the Arabidopsis thaliana glycosyl-
transferase Family 92 are functional β-1,4-galactan synthases
Background
• Pectin is a major component of primary cell walls and performs a
plethora of functions crucial for plant growth, development and plant-
defense responses.
• Despite the importance of pectin polysaccharides their biosynthesis is
poorly understood. Several genes have been implicated in pectin
biosynthesis by mutant analysis, but biochemical activity has been
shown for very few.
Approach
• We used reverse genetics and biochemical analysis to study
members of Glycosyltransferase Family 92 (GT92) in Arabidopsis
thaliana.
Outcomes and Impacts
• Biochemical analysis gave detailed insight into the properties of
GALS1 (Galactan synthase 1) and showed galactan synthase activity
of GALS2 and GALS3. All proteins are responsible for adding
galactose onto existing galactose residues attached to the
rhamnogalacturonan-I (RG-I) backbone.
• GALS activity was observed with galactotetraose as acceptor but
longer acceptors are favored.
• Overexpression of the GALS proteins in Arabidopsis resulted in
accumulation of unbranched β-1,4-galactan.
• Plants in which all three genes were inactivated had no detectable β-
1,4-galactan, and surprisingly these plants exhibited no obvious
developmental phenotypes under standard growth conditions.
• RG-I in the triple mutants retained branching indicating that the initial
Gal substitutions on the RG-I backbone are added by enzymes
different from GALS.
Ebert et al. (2018) Plant Cell Physiol, DOI: 10.1093/pcp/pcy180
FLAG-GALS1 was
expressed in
tobacco and activity
was determined
using UDP-14C-
Galactose as donor
and different
oligosaccharides as
acceptor. Elongation
of linear β-1,4-
galactooligosaccha
rides could be
detected. None of
the branched
oligosaccharides
could function as
acceptor.
Dot blots (A, B) and microarray polymer profiling (C) were used to
probe glycan epitopes. The gals triple mutant was devoid of β-1,4-
galactan according to the lack of signal with LM5, an antibody
recognizing β-1,4-galactan with DP>3.

2. A Golgi UDP-GlcNAc transporter delivers substrates
for N-linked glycans and sphingolipids
Background
• Glycosylation requires activated glycosyl donors in the form of
nucleotide sugars to drive processes such as post-translational
protein modifications and glycolipid and polysaccharide biosynthesis.
• Most of these reactions occur in the Golgi, requiring cytosolic-derived
nucleotide sugars, which need to be actively transferred into the
Golgi lumen by nucleotide sugar transporters.
Approach
• We identified a Golgi-localized nucleotide sugar transporter from
Arabidopsis thaliana that specifically transports UDP-N-acetyl-D-
glucosamine (UDP-GlcNAc).
• GlcNAc containing N-glycans and glycolipids in ugnt1 mutants were
characterized by LC-MS.
Outcomes and Impacts
• Plants carrying the ugnt1 mutation are virtually devoid of complex
and hybrid N-glycans. Instead, the N-glycopeptides exhibited high-
mannose structures, representing structures prior to the addition of
the first GlcNAc in the Golgi.
• Sphingolipid profiling revealed that the biosynthesis of GlcNAc-
containing glycosyl inositol phosphorylceramides (GIPCs) is also
reliant on this transporter.
• By contrast, plants carrying the loss-of-function alleles affecting
ROCK1, which has been reported to transport UDP-GlcNAc and
UDP-N-acetylgalactosamine, exhibit no changes in N-glycan or GIPC
profiles.
• Our findings reveal that plants contain a single UDP-GlcNAc
transporter that delivers an essential substrate for the maturation of
N-glycans and the GIPC class of sphingolipids.
Ebert et al. (2018) Nat Plants, DOI: 10.1038/s41477‐018‐0235‐5.
UGNT1 and ROCK1 were expressed in yeast and incorporated into
liposomes. Uptake of nucleotide sugars was determined by LC-MS.
UGNT1 is highly specific for UDP-GlcNAc (Km = 9 µM). Rock1 has
little activity and Km > 1 mM.
N-glycosylation patterns were determined by LC-MS/MS. Mutants in
UGNT1 accumulate high-mannose structures and lack mature
glycans in the same way as cgl1, a mutant deficient in GlcNAc
transferase. Rock1 has pattern indistinguishable from wild type.

3. Overexpression of a rice BAHD acyl-
transferase gene in switchgrass
enhances saccharification
Outcomes
• Overexpression of the rice OsAT10 gene in switchgrass altered the levels
of wall-bound ferulic acid (FA) and p-coumaric acid (p-CA).
• The engineered switchgrass lines exhibit an approximate 40% increase
in saccharification efficiency in green tissues and a 30% increase in
senesced tissues.
Switchgrass lines overexpressing
OsAT10 show alterations in cell wall-
bound phenolics. a Quantitative analysis of
cell wall-bound phenolics of green leaves of
the wild-type (WT) (3 biological replicates) and
OsAT10 overexpressing switchgrass lines,
FT2 (4 biological replicates) and FT8 (4
biological replicates). b Quantitative analysis of
cell wall-bound phenolics from senesced
tissues of the same set of switchgrass lines.
Bars indicate standard deviation, and asterisks
indicate significant differences using the
unpaired Student’s t-test (*P<0.05; **P<0.01).
Background
• Switchgrass is a promising bioenergy
feedstock.
• Recalcitrance of switchgrass biomass
impedes efficient biofuel production.
• Overexpression of the OsAT10 gene
enhances saccharification efficiency in
rice.
Significance
• We have generated two switchgrass lines with enhanced saccharification
• We have demonstrated that the OsAT10 gene is a valuable target for
improving biofuel production in bioenergy feedstocks.
Saccharification assays of green leaves (a) and
senesced tissues (b) of the wild-type (WT) and
OsAT10 overexpression switchgrass lines (FT2
and FT8). Reducing sugars released from biomass were
measured using the 3,5-dinitrosalicylic acid (DNS) method.
Bars represent mean ±standard deviation. Asterisks
indicate significant differences using the unpaired Student’s
t-test (*P<0.05).
Approach
• Overexpress the rice OsAT10 gene in
switchgrass.
• Conduct saccharification assays and other
assays on the OsAT10 overexpression
lines.
Li et al. (2018) BMC Biotechnol, DOI: 10.1186/s12896-018-0464-8

4. Mortimer, JC (2018) Exp Biol Med, DOI: 10.1177/1535370218793890
Background
• Population growth, climate change, and dwindling finite resources are
amongst the major challenges which are facing the planet.
• Requirements for food, materials, water, and energy will soon exceed
capacity.
• Green biotechnology, fueled by recent plant synthetic biology
• breakthroughs, may offer solutions.
Approach
• This review summarizes current progress towards robust and
predictable engineering of plants.
• It then discusses applications from the lab and field, with a focus on
bioenergy, biomaterials, and medicine.
Plant synthetic biology could drive a
revolution in biofuels and medicine
Above: The DBTL cycle that guides our
synthetic biology experiments. Left: Examples
of synthetic biology strategies to engineer
lignin. (a) “Zip” lignin. Introduction of
ferulated monolignols into the lignin polymer
results in alkali sensitive ester bonds. (b)
QsuB lignin. By reducing the amount of
shikimate available to the lignin biosynthesis
pathway, a lignin enriched in H‐monomers is
formed, and the soluble produces
protocatechuate accumulates.

5. Jungle Express is a versatile repressor
system for tight transcriptional control
Outcomes
• Engineered EilR promoter demonstrated over 4 order of
magnitude in terms of dynamic range
• Expression was demonstrated in E. coli, P. putida and S.
meliloti; LacZ expression was tightly controlled in E. coli.
• Crystal structures with DNA and crystal violet demonstrated
mechanism of de-repression and induction.
Significance
• Initial studies of IL tolerance mechanisms led to a new type of
promoter that demonstrated tightly regulated, high-level gene
expression in a wide range of applications.
Ruegg et al. (2018) Nature Communications, DOI: 10.1038/s41467-018-05857-3
Background
• An MFS1 pump (EilA) identified in ‘Enterobacter lignolyticus’
confers IL tolerance to E. coli (Nature Communications, 2014,
5, 490).
• EilA is regulated by EilR, a IL-inducible repressor that also is
responsive to cationic dyes.
Approach
• Intergenic regions between eilR and eilA were identified and
tested for binding using cationic dyes.
• Engineered EilR was incorporated into broad host vectors for
heterologous expression

6. Outcomes and Impacts
• We show that loss of a single monosaccharide from chain A of RG-II is
sufficient to interrupt it’s function.
• The growth phenotype of the RNAi lines can be partially rescued by boron.
• RG-II is a minor polysaccharide in the primary cell wall, but it has a critical
role in cell wall functionality.
• Since this plant has altered RG-II structure, but is unaffected in other cell
wall polysaccharides it can be a useful tool for exploring why RG-II
dimerization is so important.
Sechet et al. (2018) Plant Journal, DOI: 10.1111/tpj.14088
Background
• Rhamnogalacturonan-II (RG-II) is a highly complex pectic
polysaccharide that is essential for cell wall function.
• Like most cell wall polysaccharides, it is synthesized in the Golgi
apparatus by a complex array of (as yet mostly unidentified)
glycosyltransferases
• However, the substrates are made in the cytosol and require
nucleotide sugar transporters to selective move them into the the
Golgi lumen.
Approach
• Here we identify the Arabidopsis thaliana Golgi GDP-L-galactose
transporter, which we name GGLT1.
• We show that it is necessary for RG-II L-galactosylation, and that this,
in turn, is essential for RG-II function and plant viability.
• Using an RNAi approach, we made plants with reduced GGLT1
expression, and characterized them biochemically and physiologically.
Suppressing GGLT1 affects growth by reducing
the L-galactose content and borate cross-linking
of rhamnogalacturonan II
Top: Structure of RG‐II. The sugar that is transported
by GGLT1 is indicated with an arrow. Bottom: Effect
of partially suppressing GGLT1 expression on
Arabidopsis growth (lines #1, #2) as compared to
control Empty Vector (EV) plants

7. Natural variation in efflux pumps
underlies ionic liquid tolerance in yeast
Outcomes
• The genomic screen identified a membrane efflux pump SGE1 and
conferred IL tolerance to S. cerevisiae
• The tolerance of SGE1 was correlated with two single nucleotide
polymorphisms (SNPs) present in the IL tolerant strain but absent in
the lab strain
• These SNPs affected the stability of the SGE1 protein on the
plasma membrane and localization within the membrane
Significance
• Our results highlight the general potential for discovering useful
biotechnological functions from untapped natural sequence variation
and provide functional insight into emergent SGE1 alleles with
reduced capacities to protect against IL toxicity.
Higgins et al. (2018) Genetics, DOI: 10.1534/genetics.118.301161
Background
• In previous work at JBEI, both heterologous and homologous efflux
pumps have been used confer ionic liquid (IL) tolerance to E. coli
• Early work at JBEI had demonstrated yeast was highly sensitive to
imidazolium-based ILs; tolerance mechanisms are needed.
Approach
• In collaboration with the GLBRC, a yeast culture collection was
interrogated for IL tolerance.
• This approach yielded an environmental strain of S. cerevisiae that
displayed high levels of tolerance to multiple Ils.
• A genomic library from this strain was generated and transformed
into an IL sensitive lab strain of S. cerevisiae

8. Temporal and geographic drivers of biomass
residues in California
Background
• Utilizing organic residues offers both climate benefits, particularly when
the material would otherwise be landfilled, and economic benefits as
many wastes have a negative cost (tipping fee) upon delivery
• Understanding the regional and temporal variations in availability by
biomass type can help inform research on mixed feedstocks that will be
most prevalent
Approach
• This paper aims conducts the most detailed geospatially- and temporally-
resolved organic residue inventory to-date for California, for both high-
and low-moisture wastes, through 2050
• Estimates were based on land use data, survey data, standard
agricultural practices (timing of harvest, processing, etc.), and sector-
level activity projections for estimating future changes in biomass
availability
Outcomes and Impacts
• Biomass residue production in California could grow 16% by 2050 to 71
million tonnes of dry-matter per year
• Co-processing of diverse high-moisture residue sources and storage of
seasonally available low-moisture residues is needed to ensure
adequate steady supply to bioenergy and composting facilities
• On a total mass basis, manure is a dominant feedstock, although
adjusted to bone-dry tonnage, woody biomass is far more significant
(dead trees, mill residues, forest slash, forest shrub, forest thinnings, and
urban lumber waste)
• Almond hulls are the only non-wood biomass that matches other
categories on the basis of dry mass
Breunig et al. (2018) Resour. Conserv. Recycl., DOI: 10.1016/j.resconrec.2018.08.022

9. Rapid characterization of the activities of
lignin-modifying enzymes using NIMS
Outcomes
• Enzyme assays of a laccase and a MnP on phenolic and nonphenolic β-
aryl ether substrate revealed different primary reaction pathways due to
the availability of the phenoxy radical intermediates.
• The quantitative analysis of product formation and kinetics is rapidly
achieved by NIMS.
Representative mass spectral signatures from reactions
of laccases and MnP with phenolic β‐aryl ether
substrate.
Deng et al. (2018) Biotechnol Biofuels, DOI: 10.1186/s13068-018-1261-2
Background
• Previously, we have successfully built an assay platform based on glycan
substrates containing a charged perfluorinated tag and NIMS to study
carbohydrate active enzymes. Here we extend this approach to develop a
reliable and rapid assay to study lignin-modifying enzymes.
Significance
• This is the first time that the NIMS technology was applied to study the
activities of lignin-modifying enzymes. Our assay provides a wealth of
information on bond cleavage events not available using conventional
colorimetric assays and can easily be carried our in microliter volumes.
Our use of amphiphilic guaiacylglycerol β-O-4 substrate enables the
formation of micelles, which helps avoid product repolymerization.
Time course of laccases with phenolic β‐aryl ether substrate
(I) over 3 h time period: 955 (green diamond); 1027 (red
square); 1045 (green up triangle); 1149 (purple cross); 1151
(pale blue asterisk); 2297 (orange solid circle); 2299 (gray plus
sign)
Approach
• Two β-aryl ether bond containing model lignin dimer substrates, designed
to be suitable for studying the activities of lignin-modifying enzymes (LMEs)
by nanostructure-initiator mass spectrometry (NIMS), were successful
synthesized. Small-angle neutron scattering experiments showed that
these substrates form micelles in solution. Two LMEs, a laccase
from Trametes versicolor, and a manganese peroxidase (MnP) from
Nematoloma frowardii, were tested for catalytic activity against the two
model substrates.