JBEI Research Highlights September 2016

1. GDP-L-fucose Transport in Plants:
the Missing Piece
Outcomes
• Based on our phylogenetic analysis, it would appear that
the plant and mammalian GDP-Fuc transporters arose
independently as they share little identity.
• By bringing fungal sequences into the analysis, we were
able to hypothesize that fungal GDP-Fuc transporters
(none are currently identified) arose with their animal
counterparts but separately from plants.
Ebert et al. (2016) “GDP-L-fucose transport in plants: the missing piece” Channels. 1-3. doi: 10.1080/19336950.2016.12227
Background
• Fucose is mainly found as a component of glycan
structures such as N- and O-linked glycans, glycolipids
and in cell wall polysaccharides such as fucoidan,
xyloglucan, rhamnogalacturonan II. GDP-fucose is
biosynthesized in the cytosol, however these glycan
structures are made within the endomembrane system.
Thus a GDP-Fuc transporter is required in animals, plants
and fungi.
Significance
• This study has enabled us to identify likely fungal GDP-
Fuc homologs and outlined evidence to suggest the co-
evolution of this transporter in plant lineages and
animals/fungi lineages.
1) Phylogenetic analysis of GDP-Fuc transporters
The characterized Arabidopsis GDP-Fuc transporter forms a distinct clade with
rice candidates (Plant). This is distinct to the characterized human GDP-Fuc
transporter (Animal). Interestingly, BLAST searches enabled us to identify fungal
GDP-Fuc transporter candidates in species with evidence for fucose containing
glycans. The analysis indicates that the plant and animal/fungi GDP-Fuc lineages
arose independently.
Approach
• We recently identified the plant GDP-Fuc transporter and
were curious as to why it had not been previously
identified by sequence homology to its mammalian
counterpart.

2. 13C Metabolic Flux Analysis for systematic
overproduction of fatty acids in yeast
Outcomes
• Expression of ATP citrate lyase (ACL) to increase acetyl-CoA does
not increase fatty acid production significantly (Fig. 1).
• Flux profiles from 2S 13C MFA show that the extra acetyl-CoA
produced by ACL goes into the malate synthase (MALS) rather than
fatty acid production (ACCOACr, Fig. 2).
• Downregulating MALS and knocking out glycerol production (GPD1),
increased fatty acid production by ~70% (Fig. 1).
1) Fatty acid production for the various strains studied in this manuscript
The addition of ACL was expected to increase acetyl-CoA availability but did not
increase final production. However, the downregulation of MLS did increase
production, as suggested by flux analysis. The highest production was obtained by
knocking out glycerol production, improving production in the overall engineering
process by 70%.
Ghosh et al. (2016) “13C Metabolic Flux Analysis for Systematic Metabolic Engineering of S. cerevisiae for Overproduction of
Fatty Acids” Front. Bioeng. Biotechnol. 4:76. doi: 10.3389/fbioe.2016.00076
Background
• Tools for systematic and efficient redirection of microbial
metabolism into the abundant production of desired
bioproducts are a need in metabolic engineering.
• Previous engineering attempts with S. cerevisaie led to
a production of free fatty acids at a titer of 400 mg/L.
Significance
• The use of 2S 13C MFA provides a way to ascertain the fate of carbon
from feed to product and produces new insights to improved biofuel
yield in a systematic fashion.
2) Cytosolic acetyl-CoA balances obtained from 2S-13C
MFA
Flux of acetyl-CoA (accoa) producing and consuming reactions is
shown as a sankey diagram. Reactions on the left of the diagram
produce acetyl-coA and reactions on the right consume it, with arrow
sizes indicating total flux. Once the ACL is added (WRY2 ACL), total
acetyl-coA flux increases, but this extra flux flows into MALS, which
is consistent with the lack of fatty acid production increase (Fig. 1).
Approach
• We combined 13C labeling data with comprehensive
genome-scale models (2S 13C MFA) to shed light onto
microbial metabolism and improve metabolic
engineering efforts.

3. Proteomic Characterization of Golgi Membranes
Enriched from Arabidopsis Suspension Cell
Cultures
Outcomes
• A detailed protocol for isolating Golgi membranes with
various ‘tricks’ developed at JBEI has been outlined
• The method yields about 26 % Golgi proteins and in
combination with informatics approaches the method
could be used for proteomic surveys in plants
Hansen et al. (2016) "Proteomic Characterization of Golgi Membranes Enriched from Arabidopsis
Suspension Cell Cultures". Methods Mol Biol, 1496, 91-109. doi, 10.1007/978-1-4939-6463-5_8
Background
• The stacked membrane structures of the Golgi apparatus
along with its interactions with the cytoskeleton and ER
have historically made the isolation and purification of this
organelle difficult. Density centrifugation has typically
been used to enrich Golgi membranes from plant
microsomal preparations, and aside from minor
adaptations, the approach is still widely employed.
Significance
• Significant effort has been expended at JBEI to develop
these methods and this published protocol attempts to
distil the ‘tricks’ for the plant science community.
1) Schematic outlining the enrichment of Golgi membranes by
density centrifugation
Approach
• We outline a detailed protocol that we have extensively
refined for the enrichment of Golgi membranes from
Arabidopsis suspension cell cultures. The approach is
suitable for samples that can be used for downstream
processes such as comparative proteomic studies.
2) Validation of Golgi proteins can be undertaken by localization
with fluorescently tagged proteins
This validation process was also outlined in the published protocol. The
Golgi marker aManI-mCherry is shown as a Golgi reference signal. The
UXT3-YFP proteins clearly localizes to the Golgi (see merge).

4. Cell Wall Composition and Candidate
Biosynthesis Gene Expression During Rice
Development
Outcomes
• Through analysis of the correlations within and among the cell
wall and gene expression datasets, we establish testable
hypotheses for the potential interactions among cell wall
polymers, and the functions of putative cell wall synthesis genes
• This work successfully moves beyond the observation that
particular GTs and ATs are grass-diverged and highly expressed
to providing testable hypotheses for specific cell wall synthesis
activities, pinpointing nine uncharacterized genes with putative
functions in rice cell wall synthesis.
Lin et al. (2016) "Cell Wall Composition and Candidate Biosynthesis Gene Expression
During Rice Development". Plant Cell Physiol. doi, 10.1093/pcp/pcw125
Background
• Rice straw comprises almost one quarter of all agricultural waste
globally
• The development of rice is continuous and there is not a distinct
juvenile to adult boundary
• Significant knowledge gaps exist about the roels of cell wall
biosynthesis genes in rice correlated to changes in composition.
Significance
• These results may lead to the discovery of novel grass cell wall
synthesis enzymes, and may improve biofuel production and
other agricultural uses of cereals and grasses.
Approach
• We have developed a cell wall composition atlas that provides a
baseline for evaluating the variation of cell wall composition
across developmental time points and organs in rice, and for
comparisons to other plant species.
Abundance of cell wall components and properties measured by
chemical assays. Color intensity indicates the z-score of abundance
for each component. Red indicates relative enrichment and blue,
relative scarcity. Slashed squares are missing values due to
insufficient sample mass. The mean, un-normalized mass for each
component is shown above the heatmap. The color ribbons on the left
of heatmap indicate organs.

5. Towards Economically Sustainable
Lignocellulosic Biorefineries
Outcomes
• Given different production pathways for MA and the market price volatility of
MA, multiple scenarios were constructed (Fig. 2)
• Overall, higher MA price scenarios (‘red’) are more favorable.
• Minimizing the energy costs associated with the aerobic fermentation can
make the process more attractive from an economical standpoint
Konda et al. (2016) “Towards economically sustainable lignocellulosic biorefineries”, In Valorization of Lignocellulosic
Biomass in a Biorefinery: From Logistics to Environmental and Performance Impact. Nova publishers.
Background
• Economic viability is essential for the commercialization of cellulosic
biorefineries for the production of advanced biofuels
• Due to the low market price of fuels and market price volatility, cellulosic
biofuel production faces challenging economics
• Co-production of value added chemicals, alongside of biofuel, can
potentially enable cost-competitive production of biofuels
• Among various co-production strategies (Fig. 1), feedstock engineering to
incorporate easily extractible chemicals/intermediates is a potential option
Significance
• This study has quantified the potential impact of the co-production of
chemicals from a technoeconomic standpoint. Results help researchers to
design and develop processes that meet commercially relevant targets for
key performance metrics (e.g., yield)
Approach
• To investigate the impact of co-products on the production cost of biofuel,
we have considered the case of engineered sweet sorghum for the co-
production of Muconic Acid (MA), a precursor for Adipic Acid.
• To facilitate economic analysis, detailed biorefinery model was developed
in a commercial software platform SuperPro Designer
2) Estimated min. ethanol selling price (MESP). The ‘blue’
and ‘red’ bars represent different market scenarios with
regards to MA selling price (i.e., $1000/MT and
$1500/MT, respectively). S1 (no co-product), S2 (sugars-
to-MA pathway), S3 (PCA-to-MA pathway), S4 (MA from
both sugars and PCA)
1) A simplified superstructure representation of
integrated cellulosic biorefineries for the co-production
of chemicals together with biofuel.

6. Development of Next Generation
Synthetic Biology Tools for S. venezuelae
Outcomes
• We generated 45 expression vectors that can simultaneously
integrate at three different attB sites and characterized 15 promoters
that possess relative strengths that vary over 3 orders of magnitude.
• The tools and information generated were used to develop a
chromosomally integrated bisabolene production pathway that
produced 5.4 mg/L.
Phelan et al. (2016) “Development of Next Generation Synthetic Biology Tools for use in
Streptomyces venezuelae” ACS Synth Biol., doi: 10.1021/acssynbio.6b00202
Background
• Streptomyces have a rich history as producers of important natural
products, and this genus of bacteria has recently garnered attention
for its potential applications in the broader context of synthetic
biology.
• The dearth of genetic tools available to control and monitor protein
production precludes rapid and predictable metabolic engineering
Significance
• These results help establish a toolbox that will streamline the
introduction or modification of biosynthetic pathways in S. venezuelae
by allowing more predictable protein production and simplifying the
introduction of multiple genes or operons relevant to biofuel production.
Approach
• We developed a suite of standardized, orthogonal integration vectors
and an improved method to monitor protein production in this host
• These tools were then applied to characterize heterologous
promoters and various attB chromosomal integration sites, coupled
with a demonstration of producing the biofuel precursor bisabolene
using a chromosomally integrated expression system.
Small scale screening of bisabolene producing strains
Screen of seven differing fluorescent proteins integrated at
the VWB attB site under the control of strong, constitutive
glyceraldehyde-3-phosphate dehydrogenase promoter
(Pgapdhp(EL)) from Eggerthella lenta

7. Approaches to Understanding Protein
Hypersecretion in Fungi
Outcomes
• Case studies: (1) amylase production in Aspergillus niger, (2)
cellulase secretion in Trichoderma reesei, and (3) secretion in
Neurospara crassa.
• Traditional methods of isolation and improvement of protein
production hosts, such as mutagenesis and screening, and reverse
genetics to demonstrate the action of specific genes, will continue to
have value for the future generation of improved strains.
• The secretion pathway itself is key, as UPR and the cell’s secretion
machinery play important roles in the level of protein output from the
cell.
• The impact of the advances of the genomics era is that these
standard approaches have become more powerful and the rate of
progress has increased dramatically
Reilly et al. (2016) “Approaches to understanding protein hypersecretion in fungi” Fungal
Biology Reviews, doi: 10.1016/j.fbr.2016.06.002
Background
• Fungi play an incredibly important role in industrial biotechnology, and
applications that utilize fungal enzymes have grown to a much larger
number of industrial applications, from food and beverage production
to the digestion of plant biomass in bioenergy and bioproducts
applications
• In this review paper, we discuss three “case studies” in the area of
fungal protein hyperproduction
Significance
• The combination of traditional and advanced approaches will enable
the development of new and better fungal protein production hosts for
missions in bioenergy and beyond.
Aspergillus niger
Neurospara crassa

8. Dynamic Changes of Substrate Reactivity
and Enzyme Adsorption on Partially
Hydrolyzed Cellulose
Outcomes
• Results suggest that cellobiohydrolase CBH1 (Cel7A) and endoglucanases
EG2 (Cel5A) adsorption capacities decreased as cellulose was
progressively hydrolyzed, likely due to the “depletion” of binding sites; while
the degree of synergism between CBH1 and EG2 varied depending on the
enzyme loading and the substrates.
• Moreover, these results suggest that more oligosaccharides (DP>3
cellodextrins) than soluble sugars could be produced during cellulose
hydrolysis and influenced by both cellulose substrate and enzyme loadings
and combinations.
Shi et al. (2016) "Dynamic changes of substrate reactivity and enzyme adsorption on partially
hydrolyzed cellulose" Biotechnol Bioeng, doi, 10.1002/bit.26180
Background
• The enzymatic hydrolysis of cellulose is a thermodynamically challenging
catalytic process that is influenced by both substrate-related and enzyme-
related factors. The limited understanding of hydrolysis reaction
mechanisms and factors controlling hydrolysis effectiveness impede many
promising applications in the real world.
Approach
• A proteolysis approach was applied to recover and clean the partially
converted cellulose at the different stages of enzymatic hydrolysis to
monitor the hydrolysis rate as a function of substrate reactivity/accessibility
and investigate surface characteristics of the partially converted cellulose.
Significance
• This study provides a better understanding of the relationship between
dynamic change of substrate features and the functionality of various
cellulases during enzymatic hydrolysis.

9. A Robust Gene Stacking Method utilizing
Yeast Assembly for Plant Synthetic Biology
Outcomes
• We have demonstrated how our method can be applied to a number of plant metabolic engineering efforts.
• We have built a publicly available library of over 100 plant DNA parts to aid other plant synthetic biology efforts.
Background
• Plant synthetic biology is a
young field that has the
potential to address many
sustainability, bioenergy, and
agricultural issues
• There has been a dearth of
DNA parts libraries, versatile
transformation vectors, and
efficient assembly strategies
for plant transformations.
Approach
• We have developed a method
that utilizes yeast homologous
recombination to assemble
DNA into plant transformation
vectors.
Significance
• Our approach provides an alternative method to efficiently assembling DNA into plant transformation vectors.
• Improved DNA assembly will enable more complex plant metabolic engineering efforts to manipulate plant feedstocks to be
more sustainable and have better bioenergy traits.
2) Yeast homologous recombination vectors enable the
stacking of multiple genes together. Gene stacking can
enable yield from metabolic engineering efforts or the
introduction of entirely heterologous pathways.
1) Hierarchical assembly of plant DNA parts for plant
synthetic biology. A library of DNA parts (Level 0) can be
efficiently assemble together into gene cassettes. These
cassettes can be stitched together via yeast homologous
recombination.
Shih et al. (2016) “A robust gene stacking method uti.izing yeast assembly for plant synthetic biology”
Nature Communications. doi: 10.1038/ncomms13215

10. Glycosylation of Inositol Phosphorylceramide
Sphingolipids is Required for Normal Growth
and Reproduction in Arabidopsis
Outcomes
• Assignment of a direct role for GIPC glycan headgroups in the
impaired processes in iput1 mutants is complicated by the vast
compensatory changes in the sphingolipidome.
• However, our results reveal that the glycosylation steps of
GIPC biosynthesis are important regulated components of
sphingolipid metabolism.
1) Rescued iput1 mutants are dwarfed and have oxidative stress.
Plants from two different transformant lines are shown in comparison to wild
type. The leaves of the rescued plants show lesions indicating oxidative
stress (as confirmed with DAB staining, not shown). The plants also have high
levels of salicylic acid.
Tartaglio et al. (2016) "Glycosylation of inositol phosphorylceramide sphingolipids is required for normal growth and
reproduction in Arabidopsis". Plant J. doi, 10.1111/tpj.13382 https://www.ncbi.nlm.nih.gov/pubmed/27643972.
Background
• Sphingolipids are a major component of plant plasma membranes and
endomembranes, and mediate a diverse range of biological processes,
including cellulose biosynthesis and plant-microbe interactions.
• Study of the highly glycosylated glycosyl inositol phosphorylceramide
(GIPC) sphingolipids, has been slow due to challenges associated with
GIPCs' extractability, and their functions in the plant remain poorly
characterized.
Significance
• This study corroborates previously suggested roles for GIPC
glycans in plant growth and defense, suggests important roles
for them in reproduction, and demonstrates that the entire
sphingolipidome is sensitive to their status.
3) Sphigolipidomics of
rescued iput1 mutants.
The mutants show
accumulation of IPC, the
substrate for IPUT1
glucuronosyltransferase. The
fully glycosylated GIPC is
strongly reduced in the mutants.
Other changes in ceramides are
pleiotropic effects of the primary
defect in GIPC biosynthesis.
Approach
• We recently discovered an Arabidopsis GIPC
glucuronosyltransferase, INOSITOL PHOSPHORYL-CERAMIDE
GLUCURONOSYLTRANSFERASE 1 (IPUT1), which is the first
enzyme in the GIPC glycosylation pathway.
• Using a pollen-specific rescue construct, we have here isolated
homozygous iput1 mutants.
2) Pollen from iput1 plants has very low transmission efficiency.
Pollen tube growth appears normal, but the mutant pollen tubes
cannot efficiently be guided to the micropyle. The few efficient
pollinations do not lead to viable embryos.

11. XA-21-specific Induction of Stress-related
Genes Following Xanthomonas Infection of
Detached Rice Leaves
Outcomes
• We identified eight genes that are up-regulated in both in elf18
treated EFR:XA21:GFP rice leaves and Xoo infected XA21 rice
leaves.
• Our results show that even though the EFR:XA21:GFP-mediated
response does not confer robust resistance to Xoo, similar genes
are up-regulated during both EFR:XA21:GFP- and Myc:XA21-
mediated response
Thomas et al. (2016) “XA-21-specific Induction of Stress-related Genes Following
Xanthomonas Infection of Detached Rice Leaves” PeerJ, 4, e2446 doi: 10.7717/peerj.2446
Background
• Plant immunity is mediated, in part, by cell surface immune
receptors that recognize molecules produced by microbes..
• Studies of the XA21-mediated immune response have been limited
by the lack of rapid assays and well-characterized genetic markers.
Significance
• The discovery of RaxX and the establishment of the detached leaf
infection assay described here provide useful tools for studying
XA21-mediated immunity that will improve bioenergy crop
productivity.
Approach
• We developed a detached leaf infection assay to quickly and
reliably measure activation of the XA21-mediated immune response
using genetic markers.
• The detached leaf infection assay allows a more uniform
distribution, compared to the scissor inoculation method
• We used RNA sequencing of elf18 treated EFR:XA21:GFP plants
to identify candidate genes that could serve as markers for XA21
activation.
The stress-related marker gene PR10b is up-regulated in
Xanthomonas infected XA21 rice. PR10b expression in detached
Kitaake and Myc:XA21 rice leaves with 10 mM MgCl2 mock
treatment or infected with PXO99A or PXO99A1hrpA1 (1hrpA1)
at an O.D.600 of 0.1. Letters represent statistically significant
differences between mean expression values (p < 0:05)
determined by using a TukeyKramer HSD test. This experiment
was repeated three times with similar results.

12. Comprehensive in vitro Analysis of Acyltransferase
Domain Exchanges in Modular Polyketide Synthases
and its Application for Short-chain Ketone Production
Significance
• These results greatly enhance the mechanistic understanding of
modular PKS systems.
• As demonstrated in short-chain ketone production, our results
prove the benefits of using engineered PKSs as a synthetic
biology tool for chemical production.
Yuzawa et al. (2016) “Comprehensive in vitro analysis of acyltransferase domain exchanges in modular polyketide
synthases and its application for short-chain ketone production” ACS Synth Biol doi: 10.1021/acssynbio.6b00176.
Background
• Modular polyketide synthases (PKSs) are polymerases
that utilize acyl-CoA as substrates.
• Each polyketide elongation reaction is catalyzed by a
set of protein domains called a module.
• Individual acyltransferase (AT) domains in the PKS
modules determine the specific acyl-CoA incorporated
into each polyketide condensation reaction.
• Although successful exchange of individual AT
domains lead to the biosynthesis of a large variety of
novel compounds, hybrid PKSs often show
significantly decreased activities.
Approach
• Using model PKS systems, we have systematically
analyzed the segments of AT domains and associated
linkers in AT exchanges in vitro.
Outcomes
• We have identified the boundaries within a module that
can be used to exchange AT domains while maintaining
protein stability and enzyme activity.
• The optimized domain boundary is highly conserved,
which facilitates AT domain replacement in most PKS
modules.
New synthetic biology tool!
etc…
Acceptor PKS
AT-swapped mutant library
KAL PAL1 PAL2
Reduction
loop ACP
Reduction
loop ACP
KAL PAL1 PAL2
KS AT
KS AT
Donor PKS
KAL PAL1 PAL2
Reduction
loop ACPKS AT

13. Outcomes
• A comprehensive phylogenetic tree of Rack1 proteins
has been constructed.
• The current research status on the Rack1 protein has
been summarized.
• Future research areas regarding Rack1 are indicted.
Zhang et al. (2016) “Roles of Rack1 proteins in fungal pathogenesis” BioMed Res Int. http://dx.doi.org/10.1155/2016/4130376
Background
• Fungal pathogens cause severe losses of feedstock
biomass.
• The conserved Rack1 protein from plant pathogenic
fungi is a potential target for developing effective
fungicides.
Significance
• This study reveals the conserved and dynamic role of
Rack1 proteins in different fungal species.
• This study facilitates the development of fungicides
targeting the fungal Rack1 protein to improve biofuel
feedstock production.
Approach
• Phylogenetic analysis
• Compare functions of Rakc1 protein from different
organisms
Roles of Rack1 Proteins in Fungal
Pathogenesis

14. From Lignin Association to Nano-/Micro-particle
Preparation: Extracting Higher Value of Lignin
Outcomes
• Literature studies on preparation, functionalization
and application of lignin nanoparticles are still limited.
• Fundamental understandings of lignin solution structures, their relation to nanoparticle preparation and properties,
nanoparticles stability and dispersion will certainly lead to advancement in synthesizing lignin-based functional materials.
• From an industrial point of view, it is desirable to minimize utilization of hazardous solvents or to replace them with green
solvents such as water, supercritical CO2 and room temperature ionic liquids (RTILs), etc.
Zhao et al. (2016) “From Lignin Association to Nano-/Micro-particle Preparation:
Extracting Higher Value of Lignin” Green Chemistry, doi: 10.1039/c6gc01813k
Background
• Value-added applications of lignin are essential
for economic viability for future bio-refineries.
• Conversion of lignin stream to value-added
components still remains a challenge which casts a
shadow over the fate of future biorefineries
Significance
• This work provides a perspective that would prompt the development of new processes for manufacturing lignin
nanoparticles with designed properties.
Approach
• Lignin nanoparticles emerge as novel building
blocks with unique and tunable properties, which
is expected to play a vital role in value-added
applications.
• Methods for preparation of lignin nanoparticles
are essentially adopted from those used in
manufacturing polymer nanoparticles.