JBEI Research Highlights November 2016

1. Exploiting members of the BAHD acyltransferase
family to synthesize multiple hydroxycinnamate
and benzoate conjugates in yeast
Outcomes
• The synthesis of valuable products such as rosmarinic acid, chlorogenic acid, glycerol, polyamine, monolignol, malate and fatty
alcohol hydroxycinnamates was achieved for the first time in yeast.
Aymerick Eudes et al.(2016). “Exploiting members of the BAHD acyltransferase family to synthesize multiple
hydroxycinnamate and benzoate conjugates in yeast”. Microb. Cell Fact. doi: 10.1186/s12934-016-0593-5
Background
• The production of renewable
chemicals using engineered
microbes is an alternative route
to energy-intensive chemical
syntheses that consume
petroleum-based precursors.
• Identification of appropriate
enzymes is a prerequisite for
the design and implementation
of metabolic pathways in
microbes and bioenergy crops.
Approach
• BAHD acyltransferases that use
hydroxycinnamoyl-CoA lignin
precursors and/or benzoyl-CoA
as donors were characterized
for the synthesis of valuable
metabolites in S. cerevisiae.
Significance
• The BAHD acyltransferases characterized in the work can be expressed in bioenergy crops for the
production of co-products and to add value to plant biomass.
1) Mechanism of acylation catalyzed by BAHD acyltransferases.
BAHD acyltransferases using 4-hydroxycinnamoyl-CoAs (R = 4-
hydroxystyrene) and benzoyl-CoAs (R = benzene) as donors were
used for this study. R’ = BAHD acceptor.
Hydroxycinnamoyl-CoA:glycerol
transferase
Hydroxycinnamoyl-CoA:spermidine
transferase
Hydroxycinnamoyl-CoA / benzoyl-CoA:alcohol transferase
Hydroxycinnamoyl-CoA:fatty alcohol
transferase
Hydroxycinnamoyl-CoA:monolignol
transferase
Rosmarinic acid synthase Hydroxycinnamoyl-CoA:Quinate
transferase
+
BAHD
CoA-activated
acyl donors
Acyl acceptors
Ester bond
Amide bond
Cinnamates
BAHDs
Acceptor
Cinnamoyl-CoAs + Acceptor
Supplied Produced
4CL
Cinnamoyl
conjugates
2) Strategy for the synthesis of cinnamate and benzoate
conjugates. Yeast strains expressing various BAHDs are fed with
cinnamate or benzoate donors in combination with adequate
acceptor molecules. The produced conjugates are detected
from the culture medium. 4CL, 4-coumarate:CoA ligase.
3) Name of the BAHDs synthesized for expression in yeast and examples
of hydroxycinnamate and benzoate conjugates produced in this work.
Hydroxycinnamoyl-CoA:malate
transferase

2. Loss of inositol phosphorylceramide sphingolipid
mannosylation induces plant immune responses
and reduces cellulose content in Arabidopsis
Outcomes
• GMT1 mannosylates a class of sphingolipids (GIPCs) which
are abundant in the plant plasma membrane (Figure 1).
• Disruption of this glycosylation in gmt1 causes a specific
decrease in the plasma-membrane synthesized cellulose.
Golgi-synthesized polysaccharides, including pectin, were
unaffected.
• gmt1 plants also have a constitutively active defense response.
Fang et al. (2017). "Loss of Inositol Phosphorylceramide Sphingolipid Mannosylation Induces Plant Immune Responses
and Reduces Cellulose content in Arabidopsis". Plant Cell. doi, 10.1105/tpc.16.00186
Background
• Plant Golgi-localized glycosyltransferases (GTs) are involved in
non-cellulosic polysaccharide biosynthesis, as well as lipid and
protein glycosylation.
• Previous work had suggested that GMT1, from CAZy family
GT64, was Golgi localized and involved in pectin biosynthesis,
but its function was unknown.
Significance
• These results describes the function of the first plant GT64,
and only the second GT involved in GIPC biosynthesis. It
uncovers a new potential role for GIPCs in regulating cellulose
content. The mechanism by which this could occur is unknown
and will be explored further to applications in bioenergy.
Approach
• Here, we fully characterized mutants in GMT1, as well as
heterologously expressed the protein in yeast and tobacco.
Above: GIPCs, a class of sphingolipid, are an abundant component
of the outer leaflet of the plant plasma membrane. Loss of a
single mannose on the GIPC headgroup has a major impact on
plant development. Below: 13C CP-MAS solid state NMR (left) and
the subtraction spectra (WT-gmt1; right) reveals that cellulose
signals are specifically lost in the gmt1 stem (labelled C1-C6,
where C1 is the anomeric carbon of glucose in cellulose).

3. Enrichment of the Plant Cytosolic Fraction
Outcomes
• A detailed protocol for enriching the cytosolic fraction from
cell cultures developed at JBEI.
• The method highlights the necessity for protoplasting to
ensure high levels of purity
Lao et al. (2017). “Enrichment of the Plant Cytosolic Fraction”. In N. L. Taylor & A. H. Millar (Eds.), Isolation of Plant
Organelles and Structures: Methods and Protocols (pp. 213-232). New York, NY: Springer New York.
Background
• The cytosol is at the core of cellular metabolism and
contains many important metabolic pathways, such as
glycolysis, gluconeogenesis and the pentose phosphate
pathway. Despite the importance of this matrix, few
attempts have sought to specifically enrich this
compartment from plants.
Significance
• The approach yields samples suitable to define the
proteome of the cytosol, a much understudied fraction
within the plant cell. The approach is high suitable for
comparative proteomic approaches.
1) Schematic outlining the enrichment of the cytosolic fraction
from rice cells using a protoplast rupture method.
Approach
• We outline a detailed protocol for the enrichment of the
cytosolic fraction from rice cell cultures. The approach
highlights the requirement for protoplasting cells to
ensure reduced contamination from organelles. This
approach results in a highly enriched fraction and
expands work conducted at JBEI focussing on
Arabidopsis cytosolic fractions.
2) Immunoblotting with organelle marker antibodies demonstration
the removal of contaminating organelles by centrifugation.

4. Structure and mechanism of NOV1, a
resveratrol-cleaving dioxygenase
Outcomes
• The structure of NOV1 was determined in complex with a
representative substrate (resveratrol), a representative product
(vanillin), and without ligand bound.
• These structures, along with EPR analysis, allow us to propose a
mechanism in which a ferric-superoxide reacts with substrate
activated by deprotonation of a phenol group at position 4 of the
substrate.
McAndrew et al. (2016) ”Structure and mechanism of NOV1, a resveratrol-cleaving
dioxygenase”. Proc Natl Acad Sci U S A. DOI: 10.1073/pnas.1608917113
Background
• NOV1 is a stilbene cleavage oxygenase (SCO). SCOs cleave the
central double bond of stilbenes, forming two phenolic aldehydes.
Stilbenes, such as resveratrol, are produced by plants and they
are also formed from lignin during kraft pulping.
• SCOs are related to carotenoid cleavage oxygenases (CCOs),
which cleave β-carotene or apocarotenoids. Carotenoids play
important roles in photosynthesis and light perception in the eye.
Significance
• The conversion of lignin, which accounts for ∼30% of plant cell
wall carbon, into chemicals or fuels could have a significant
impact on the economics of processing lignocellulosic biomass.
Ultimately, enzymes like NOV1 could assist in the biological
valorization of dimeric fragments derived from lignin and so
contribute to the sustainable operation of a biorefinery for the
production of biofuels and other bioproducts.
Approach
• We used X-ray crystallography to determine the first structure of
an SCO. EPR analysis was also used to help determine the
enzyme mechanism.
• Collaboration with Brian Fox @ GLBRC
Overall Structure of NOV1. (A) NOV1 is a seven-bladed β-propeller. An
iron coordinated by four histidines is located down the central axis. Dioxygen
binds to the iron, and resveratrol binds with its central double bond proximal
to the oxygen. (B) A surface slice representation, oriented perpendicular to
the β-propeller axis shows the shape of the active site cavity.
A B
Mechanism of NOV1. (A) Formation of ternary complex of Fe(III)-
superoxo NOV1, O2 and alkene substrate, and contributions of
deprotonation of 4′-OH by Y101 and K135 in activation of the substrate. (B)
Intermediate formed prior to formation of a C–O bond. (C) Intermediate
formed after formation of the first C–O bond. (D) Cleavage of the O–O
bond and formation of the second C–O bond. (E) Cleavage of the C–C
bond and restoration of the Fe(II) enzyme. (F) Reaction products.
A B C
F E D

5. Sequential enzymatic saccharification and
fermentation of ionic liquid and organosolv
pretreated agave bagasse for ethanol production
Outcomes
• IL pretreatment reduced lignin by 28% and xylan by 50%
while OV solubilizes 86% xylan and 45% of lignin.
• High glucan (>90 %) and xylan (>83 %) conversion was
obtained with both pretreated samples that lasted 18h.
• During the fermentation stage (48 h), 12.1 and 12.7 kg of
ethanol were produced per 100 kg of untreated AGB for
IL and OV, respectively.
A) Mass balance per 100 kg of
untreated AGB during sequential
enzymatic saccharification and
fermentation (SESF) for IL and
OV pretreatments.
When the Agave productivity is
considered, 3067-7082 L/ha year is
obtained, favorable when compared to
corn (2050 L/ha year) or sugarcane
(4900 L/ha year).
Pérez-Pimienta et al. (2016). “Sequential enzymatic saccharification and fermentation of ionic liquid and organosolv
pretreated agave bagasse for ethanol production”. Bioresource Technology (2017) 2225, pp. 191-198.
Background
• Agave bagasse (AGB) has gained recognition
as a drought-tolerant biofuel feedstock with
high productivity in semiarid regions.
• Studies on AGB for ethanol production have
used only the separate hydrolysis and
fermentation (SHF) strategy.
Significance
• These comparative analyses showed the advantages of SESF using IL
and OV in a biorefinery configuration where a better understanding of AGB
recalcitrance is key for future applications.
B) XRD spectra of untreated and
pretreated AGB.
1) The crystallinity index (CrI) of
untreated AGB was 39.0% whereas IL
decreased to 22.6% and OV increased
to 44.6%. 2) Spectrum of untreated
AGB shows distinctive peaks at 2θ =
14.8 °, 24.2 °, 30.0 ° and 38.0 °
from calcium oxalate.
Approach
• Comparative analysis of ionic liquid (IL) and
organosolv (OV) pretreatment technologies in
AGB was performed using a sequential enzymatic
saccharification and fermentation (SESF) strategy
with cellulolytic enzymes and metabolic
engineered Escherichia coli strain MS04.
A
B
C) Scanning electron (top) and confocal fluorescence
(bottom) of untreated and pretreated AGB.
Intact plant cell wall structure of AGB is shown while
pretreatment with [C2C1Im][OAc] in AGB resulted in a structural
modification into the organization of macrofibrils with a rough
and swollen surface.
C

6. Nitrogen amendment of green waste impacts
microbial community, enzyme secretion and
potential for lignocellulose decomposition
Outcomes
• Carbon/nitrogen ratios of 25–30 are considered ideal for thermophilic biological
deconstruction.
• Carbon/nitrogen ratios of the green waste after nitrogen amendment ranged between 25 and
32, however, even within this range, significant shifts in the microbial community, enzyme
production and potential for lignocellulose hydrolysis were observed.
Yu et al. (2016). "Nitrogen amendment of green waste impacts microbial community, enzyme secretion and potential
for lignocellulose decomposition". Process Biochemistry. doi, 10.1016/j.procbio.2016.11.002
Background
• Each year, 167 million metric tons of municipal
solid wastes are sent to landfills in the U.S and
about 44.9% of these wastes are organic
• Microorganisms involved in biomass
deconstruction are an important resource for
organic waste recycling and enzymes for
lignocellulose bioconversion.
Significance
• The results suggest nitrogen levels present in green waste bioconversion processes could
be better tuned to achieve more efficient deconstruction of recalcitrant polysaccharides and
discovery of enzymes for bioconversion.
Approach
• The goals of this study were to elucidate the
biological mecha-nisms of enhanced green
waste decomposition with nitrogen amendment
through examination of xylanase and
endoglucanase secretion, microbial community
restructuring and potential for green waste
deconstruction.
Mean relative abundance (%) of phyla in bacterial
communities and classes in fungal communities

7. Ionic liquid-tolerant microorganisms and microbial
communities for lignocellulose conversion to
bioproducts
Outcomes
• IL tolerant microorganisms from environmental
samples by generating less complex lignocellulolytic
microbial communities and facilitating the discovery
of potential enzymes and microorganisms for
biomass deconstruction.
Yu at al. (2016). "Ionic liquid-tolerant microorganisms and microbial communities for lignocellulose conversion to
bioproducts". Appl Microbiol Biotechnol, 100(24), 10237-10249. doi, 10.1007/s00253-016-7955-0
Background
• While effective at pretreatment, certain ionic liquids
inhibit not only the enzyme activities but also the
growth and productivity of microorganisms used in
downstream hydrolysis and fermentation processes
• The discovery of IL tolerance in environmental
microbial communities and individual microbes has
lead to the proposal of molecular mechanisms of
resistance.
Significance
• A combined approach that includes ILs designed for
reduced toxicity and robust IL-tolerant
microorganisms will ultimately result in more efficient
and economical IL-pretreatment based bioconversion
processes.
Approach
• This is a review paper on recent progress on
discovering IL-tolerant microorganisms, identifying
metabolic pathways and mechanisms of tolerance,
and engineering microorganisms for IL tolerance
Examples of ionic liquid tolerance organisms

8. Bio-based production of fuels and industrial
chemicals by repurposing modular polyketide
synthases: opportunities and challenges
Yuzawa, et al. (2016). “Bio-based production of fuels and industrial chemicals by repurposing antibiotic-producing type I
modular polyketide synthases: challenges and opportunities” J Antibiot (Tokyo) doi: 10.1038/ja.2016.136.
Background
• Complex polyketides comprise a large number of
natural products that have broad application in
medicine and agriculture.
• They are produced in bacteria and fungi from enzyme
complexes named type I modular polyketide synthases
(PKSs) that contain discrete enzymatic domains
organized into modules.
• The modular nature of PKSs has enabled a multitude
of efforts to “repurpose” the PKS genes to produce
fuels and industrial chemicals in a predicted manner.
Significance
• These results greatly enhance the mechanistic
understanding of PKS and pave the way for
exploitation of PKS as a platform to produce fuels
and industrial chemicals.
(C)
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LipPks1+TE
ACP KS AT KR ACP TE
R
O
S
(A)
Load Module 1
AT
AT-swapped LipPks1+TE
ACP KS KR ACP TE
R
O
S
Load Module 1
AT
S
O
R
HO
R
O
OH
OH
AT KS ATACP KR ACP TEER
O
HO
O
OH
S
O
O
HO
O
HO
O
S
DH
KR-swapped BorA2+TE
Module 1
BorA1
Load
AT
KR-inactivated LipPks1+TE
ACP KS AT KR ACP TE
R
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(B)
Load Module 1
AT
KR-inactivated, AT-swapped LipPks1+TE
ACP KS KR ACP TE
R
O
S
Load Module 1
AT
S
O
R
O
R
O
R
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R =
Starter acyl-CoA= Propionyl n-Butyryl Isobutyryl 2-Methylbutyryl Isovaleryl
R =
Starter acyl-CoA= Propionyl n-Butyryl Isobutyryl 2-Methylbutyryl Isovaleryl
R
O
OH
OH
S
O
R
HO
S
O
R
O
Adipic acid
Opportunities
• We have repurposed PKSs to produce 3-hydroxy
acids, ketones, and diacids that could have
applications as fuels or industrial chemicals.
Challenges
• The challenge is to make these compounds at
reasonable cost. We need to find the ‘best’ host for
production of type I modular PKS-based fuels and
industrial chemicals.

9. Enrichment of Golgi Membranes from Triticum
aestivum (Wheat) Seedlings
Outcomes
• A detailed protocol for enriching Golgi membranes from
plant material (seedlings) using approaches developed at
JBEI
• The method results in an enrichment rather than an
organelle purification
Zeng et al. (2017). “Enrichment of Golgi Membranes from Triticum aestivum (Wheat) Seedlings”. In N. L. Taylor & A. H. Millar
(Eds.), Isolation of Plant Organelles and Structures: Methods and Protocols (pp. 131-150). New York, NY: Springer New York.
Background
• The complex collection of membrane structures
comprising the Golgi apparatus has historically been
difficult to purify from plant material. Density
centrifugation has typically been used to enrich Golgi
membranes from microsomal preparations, and aside
from minor adaptations, the approach is still widely
employed by the field.
Significance
• The approach yields an adequate enrichment of Golgi
membrane to enable a variety of biochemical analyses.
Knowledge of the published Golgi proteome can be
leveraged to enable proteomic surveys of resultant
samples.
1) Schematic outlining the enrichment of Golgi membranes from
wheat by density centrifugation
Approach
• We outline a detailed protocol that we have extensively
refined for the enrichment of Golgi membranes from
wheat seedlings. While the approach results in a
relatively impure preparation, it is suitable for downstream
processes such as comparative proteomic or biochemical
assays.
2) Validation of the Golgi enrichment process using
immunoblotting with organelle marker antibodies and enzyme
assays (UDPase).

10. Significance
• We provide the largest, most comprehensive and well-characterized toolkit for S. cerevisiae strain
engineering to date, allowing for quick and easy metabolic pathway construction.
• Using our tools, we improve taxadiene titers in S. cerevisiae to the highest levels reported to date.
Providing better Cas9-based tools for genetic
engineering in Saccharomyces cerevisiae
Background
• Strain development remains slow and
laborious because of difficulties anticipating the
combined effect of different expression parts
and conditions in S. cerevisiae.
Approach
• We developed a Cas9-based toolkit to quickly
institute genetic changes in S. cerevisiae for
optimizing heterologous gene expression.
C
B
720CAN1
911
1114
1309
1414
YOLCdelta1
416
C
Constructed many high-efficiency Cas9-
sgRNA plasmids for easy integration
A diversity context library on taxadiene
synthase, using several of our parts, resulted
in a 25-fold improvement in taxadiene titer
Outcomes
• Constructed high-efficiency, Cas9-sgRNA
plasmids targeting 23 characterized integration
loci.
• Characterized 37 standardized promoters in
different grow phases and media
• Validated functions for 10 protein tags conferring
specific protein localization, turnover or
solubility.
• Provide a software tool to allow for easy utilization
of parts.
A thorough single-cell fluorescent
evaluation of promoters across time and
media discovered a range of activation
profiles including stationary phase and
exponential phase induction.
Reider Apel et al. (2016). “A Cas9-based toolkit to program gene expression in
Saccharomyces cerevisiae”. Nucleic Acid Research, doi: 10.1093/nar/gkw1023.