JBEI June 2019 highlight slides

1. Production of clovamide and its analogs in
Saccharomyces cerevisiae and Lactococcus lactis.
Background
• N-hydroxycinnamoyl-L-amino acids (HAA) such as clovamide (N-
caffeoyl-L-Dopa) are bioactive plant-derived phenolic compounds
with health-beneficial effects.
• Relying on chemical synthesis or direct extraction from plant
sources for the supply of these valuable molecules poses
challenges to environmental sustainability.
• Methods to synthesize HAA biologically using microorganisms are
needed.
Approach
• Arabidopsis 4-coumarate:CoA ligase (4CL5) and red clover
hydroxycinnamoyl-CoA:L-DOPA/tyrosine hydroxycinnamoyl
transferase (HDT1) were expressed in S. cerevisiae and L. lactis
for the production of clovamide and derivatives (Figure A).
• Feeding the engineered microorganisms with various
combinations of cinnamates and amino acids allowed synthesis of
several HAA (Figure B).
Outcomes and Impacts
• The production of HAA in GRAS microorganisms such as S.
cerevisiae and L. lactis provides new options for their delivery to
the human body as therapeutics.
• This study also highlights the substrate promiscuity of HDT1.
• HAA produced in this work will be used as standards for the
metabolomic characterization of plants engineered with HDT1
towards lignin valorization and reduced biomass recalcitrance.
Bouchez
et
al.
(2019)
Le$
Appl
Microbiol,
doi:
10.1111/lam.13190.
Structure of the HAA produced in this study. (A) Strategy
and reactions catalyzed by 4CL5 and HDT1 for the
synthesis of HAA from hydroxycinnamates (blue) and L-
amino acids (black). R1 and R2 = H, OH, or OMe. R =
amino acid functional group (side chain). SCoA: Coenzyme
A. (B) Conjugates of hydroxycinnamates with
phenylalanine, tyrosine, L-dopa, and tryptophan are
represented. Hydroxycinnamoyl moieties are p-coumarate
(R1 = R2 = H), caffeate (R1 = OH, R2 = H), ferulate (R1 =
OMe, R2 = H), or sinapate (R1 = R2 = OMe).
(A) (B)

2. Sphingolipid biosynthesis modulates plasmodesmal
ultrastructure and phloem unloading
Background
• Phloem loading/unloading is critical for the transport of the
products of photosynthesis from the source tissue (e.g. leaves)
to sink tissue (e.g. roots).
• Plasmodesmata are channels which cross the plant cell wall
and allow cell-to cell transport and communication. These are
lined with plasma membrane, and contain the desmotubule – a
narrow tube that connects the smooth endoplasmic reticulum of
the two cells. They come in two forms (Type I and Type II).
• The cytoplasm between the desmotubule and the plasma
membrane allows for symplastic transport. It is not clear how
this is regulated.
Approach
• In work led by researchers at the Universities of Cambridge
(UK) and Bordeaux (France), a mutant in sphingolipid
biosynthesis (PHLOEM UNLOADING MODULATOR, PLM) was
identified via a suppressor screen of a callose gain-of-function
mutant (cals3-d) with aberrant plasmodesmata function.
Outcomes and Impacts
• plm had decreased GIPCs and ceramides (sphingolipids) in the
plasma membrane
• PLM has a role in regulating plasmodesmata membrane
composition, and leads to a change in plasmodesmata type.
• In turn, this alters plasmodesmata permeability.
• This demonstrates that plasmodesmata are not regulated by
the cell wall (or callose) alone.
Yan
et
al.
(2019)
Nature
Plants,
5:604-­‐615,
doi:
10.1038/s41477-­‐019-­‐0429-­‐5
pSUC2::GFP
expressed
in
Arabidopsis
roots,
which
is
used
as
a
measure
of
phloem
unloading.
GIPC
composiRon
in
wild
type
(Col-­‐0)
and
plm-­‐1

3. A web-based tool for the prediction of rice
transcription factor function
Background
• Only a small number of genes encoding transcription factors
(TFs) have been characterized in Oryza sativa (rice).
• Ancient genome duplications indicate that ~50% of all genes
related to non-transposable elements in rice are functionally
redundant.
• Gene duplication and functional redundancy complicate the
analysis of TFs.
Approach
• Focused on rice transcription factors (TFs) and transcriptional
regulators (TRs).
• Developed a web-based tool called the Rice Transcription Factor
Phylogenomics Database (RTFDB) and demonstrated its
application for predicting TF function and to identify tissue-specific
and stress-related gene expression.
Outcomes and Impacts
• 273 genes preferentially expressed in specific tissues or organs,
455 genes showing a differential expression pattern in response
to 4 abiotic stresses, 179 genes responsive to infection of various
pathogens and 512 genes showing differential accumulation in
response to various hormone treatments were identified.
• Estimated a predominant role for 83.3% (65/78) of the TF or
transcriptional regulator genes that had been characterized via
loss-of-function studies.
• Method is applicable for functional studies of other bioenergy crop
species with annotated genomes.
Chandran
et
al.
(2019)
Database,
doi:
10.1093/database/baz061
Heatmaps
of
featured
gene
expression
groups
that
are
derived
from
meta-­‐expression
analysis
of
(A)
anatomical
samples,
(B)
abioRc
stress
samples,
(C)
bioRc
stress
samples
and
(D)
hormone-­‐treated
samples.
The
number
of
genes
per
group
is
shown
in
parentheses
aXer
the
group
name.
For
stress
and
hormone-­‐treated
featured
groups,
a
staRsRcal
cut-­‐off
of
>2
log2
fold-­‐change
at
P < 0.05
was
used.

4. Integration of renewable deep eutectic solvents with
engineered biomass to achieve a closed-loop biorefinery
Background
• Deep eutectic solvents (DESs) have gained increasing attention
due to their properties including universal solvating capabilities
and wide tunability.
• Ease of synthesis and broad availability from inexpensive
renewable source such as lignin could render DESs more versatile
solvents for biomass pretreatment.
• Certain lignin mutants accumulate unusual aromatics that can be
potentially converted into valuable DESs for a closed-loop
biorefinery.
Approach
• We developed a process that integrates the use of low-recalcitrant
engineered biomass with a pretreatment consisting of lignin-
derived DESs.
• Arabidopsis engineered plants deficient for cinnamyl alcohol
dehydrogenase (CAD) accumulate lignin enriched in
cinnamaldehydes instead of cinnamyl alcohols.
• Renewable DESs can be synthesized from such phenolic
aldehydes.
Outcomes and Impacts
• New DESs derived from vanillin and 4-hydroxybenzaldehyde were
developed (ChCl-VAN and ChCl-HBA).
• ChCl-VAN and ChCl-HBA are efficient for the pretreatment of
biomass prior enzymatic saccharification.
• In addition to exhibit reduced biomass recalcitrance, CAD mutants
represent an ideal source of vanillin and 4-hydroxybenzaldehyde
for DES synthesis.
Kim
et
al.
(2019)
Proc.
Natl.
Acad.
Sci.
U.S.A.,
doi:
10.1073/pnas.1904636116.
Complex formation in two new DESs ChCl-HBA and
ChCl-VAN synthesized for biomass pretreatment.
Glucan conversion from CAD mutant and wild-type (WT)
biomass pretreated with the new DESs, ChCl-VAN and ChCl-
HBA.
R = H, hydroxybenzaldehyde (HBA)
R = OCH3, vanillin (VAN)

5. A dominant negative approach to reduce
xylan in plants
Background
• After cellulose, the hemicellulose xylan make up the majority
of the carbohydrate content of plant biomass.
• Xylan is composed of pentoses, which are difficult to convert
into biofuels and bioproducts, and reduced xylan is a
desirable trait of bioenergy crops.
• Synthesis of the xylan backbone requires the proteins IRX9,
IRX14 and IRX10 and their partially redundant homologs.
Mutants in the corresponding genes have reduced xylan but
compromised growth properties.
Approach
• Conserved residues of IRX10 xylan synthase were mutated
in order to generate non-functional versions of IRX10.
• Plants were transformed with the mutated IRX10 proteins
under 35S promoter, with the hypothesis that a non-functional
enzyme would serve as a dominant negative.
Outcomes and Impacts
• Mutations in glycine-238 or glutamate-293 caused strong
reduction in growth.
• The plants had up to 55% reduction in cell wall xylose, and
strongly reduced xylan content, confirming the hypothesis
• The results confirm that IRX10 functions in a protein complex
• The approach can be used to generate healthy plants with
decreased xylan by restricting expression of the mutated
IRX10 to fiber cells
• A similar dominant negative approach can be employed as a
biosystems design tool in other cases where a protein
complex is required for a biosynthetic reaction
Brandon
et
al.
(2019)
Plant
Biotechnol.
Journal,
doi:
10.1111/pbi.13198
Some
of
the
plants
expressing
mutated
IRX10
proteins
have
reduced
growth.
The
residue
G283
is
conserved
in
human
EXT1
protein
and
mutaRons
are
known
to
cause
disease.
It
may
be
a
catalyRc
residue
but
the
structure
of
IRX10
or
EXT1
is
not
known.
Xylan
synthase
acRvity
is
reduced
in
plants
with
mutated
IRX10.
AcRvity
was
determined
in
microsomes
isolated
from
stems
and
using
xylohexaose-­‐ANTS
as
substrate.
Products
were
analyzed
by
PACE.
Significant
differences
from
EVC
are
indicated
(p
<
0.01,
ANOVA
and
Dunnef’s
test).
EVC IRX10 H146D F277A C278A G283D E293Q
0
100
200
300
400
500
600
700
800
900
Rha Ara Gal Glc Xyl Man GalA GlcA
nmol/mgAIR
Col-0 P-value
IRX10 .321
H146D .012
C278A .627
G283D .002
E293Q .001
EVC
0
25
50
75
100
XylTactivity
(%ofEVC)
EVC IRX10 G283D E293Q
Cell
walls
(AIR)
from
mutated
plants
have
less
xylose
compared
to
empty
vector
control
(EVC).
Bars
show
average
±
SD
(n=3-­‐5).
The
p-­‐values
indicate
significance
of
difference
from
EVC
(t-­‐test).
**
**

6. Toolset of constitutive promoters for metabolic
engineering of Rhodosporidium toruloides
Integration at CAR2 locus
(A) Strain & Constructs
Selection of white colonies
EGFP
mRuby
CellCountCellCount
(B) Transformation (C) Growth (D) Analysis
R. toruloides
IFO0880
∆ku70
Promoter EGFP
mRuby2 EGFPPromoter
58 constructs
SD
1% glucose
SD
1% xylose
SD
1% xylose
1% glucose
Growth in 24-well plates
YPD
mRuby21.
2.
mRuby EGFPPROMOTER
CAR2
CAR2CAR2
Bidirec'onal
Background
• Rhodosporidium toruloides is a promising host for the
production of biofuels and bioproducts from lignocellulosic
biomass.
• there is a lack of characterized promoters to drive
expression of heterologous genes for strain engineering in
R. toruloides.
Approach
• Candidates selected based on RNAseq data.
• 58 dual reporter constructs synthesized at the JGI.
• Site-specific integration into Rhodosporidium toruloides.
• Promoter expression strength was determined by
measurement of EGFP and mRuby2 reporters by flow
cytometry.
Outcomes and Impacts
• Established A set of robust and constitutive promoters to
facilitate genetic engineering of R. toruloides is presented
here, ranging from a promoter previously used for this
purpose (P7, glyceraldehyde 3-phosphate dehydrogenase,
GAPDH) to stronger monodirectional (e.g., P15,
mitochondrial adenine nucleotide translocator, ANT) and
bidirectional (e.g., P9 and P9R, histones H3 and H4,
respectively) promoters.
• This set of characterized promoters significantly expands
the range of engineering tools available for this yeast and
can be applied in future metabolic engineering studies for
the production of biofuels and bioproducts.
Nora
et
al.
(2019)
Microbial
Cell
Factories,
doi:
10.1186/s12934-­‐019-­‐1167-­‐0

7. Early cyanobacteria and the innovation of
microbial sunscreens
Background
• Understanding when cyanobacteria
evolved the ability to produce small
molecules that function as sunscreens
provides and understanding of when basic
photoprotection mechanisms evolved.
• UV photoprotection is a common
adaptation across microbes and plants,
with different compounds that are able to
function as sunscreens.
Shih
et
al.
(2019)
mBIO,
doi:
10.1128/MBIO.01262-­‐19
Approach
• We provide a review and commentary piece on the
field of microbial evolution and secondary metabolism
of the cyanobacterial sunscreen, scytonemin.
Outcomes and Impacts
• Understanding when scytonemin biosynthesis
originated provides a constraint key insight on the
origins of oxygenic photosynthesis in cyanboacteria
and how early phototrophic organisms had to evolve to
both oxygen and light.

8. Xyloglucan endotransglucosylase-hydrolase 30
negatively affects salt tolerance in Arabidopsis
Background
• Plants have evolved various strategies to sense and
respond to saline environments that severely reduce
plant growth and limit agricultural productivity. Alteration
to the cell wall is one strategy that helps plants adapt to
salt stress. However, how cell wall components respond
to salt stress is not fully understood.
Approach
• Performed salt sensitivity assay of xth30 mutant plants.
• A series of analysis was performed, including H2O2 level
measurement, antioxidant enzyme activity measurement,
subcellular localization of XTH30, cellulose content,
structural analysis of XyG oligosaccharides and
microtubule observation.
Outcomes and Impacts
• XTH30, encoding a xyloglucan endotransglucosylase-
hydrolase, negatively affected salt tolerance.
• We revealed that XTH30 negatively affected salt
tolerance, probably through modulating XyG side chains
composition, affecting XLFG accumulation, cellulose
synthesis and cortical microtubule stability.
• The study showed that down regulation of XTH30 can be
a potential strategy to increase plant drought resistance.
Yan
et
al.
(2019)
Journal
of
Experimental
Botany,
doi:
10.1093/jxb/erz311
XTH30 negatively affects salt stress tolerance
A potential model for XTH30 regulation in salt stress responses

9. A screening method to identify efficient sgRNAs
in Arabidopsis, used in conjunction with cell-
specific lignin reduction
Background
• Single guide RNA (sgRNA) selection is important for the
efficiency of CRISPR/Cas9 mediated genome editing.
However, in plants, the rules governing selection are not
well established.
Approach
• Developing an assay to test sgRNA efficiency in vivo.
• In case study I, use the selected sgRNA from the assay to
knockout HCT, an essential gene involved in lignin
biosynthesis, in a tissue-specific manner
• In case study II, use the selected sgRNA from the assay
to generate mutant of GONST2, a nucleotide sugar
transporter involved in GIPC synthesis
Outcomes and Impacts
• A transient assay was developed, which is widely
applicable to evaluate sgRNA efficiency before applying
CRISPR genome editing in stable transgenic plants.
• Using highly efficient sgRNA, chimeric hct plants were
generated for the first time. The plants showed decreased
lignin content and increased saccharification rate, while
maintaining plant integrity.
• The study also indicated that the use of highly efficient
sgRNA may accelerate the process of expanding
germplasm for both molecular breeding and research.
Liang
et
al.
(2019)
Biotechnology
for
Biofuels,
12:
130,
doi:
10.1186/s13068-­‐019-­‐1467-­‐y
Development
of
a
transient
assay
to
test
sgRNA
efficiency
for
CRISPR
ediRng
20-­‐30%
reducRon
in
total
lignin
content
(A)
and
30-­‐50%
increase
in
saccharificaRon
rate
(B)
were
observed
in
chimeric
hct
plants
A B