JBEI Research Highlights - July 2018

1. Insulator nanostructure desorption ionization
mass spectrometry
Background
• Surface-assisted laser desorption ionization (SALDI) is an
approach for gas-phase ion generation for mass spectrometry
using laser excitation on typically conductive or
semiconductive nanostructures
• Here, we introduce insulator nanostructure desorption
ionization mass spectrometry (INDIMS), a nanostructured
polymer substrate for SALDI-MS analysis of small molecules
and peptides
Approach
• INDI-MS surfaces are produced through the self-assembly of
a perfluoroalkyl silsesquioxane nanostructures in a single
chemical vapor deposition silanization step
• Used this approach to analyze a 288 compound secondary
metabolite library from Enzo life sciences, as well as glycosyl
hydrolase probes to compare the performance of two
commercial cellulase and hemicellulase enzyme cocktails
Outcomes and Impacts
• Surfaces formed from the perfluorooctyltrichlorosilane monomer
assemble semielliptical features with a 10 nm height, diameters
between 10 and 50 nm
• Self-desalting occurs passively as a sample dries with hydrophobic
molecules of interest assembling on the INDI regions and salts
depositing on the silicon
• Detected all compounds in the Enzo library and could effectively
tracked GH activity
• INDI-MS substrates represent a new SALDI surface for the analysis
of small molecules that can be used by scientists in multiple fields,
including bioenergy R&D
Duncombe et al. (2018) Analytical Chemistry, DOI: 10.1021/acs.analchem.8b01989
Insulator nanostructure desorption ionization mass spectrometry
(INDI-MS) (A) INDI substrates are generated in 20 min using silicon
oxide surface to form (III) nanostructures with a siloxane backbone
(black) decorated with fluorocarbon side groups (yellow). (B) AFM
and (C) SEM demonstrate the 10 to 50 nm diameter semielliptical
nanostructures. (D) LDI-MS is performed directly on the INDI surface
to analyze adsorbed molecules. (E) INDI-MS mass spectra of a
sample containing 250 fmols of dextromethorphan, verapamil, and
palmitoyl carnitine, 2.5 pmol mastoparan, and background.

2. A mosaic monoploid reference sequence for
the highly complex genome of sugarcane
Background
• Sugarcane (Saccharum spp.) is a major crop for sugar and
bioenergy production
• Its polyploid, aneuploid, heterozygous, and interspecific
genome poses major challenges for producing a reference
sequence
Approach
• This international team exploited colinearity with sorghum to
produce a BAC-based monoploid genome sequence of
sugarcane
• A minimum tiling path of 4660 sugarcane BAC that best
covers the gene-rich part of the sorghum genome was
selected based on whole-genome profiling, sequenced, and
assembled in a 382-Mb single tiling path of a high quality
sequence
Outcomes and Impacts
• A total of 25,316 protein-coding gene models are predicted,
17% of which display no colinearity with their sorghum
orthologs
• The two species analyzed, S. officinarum and S. spontaneum,
differ by their transposable elements and by a few large
chromosomal rearrangements, explaining their distinct
genome size and distinct basic chromosome numbers while
also suggesting that polyploidization arose in both lineages
after their divergence
• This first-ever published analysis of the sugarcane genome
provides bioenergy researchers increased knowledge that can
aid in targeted genetic engineering strategies to enhance
desired biofuel traits
Garsmeur et al. (2018) Nature Communications, DOI: : 10.1038/s41467-018-05051-5
Distribution of the 11,732 sugarcane BACs aligned onto the sorghum
genome through WGP. Sugarcane BAC clones are represented by
orange bars. Sorghum gene and transposable element densities are
represented in green and gray, respectively.
Sequencing strategy targeting the sugarcane monoploid genome
based on the overall synteny and colinearity conservation within
sugarcane homologs and with sorghum. a WGP sequence tags were
produced from R570 sugarcane BACs. b WGP sequence tags were
aligned onto the sorghum sequence. c A minimum tiling path of a
BAC (MTP) corresponding to a monoploid sugarcane genome d
Overlapping BAC sequences were trimmed to construct the single
tiling path (STP) B The STP sequence contains BAC contigs that
belong to distinct homologous chromosomes.

3. Natural variation in the multidrug efflux pump SGE1
underlies ionic liquid tolerance in yeast
Background
• Certain ionic liquids (ILs) are deconstruction solvents that
show great promise as a biomass pretreatment, but residual
solvent can be toxic to microbes and inhibit biofuel production
• The natural genetic diversity of Saccharomyces cerevisiae
isolates may help identify genes that contribute to ionic liquid
(IL) tolerance
• elucidate defense mechanisms that cells employ to improve
performance of bioconversion microbes
Approach
• Examined over 100 S. cerevisiae yeast isolates from diverse
ecological niches for growth in [C2C1im]Cl, identifying strains
with exceptional tolerance to that IL
• Screened DNA libraries from top performing strain to identify
genes that contribute to IL resistance
Outcomes and Impacts
• Two genes were associated with increased IL tolerance:
SGE1 and ILT1, encoding a multidrug efflux pump and a
predicted membrane protein, respectively
• Two major sequence variants of the SGE1 gene were found to
occur among yeast lineages that confer tolerance or sensitivity
to IL; alterations in Sge1 protein stability and cell surface
localization may impact the amount of IL that cells can pump
out of the cell
• Natural variation among diverse microbial isolates is an
important resource that can contribute to identification of
biological mechanisms of interest for biofuels conversion
Higgins et al. (2018) Genetics, DOI: 10.1534/genetics.118.301161
Schematic depiction of the multidrug efflux pump
SGE1 in the presence of ionic liquids.
BY strains containing GFP fused
to the indicated genes were
cultured in YPD medium. GFP
fluorescence (A) from
representative cells was
visualized with 100X
magnification. Insets in the lower
right corners display a single
representative cells with
additional 50% higher
magnification. ILT1 was deleted
from BY strains containing
SGE1SLS or sge1PLL alleles (B).
Resulting strains were cultured in
YPD pH 5 medium containing
62.5 mM [C2C1im]Cl. Cell growth
is reported as average cell
densities ± SEM from 3
independent biological replicates.

4. Microbial community structure and functional
potential along a hypersaline gradient
Background
• Microbial communities isolated from extreme environments
are an important source of new microbes and enzymes with
desired traits, including tolerance to salts and ionic liquids
• Salinity is one of the strongest environmental drivers of
microbial evolution and community composition
Approach
• We aimed to determine the impact of salt concentrations (2.5,
7.5, and 33.2%) on the microbial community structure of
reclaimed saltern ponds near San Francisco
• Community compositions were determined by 16S rRNA
amplicon sequencing revealing both higher richness and
evenness in the pond sediments compared to the water
columns
Outcomes and Impacts
• Functional annotation of shotgun metagenomic DNA showed
different capabilities of the microbial communities at different
salinities for methanogenesis, amino acid metabolism, and
carbohydrate-active enzymes
• There was an overall shift with increasing salinity in the
functional potential for starch degradation, and a decrease in
degradation of cellulose and other oligosaccharides
• Many of the carbohydrate-active enzymes identified have
acidic isoelectric points that have potential biotechnological
applications, including deconstruction of biofuel feedstocks
under high ionic conditions
Kimbrel et al. (2018) Frontiers in Microbiology, DOI: 10.3389/fmicb.2018.01492
OTU abundance, diversity, and phyla of sampled ponds. Pond sample
types are denoted with an “L” or “S” for water or sediment. (A) Richness
and Evenness metrics for triplicates at each sample site (Pond A23
sediments has only a single replicate) (B) Bray-Curtis Dissimilarity
dendrogram of all sample sites, color coded by sampled pond with
assignment into seven groups at a cut-off of 0.6. (C) Relative abundance
of the 8 most abundant phyla grouped by sample site.

5. Increased drought tolerance in plants engineered
for low lignin and low xylan content
Background
• We previously developed several strategies to engineer plants in order
to produce cost-efficient biofuels from plant biomass
• Engineered Arabidopsis plants with low xylan and lignin content
showed normal growth and improved saccharification efficiency under
standard growth conditions
• However, it remains to be determined whether these engineered plants
perform well under drought stress, which is the primary source of
abiotic stress in the field
Approach
• Upon exposing engineered Arabidopsis plants to severe drought, we
observed better survival rates in those with a low degree of xylan
acetylation, low lignin, and low xylan content as compared to those in
wild-type plants
• The drought tolerant plants exhibited low water loss from leaves, and
drought-responsive genes (RD29A, RD29B, DREB2A) were generally
up-regulated under drought stress, which did not occur in the well-
watered state
• When compared with the wild type, plants with low lignin showed a
stronger response to abscisic acid (ABA) in assays for seed
germination and stomatal closure
Outcomes and Impacts
• This study shows that plants engineered to accumulate less lignin or
xylan are more tolerant to drought and activate drought responses
faster than control plants
• This finding demonstrates that modification of secondary cell walls
does not necessarily render the plants less robust in the environment,
and it shows that substantial changes in biomass composition can be
achieved without compromising plant resilience.
Yan et al. (2018) Biotechnol Biofuels, DOI: 10.1186/s13068-018-1196
Plants with low xylan (XE#55 and X4) or low lignin (QsuB, W4, X4)
show increased tolerance to drought stress. Plants with secondary
cell wall galactan (W2) are not different from the Col-0 control.
Abscisic acid (ABA) is a hormone
induced by drought. The engineered
plants have normal, low ABA
content in well-watered conditions.
All the plants induce ABA in
response to drought stress, but the
low lignin plants (QsuB, W4, X4)
have much higher ABA levels after
induction. The low xylan plants
XE#55 do not show this change,
indicating an ABA-independent
improved drought tolerance in those
plants.