JBEI May 2020 Highlights

1. The effect of continuous tubular reactor
technologies on the pretreatment of lignocellulosic
biomass at pilot-scale for bioethanol production
Background
• Effective pretreatment of biomass is a critical unit operation in the
production of biofuels and bioproducts.
• Most pretreatment technologies used today operate in batch
mode.
• Continuous operation is preferred, since productivity is a key
aspect in industrial processes at commercial scale..
Approach
• A pilot-scale continuous tubular reactor (PCTR) was employed for
the isothermal pretreatment of agave bagasse (AG), corn stover
(CS), sugarcane bagasse (SC), and wheat straw (WS) with three
residence times.
Outcomes and Impacts
• Deformation in cellulose and hemicellulose linkages and xylan
removal of up to 60% were achieved after pretreatment.
• The shortest residence time tested (20 min) resulted in the highest
glucan to glucose conversion in the low solid loading (4% w/v)
enzymatic saccharification step for AG (83.3%), WS (82.8%), CS
(76.1%) and SC (51.8%).
• Final ethanol concentrations after SSF from PCTR-pretreated
biomass were in the range of 38 to 42 g/L (11.0–11.3 kg of ethanol
per 100 kg of untreated biomass).
• Approach can be readily adapted to include organisms that can
produce more advanced biofuels and bioproducts from pretreated
biomass.
Perez-Pimienta et al. (2020) RSC Advances, doi: 10.1039/d0ra04031b
Plot of sugar consumption and ethanol production levels
from SSF of four pretreated samples (AG = agave bagasse,
CS = corn stover, SC = sugarcane bagasse, and WS = wheat
straw) loaded at 20% solids.

2. Synthesis and function of complex sphingolipid
glycosylation
Background
• Glycosylinositol phosphorylceramides (GIPCs) constitute up
to 40% of the plasma-membrane lipids in plants.
• The complex glycan headgroups of GIPCs vary between
plant species and tissues and has importance for
development, abiotic stress tolerance, and interactions with
pathogenic and symbiotic microorganisms.
Approach
• The paper reviews recent studies of the biosynthesis and
function of GIPC glycan headgroups.
Outcomes and Impacts
• The headgroup is synthesized in the Golgi lumen by
glycosyltransferases similar to those involved in cell wall
biosynthesis.
• Biosynthesis depends on the same nucleotide sugar
transporters in the Golgi that are required for cell wall
biosynthesis and protein glycosylation.
• All plant GIPCs have glucuronic acid as the first sugar. It is
added by an enzyme related to xylan
glucuronosyltransferases and is essential for plant growth.
• Specific GIPC headgroups are essential for symbiosis with
mycorrhizal fungi nitrogen fixing bacteria in root nodules.
• Modulation of GIPC headgroups may be a way to improve
stress tolerance and biotic interactions in bioenergy crops.
Mortimer J.C., Scheller H.V. (2020) Trends in Plant Science, doi: 10.1016/j.tplants.2020.03.007
Structure of GIPCs. All plant GIPCs have glucuronic acid as the
first sugar. It is added by an enzyme related to xylan
glucuronosyltransferases. Subsequent sugars and chain length
vary between species and tissues.
(B)
IPC
IPC
IPC
IPC
GlcAHex GlcNAcMan
IPC
IPC
GlcN
(A)
Celluose synthase
complex (CSC)
Ion
transporter Receptors
Cytosol
Apoplast
GIPC
Sterol
Glucosylceramide Phospholipid
GPI-anchored protein
Schematic view of the plant plasma membrane. The figure highlights
the asymmetry between the inner and outer leaflet, and how the GIPC
glycan headgroups can be tightly packed on the surface. Examples of
classes of membrane proteins are shown, whose functions have been
proposed to be affected by GIPC headgroup structure.

3. Chemoinformatic-guided engineering of
polyketide synthases
Background
• Polyketide synthase (PKS) engineering is an attractive method to
generate new molecules such as commodity, fine and specialty
chemicals.
• A significant challenge is re-engineering a partially reductive PKS
module to produce a saturated β-carbon through a reductive loop (RL)
exchange.
Approach
• We sought to establish that chemoinformatics, a field traditionally used
in drug discovery, offers a viable strategy for RL exchanges.
• We introduced a set of donor RLs of diverse genetic origin and
chemical substrates into the first extension module of the lipomycin
PKS (LipPKS1).
• We then introduced RLs of divergent chemosimilarity into LipPKS2 and
determined triketide lactone production.
Outcomes and Impacts
• Product titers of these engineered unimodular PKSs correlated with
chemical structure similarity between the substrate of the donor RLs
and recipient LipPKS1, reaching a titer of 165 mg/L of short-chain fatty
acids produced by the host Streptomyces albus J1074.
• Our results determined statistical significance in the correlation between
production and the chemosimilarity of the substrate between the donor
and recipient modules.
• These design principles may accelerate the combinatorial approach
currently used for de novo biosynthesis and help provide a framework
to more rapidly produce valuable biofuels, bioproducts and
biochemicals.
Zargar et al. (2020) J Am Chem Soc, doi: 10.1021/jacs.0c02549
A chemoinformatic approach to reductive loop exchanges.
(A) ClusterCad search revealed the closest substrates to
LipPKS1 containing full RLs. (B) Production levels of
junction B. RL exchanges are ordered from highest KR
substrate similarity with LipPKS1 (MonA2, LaidS2, and
NanA2) to progressively less similarity (IdmO, AurB, and
SpnB) in biological triplicate (error bars denote standard
deviation).

4. Design of orthogonal regulatory systems for
modulating gene expression in plants
Background
• Current plant-based green technologies are limited by a dearth of tools
and components available for synthetic plant biology.
• This study developed a novel method for the assembly and
characterization of synthetic elements for modulating gene expression
strength. Additionally, this system allows for tissue-specific and
environmentally responsive gene expression in plants.
Approach
• We developed an orthogonal system that harnesses transcription
factors (TFs) and their associated binding motifs from yeast along with
minimal promoters from plants. By leveraging the inherent variability of
each element we constructed over 500 unique TF/promoter pairs, each
with a distinct output profile, and tested them in varying plant systems.
Outcomes and Impacts
• Our orthogonal system allows for the integration of synthetic gene
circuits that operate in parallel to the endogenous systems of the host.
• The sequence variability of our promoter elements will help limit the
phenomenon of gene-silencing often observed in transgenic lines.
• We developed a method that can theoretically be applied to any
eukaryotic host by introducing host-specific minimal promoters.
• This system can now be tested in our Sorghum protoplast pipeline with
the introduction of Sorghum spp. specific minimal promoters.
• Our parts can be applied for more targeted and elegant engineering of
any bioenergy crop in future studies and will assist in the development
and refinement of plant-based green technologies.
Belcher et al. (2020) Nature Chemical Biology; https://doi.org/10.1038/s41589-020-0547-4
Synthetic chimeric transcriptional regulators were
developed with elements from both yeast and plants.
These regulators can be leveraged for the modulation of
gene expression in both a tissue-specific and
environmentally responsive manner.

5. Structural changes in bacterial and fungal soil
microbiome components during biosolarization as
related to volatile fatty acid accumulation
Background
• Biosolarization, where moist soil is covered with clear tarp to
induce elevated soil temperatures through passive solar heating,
is an integrated pest management strategy that combines soil
solarization with organic amendment application.
• There is a gap in understanding the changes in the bacterial and
fungal microbiome that occur during biosolarization.
Approach
• Soil samples taken after biosolarization using tomato pomace and
green waste compost (GWC) amendments were analyzed via
sequencing and bioinformatic analysis of 16S rRNA gene and ITS2
amplicons to elucidate changes to the soil microbiome, including
both fungal and bacterial communities.
Outcomes and Impacts
• Structural and network analyses were used to quantify differences
in microbiota between treatments and these results were compared
against measured levels of volatile fatty acids in the soils.
• The results showed that biosolarization had a stronger impact on
the bacterial community relative abundance profile than on the
fungal community at the phyla and order levels.
• Network analysis established microbial clusters and correlation
between the clusters and soil volatile fatty acid (VFA) production
suggested the bacterial Clostridium, Weissella and Acetobacter
genera tolerate, and perhaps drive, VFA accumulation.
Achmon et al. Applied Soil Ecology 153 (2020): 103602., doi.org/10.1016/j.apsoil.2020.103602
0
10
20
30
40
50
60
70
80
90
100
Soil100%str
TomatoStr
Tomato+GWCstr
Soil100%RT
TomatoRT
Tomato+GWCRT
Soil100%0-7.5cm
Soil100%7.5-15cm
Soil100%15-22.5cm
Tomato0-7.5cm
Tomato7.5-15cm
Tomato15-22.5cm
Tomato+GWC0-7.5cm
Tomato+GWC7.5-15cm
Tomato+GWC15-22.5cm
Relativeabandenc(%)
p__Firmicutes p__Proteobacteria p__Acidobacteria
p__Bacteroidetes p__Cyanobacteria p__Actinobacteria
p__Gemmatimonadetes p__Chloroflexi p__Verrucomicrobia
Others
Bacterial community as a function of soil solarization
Trichurus
Fungi
Hydropisphaera
RhodocyclalesOR
Steroidobacter
SphingobacterialesOR
Xanthomonadaceae
Talaromyces
Methylobacterium
Balneimonas
Microbial co-occurrence networks