JBEI Research Highlights - March 2019

1. Multiplexed CRISPR-Cas9-Based Genome
Editing of Rhodosporidium toruloides
Background
• The yeast R. toruloides is a promising host for converting
lignocellulosic material into valuable fuels and chemicals. It
naturally accumulates high levels of lipids and carotenoids, and
is known to grow robustly, even in stressful environments
• However, efforts to enhance this non-model organism’s
production capabilities have been impeded by a lack of tools for
genome engineering
• This study establishes a framework for utilizing CRISPR-Cas9 to
enact targeted gene disruption in R. toruloides
Approach
• We designed and optimized cassettes for expressing Cas9 and
sgRNAs targeting URA3 and CAR2 individually or in tandem
• The capacity of these cassettes to cause gene disruption in a
single transformation was assayed using CFUs and sequencing
Outcomes and Impacts
• Cas9-based disruption of URA3 was successfully enacted by
integrating an expression cassette into R. toruloides’ genome
• Initial low editing efficiencies of 0.0017% ±0.0011% were
increased 364-fold to 0.62% ±0.50% by optimizing sgRNA
promoters, target sequences, and removing the ribozyme
• An array of four sgRNAs separated by tRNAs was used to enact
multiplexed gene-editing of URA3 and CAR2 in a single
transformation, as well as demonstrating successful excision of
intergenic DNA between nearby cut sites
• These results significantly expand the capacity to rapidly edit the
genome of R. toruloides, and is an essential step towards
development of industrially-relevant variants of the yeast
Otoupal et al. (2019) mSphere, doi: 10.1128/mSphere.00099-19
0
50
100
150
Transformantswith
GeneDisruption
…TGCG TCTCGGTTGACGTG…
…TGCGGTCTCGGTTGACGTG…
…TGC- -----------GTG…
…TGC- TCTCGGTTGACGTG…
Wild Type
Sequencing Confirms
Editing at Cas9 Cut Site
P=0.02
-CRISPR
Cassette
+CRISPR
Cassette
+CRISPR 1
+CRISPR 2
+CRISPR 3
sgRNA target PAM
Cut Site
Expression Cassette for CRISPR-Cas9 Editing in R. toruloides:
KO-CAR2 (in KO-URA3)
1.1% ± 0.8% 3.2% ± 0.5%
KO-URA3 KO-CAR2
30% ± 8%
Editing Efficiencies:
Single
KO
Double
KO
sgRNA Cassette for Multiplexed CRISPR-Cas9 Editing:
Sequencing 8 Double KOs
Reveals Cas9-Editing at:
1 2 3 4 5 6 7 8
URA3a ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
URA3b ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
CAR2c ✓ ✓ ✓ ✓ ✓ ✓ ✓ ✓
CAR2d ✓ ✓ ✓ ✓ ✓

2. Glycome and proteome components of Golgi membranes
are common between two angiosperms with distinct cell
wall structures
Background
• The plant ER-Golgi apparatus is the site of synthesis and assembly
of all non-cellulosic polysaccharides, proteoglycans, and proteins
destined for the cell wall
• As grass species make cell walls distinct from other angiosperms, it
has been assumed that the differences in cell wall composition
stem from differences in biosynthetic capacities of their respective
Golgi
Approach
• Golgi membranes were prepared from coleoptiles of maize and
leaves of Arabidopsis
• Glycans in the Golgi preparations were analyzed by
immunosorbent-based screens and carbohydrate linkage analysis
• Golgi membranes were further purified by free-flow electrophoresis
and analyzed by proteomics
Outcomes and Impacts
• Arabinogalactan-proteins and arabinans represent substantial
portions of the Golgi-resident polysaccharides not typically found in
high abundance in cell walls of either species
• Proteomics yielded over 200 proteins known to function in the
biosynthesis and metabolism of cell wall polysaccharides common
to all angiosperms, and not just those specific to wall type
• We propose that the distinctive compositions of grass primary cell
walls result from differential gating or metabolism of secreted
polysaccharides post-Golgi by an as yet unknown mechanism, and
not by differential expression of genes encoding specific synthase
complexes
Okekeogbu et al. (2019) Plant Cell, doi: 10.1105/tpc.18.00755.
Glycans were analyzed by glycome profiling using specific
antibodies. The profiles show many unexpected differences
between the Golgi vesicles and the cell walls. For example,
xyloglucan is very abundant in maize Golgi but has very low
abundance in the wall. The opposite is true for Arabidopsis.
Likewise, pectic RGI is very abundant in maize Golgi but has
low abundance in the wall.

3. Techno‐economic and greenhouse gas analyses of
lignin valorization to eugenol and phenolic products in
integrated ethanol biorefineries
Background
• Valorizing lignin is key to improving the economics of lignocellulosic
biofuel production
• One option for upgrading lignin is to employ catalytic hydrogenolysis
assisted by isopropanol (IPA) to convert the lignin recovered after ionic
liquid (IL) pretreatment, saccharification, and fermentation
• However, a detailed analysis is required to better understand whether
this process offers an economic and environmental advantage relative
to using lignin for onsite heat and power
Approach
• Develop a process model for an IL-based biorefinery with lignin
upgrading to eugenol, linked with a feedstock availability and logistics
model for Mexico
• We evaluated the feasibility of this lignin-valorizing biorefinery using
locally-available combinations of corn stover, sorghum stubble,
and Jatropha fruit shells
Outcomes and Impacts
• The minimum ethanol selling price (MESP) for this case was
$2.02/gal. The resulting cradle‐to‐gate GHG footprint of bioethanol
was 21 g CO2‐eq/MJ, a 78% reduction with respect to gasoline when
system expansion is used for allocation
• Research should target a reduction in IL input by 30% and IPA input by
40%, together with more energy‐efficient separation processes
• Sensitivity analysis showed that, for biomass prices higher than $45/t,
biorefinery capacities must exceed 5000 t/d biomass input
Martinez-Hernandez et al. (2019) Biofuel. Bioprod. Biorefin., doi: 10.1002/bbb.1989)

4. The multi-scale challenges of biomass fast
pyrolysis and bio-oil upgrading
Background
• Biomass fast pyrolysis is potentially one of the cheapest
routes toward renewable liquid fuels
• Crude pyrolysis oil is not immediately usable in the
current energy infrastructure, due to undesirable
properties such as low energy content and
corrosiveness as a result of its high oxygenate content
Approach
• While various types of pyrolysis reactors and upgrading
technologies are under development, knowledge
transfer and closing the gap between theory and
application requires model development
• The conceptual process design including the choice of
feedstock, pretreatment technologies, design and
operation of pyrolysis and upgrading reactors, and the
role of phenomenological modeling in technology
transfer was elaborated in this review
Outcomes and Impacts
• Underpinning economic and environmental impacts of
biofuel production requires expanding the system
boundaries to include the overall process and supply
chain
• Design of multi-functional catalysts which can telescope
the reaction networks toward high-value fuels and
chemicals will enhance the energy conversion efficiency
Sharifzadeh et al. (2019) Progress in Energy and Combustion Science, https://doi.org/10.1016/j.pecs.2018.10.006
The multi-scale nature of research
into biomass pyrolysis

5. Linking ‘omics’ to function unlocks the biotech
potential of non-model fungi
Background
• Non-model fungi are increasingly used in biotechnology, spanning
medical, industrial, and agricultural applications, but few have been
sequenced compared to yeasts and prokaryotes
• This study focuses on the barriers that must be overcome to to
translate in silico discoveries enabled by new fungal genomes to
applications in biotechnology and biomass hydrolysis
Approach
• This review highlights recent examples where bioinformatics was
used to identify genes and pathways of interest from fungi that
were exploited to produce biotechnologically important secondary
metabolites, transporters, and lignocellulose-active enzymes
Outcomes and Impacts
• Implementation of long-read sequencing technologies has
accelerated the the number of high-quality fungal genomes and
transcriptomes available for study
• It is critical to combine sequencing information and systems biology
to guide both genetic engineering and heterologous expression
strategies to harness the biotech potential of non-model fungi
• Direct experimental validation and exploitation of fungal systems
lag behind the rate at which novel enzymes and biosynthetic
pathways are identified in silico
• Additional genome scale models and genome editing techniques
must be developed to enable computationally-driven metabolic
engineering of non-model fungi
Wilken et al. (2019) Current Opinion in Systems Biology, doi: 10.1016/j.coisb.2019.02.001
Vast amount of sequencing data have been recently collected
for non-model fungi. Systems and bioinformatic tools are used
to identify enzymes and pathways of biotech relevance. In
silico analyses and predictions must be coupled with
experimental techniques to validate the gene annotation.

6. Harnessing Nature’s Anaerobes for Biotechnology
and Bioprocessing
Background
• Industrial application of microbial biotechnology is typically
biased toward model microbes with straightforward culturing
requirements, genetic engineering tools, and production
scaling.
• Anaerobes are microorganisms that thrive in the absence of
oxygen, and they have widely different culture requirements
and a unique metabolism compared to model microbes.
• This study considers the opportunities and challenges
associated with implementing anaerobes in scalable
bioprocessing strategies.
Approach
• We review current and future uses for anaerobes in
biotechnology and bioprocesing in the post-genomics era,
with a focus on lignocellulose degradation.
Outcomes and Impacts
• Early-branching anaerobic fungi (Neocallimastigomycota)
have recently been sequenced to reveal a wealth of
carbohydrate active enzymes, carbohydrate transporters, and
secondary metabolite biosynthesis pathways.
• Enzymatic cocktails of secreted enzymes from anaerobic
fungi have demonstrated activity on par with many industrial
formulations.
• Resolving current genetic intractability, scale-up, and
cultivation challenges will unlock the potential of
lignocellulolytic anaerobes to accelerate bio-based
production.
Podolsky et al. (2019) Annual Review of Chemical and Biomolecular
Engineering., doi: 10.1146/annurev-chembioeng-060718-030340
Anaerobic gut fungi (AGF) biotechnological applications exploit
unique features: (a) robust cellulose-, hemicellulose-, and possibly
lignin-degrading enzymes; (b) biohydrolysate transporters for
heterologous expression; (c) novel secondary metabolite clusters;
and (d) lignocellulose fermentation by consortia that lead to the
production of sustainable chemicals and fuels.