This study optimized terpene production in the yeast Rhodosporidium toruloides by engineering enzymes in the mevalonate pathway. Multi-omic analysis of bisabolene-producing strains identified specific enzymes for optimization, including HMG-CoA reductase and mevalonate kinases. Optimization of these enzymes for two terpenes, 1,8-cineole and bisabolene, improved titers to 1.4 g/L and 2.6 g/L respectively. The study demonstrates improving terpene production from lignocellulosic biomass in R. toruloides, an efficient host for converting renewable carbon sources.

1. Use of ensiled biomass sorghum increases ionic liquid
pretreatment efficiency and reduces biofuel production
cost and carbon footprint
Background
• Efficient biomass deconstruction is essential for economical
conversion of lignocellulose into biofuels
• This study examines the impact of using the common practice of
ensiling grassy biomass as a form of biomass conditioning prior to
pretreatment and deconstruction
Approach
• Ensiled sorghum was used as a feedstock for a one-pot ionic liquid
pretreatment and deconstruction process using the biocompatible
ionic liquid cholinium lysinate and compared to non-ensiled dry
sorghum biomass
Outcomes and Impacts
• We found that ensiled biomass was more amenable to biomass
deconstruction, resulting on higher sugar yields under more mild
pretreatment conditions compared to dry biomass.
• The ensiled biomass hydrolysates were determined to be
biocompatible and were converted into the biofuel precursor
bisabolene using the yeast Rhodosporidium toruloides
• Technoeconomic analysis and lifecycle analysis shows that the use
of ensiled biomass reduces the minimum selling price and
greenhouse footprint of biofuel production compared to dry
biomass, indicating that this process can help enable cost
competitive production of biofuels
• Ensile biomass can also be sold as animal feed, giving farmers
more options and more incentive to produce bioenergy crops
Magurudeniya et al. (2021) Green Chemistry, doi: 10.1039/d0gc03260c

2. A SWEET surprise: Anaerobic fungal sugar
transporters and chimeras enhance sugar uptake in yeast
Background
• Membrane-embedded transport proteins govern carbon uptake into
cell factories such that the activity of these transporters can dictate
the productivity of production strains.
• This study addresses the transport challenge of lignocellulosic
hydrolysate fermentation by the yeast Saccharomyces cerevisiae,
namely poor co-utilization of the abundant xylose sugar, by
evaluating the performance of heterologously expressed anaerobic
fungal SWEETs.
Approach
• Anaerobic fungal SWEET transporters were heterologously
expressed in a transporter-deficient strain to screen sugar transport
activity by growth and sugar consumption.
• Single crossover chimeras sampled the functional qualities of
SWEETs that failed to functionally expressed.
Outcomes and Impacts
• The anaerobic fungal transporter NcSWEET1 was functionally
expressed in S. cerevisiae, demonstrating broad activity on hexose
sugars and novel activity on xylose.
• A leading NcSWEET1 expression enabling domain recovered
robust chimeras that improved performance on hexose sugars
• By manipulating the crossover junction location, a narrow set of
residues that control substrate specificity were identified.
• NcSWEET1 mediated co-utilization of glucose and xylose
outperformed the engineered HXT7(F79S) control, highlighting the
utility of SWEETs as yeast strain engineering tools.
Podolsky et al. (2021) Metabolic Engineering, doi: https://doi.org/10.1016/j.ymben.2021.04.009
SWEET chimeras sampled characteristics of poorly trafficked
homologues by using a leading NcSWEET1 domain. The wild-
type NcSWEET1 demonstrated robust xylose consumption.
1 3 2 5 7 6
4
Domain 1 Domain 2
5 µm
NcSWEET1 PfSWEET2
10
20
30
0
4
8
12
OD600 [Glucose] [Xylose]
10
20
30
0
4
8
12
0 24 48 72 96 120
Concentration
(g/L)
OD
600
Time (h)
NcSWEET1

3. Integrating systems and synthetic biology to
understand and engineer microbiomes
Background
• Microbiomes, diverse communities of microorganisms,
inhabit every known environment, from oceans and soil to
the intestines of humans and animals, and perform myriad
functions including biogeochemical nutrient cycling and
conversion of dietary substrates within multicellular hosts.
• Leveraging microbiomes for environmental and societal
benefit in agriculture, medicine, and bioproduction
demands holistic understanding of the various biochemical,
molecular, spatial, and ecological factors that dictate their
composition and function.
Approach
• This review highlights recent developments in meta-omics,
modeling, and engineering tools that enable us to
understand and manipulate microbiome composition and
function, with emphasis on the ecological parameters that
drive community behavior and must be considered for
predictive microbiome design.
Leggieri et al. (2021) Annual Review in Biomedical Engineering, doi: 10.1146/annurev-bioeng-082120-022836
Microbiome engineering tools can be used to modify
existing functions within the community or to introduce
novel functions on many different scales, from editing a
single genome to an introducing an entirely new
engineered community. Development and deployment
of these tools is facilitated by integrating multiple meta-
omics analyses to develop causal links between
microbiome composition and function.
Outcomes and Impacts
• Continuously improving meta-omics tools allow comprehensive characterization of environmental and cultured
samples and elucidate strategies for tailoring the performance of microbial communities.
• Computational models of microbiomes synthesize vast empirical data to predict new strategies to accentuate
desired microbiome functions. Experimental validation is irreplaceable for model validation and improvement;
obtaining key metrics such as temporal stability, growth rates, and fluxes remains a challenge.
• Owing to its complexity, microbiome engineering requires versatile tools that allow the precise introduction of
targeted traits into complex communities with predictable outcomes.

4. Reprogramming sphingolipid glycosylation is required
for endosymbiont persistence in Medicago truncatula
Background
• Plant endosymbiosis relies on the development of specialized
membranes that encapsulate the endosymbiont and facilitate
nutrient exchange.
• However, the identity and function of lipids within these
membrane interfaces is largely unknown.
Approach
• Here, we identify GLUCOSAMINE INOSITOL
PHOSPHORYLCERAMIDE TRANSFERASE1 (GINT1) as a
sphingolipid glycosyltransferase highly expressed in Medicago
truncatula root nodules and roots colonized by arbuscular
mycorrhizal (AM) fungi and further demonstrate that this enzyme
functions in the synthesis of N-acetyl-glucosamine-decorated
glycosyl inositol phosphoryl ceramides (GIPCs) in planta.
Outcomes and Impacts
• MtGINT1 expression was developmentally regulated in symbiotic
tissues associated with the development of symbiosome and
periarbuscular membranes.
• RNAi silencing of MtGINT1 did not affect overall root growth but
strongly impaired nodulation and AM symbiosis, resulting in the
senescence of symbiosomes and arbuscules.
• Our results indicate that, although M. truncatula root
sphingolipidome predominantly consists of hexose-decorated
GIPCs, local reprogramming of GIPC glycosylation by MtGINT1
is required for the persistence of endosymbionts within the plant
cell.
• Understanding communication between plants and AM fungi is
important for the engineering of bioenergy crops with
environmental resilience and increased carbon sequestration
Moore et al. (2021) Current Biology, doi: 10.1016/j.cub.2021.03.067
Glycosylinositol phosphoryl
ceramides (GIPCs) are major plasma
membrane lipids in plants. GINT1
adds N-acetylglucosamine to GIPCs.
In M. truncatula this form of GIPC is
found in endosymbiotic tissues
whereas other tissues have GIPCs
with mannose. Additional sugars
can be added to GIPCs (not shown)
Cer
Ins
IPUT1
GINT1 GMT1
P Ceramide
Ins
Cer
Ins P
Cer
Ins P
GlcN(Ac) Man
GlcA
P
Dowregulation of GINT1 results in inability of the plant to
develop functional symbioses with N-fixing Rhizobia (left) and
arbuscular mycorrhizal fungi (right). The symbionts will in
both cases enter the roots, i.e. the initial signaling processes
are functioning, but the symbiotic structures do not fully
develop. Nodules cannot fix nitrogen (measured as C2H4
production from acetylene), and arbuscles are few and
senesce quickly.
*
*
*
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
14 dpi 28 dpi
C
2
H
4
(nmol/h)
GUS-RNAi
MtGINT1-RNAi

5. In-planta production of the biodegradable polyester
precursor 2-pyrone- 4,6-dicarboxylic acid (PDC): Stacking
reduced biomass recalcitrance with value-added co-product
Background
• 2-Pyrone-4,6-dicarboxylic acid (PDC) is a promising building block
for the manufacturing of performance-advantaged polymers.
• Reduction of lignin and in-planta production of chemicals in
bioenergy crops are two desired traits to improve the economics of
second-generation biofuels and bioproducts.
• This study demonstrates an approach to concomitantly reduce lignin
and produce PDC in the model Arabidopsis.
Approach
• A five-gene construct was designed for overproduction of
protocatechuate (PCA) and its conversion into PDC at the expense
of lignin.
• Bacterial genes encoding AroG, QsuB, PmdA, PmdB, and PmdC
were fused to signal peptide sequences for targeting to plastids
where the shikimate pathway occurs.
• Several independent transgenic lines were obtained and analyzed for
lignin content, PDC production, and yields of sugars after
saccharification.
Outcomes and Impacts
• Most transgenic lines are healthy and show no yield penalty.
• These lines accumulate PDC (up to 3% DW), have 30-45% less
lignin, and release more sugars upon cellulase and xylanase
treatment (up to +75%).
• A construct containing the same bacterial genes placed under the
control of monocot-specific promoters has been designed for
sorghum transformation.
• This study will guide the design of bioenergy crops with reduced
recalcitrance and accumulation of co-products
Lin et al. (2021) Metabolic Engineering, doi: 10.1016/j.ymben.2021.04.011
WT Transgenic lines
High PCA / PDC
WT Transgenic lines
Low lignin
PDC de-novo pathway
WT Transgenic lines
No yield penalty
**P < 0.001

6. Background
• Lignin peroxidases (LiP) catalyze cleavage of both β-O-4’ ether
and C–C bonds in lignin and are essential for depolymerizing
lignin into fragments amendable to biological upgrading to
valuable products.
• This study reported on the effect of pH on the LiP-catalyzed
cleavage of β-O-4’ ether C–C bonds in LiP isozyme H8 from
Phanerochaete chrysosporium and an acid stabilized variant of it.
Approach
• Nanostructure initiator mass spectrometry assay to quantify bond
breaking in a phenolic model lignin dimer (LigNIMS assay).
• Ab initio molecular dynamics simulations and climbing-image
nudge elastic band based transition state searches to test
hypothesis that the effect of lower pH is via protonation of
aliphatic hydroxyl groups of the lignin-like dimer.
Outcomes and Impacts
• Degradation of the lignin like dimer to products by an acid-
stabilized variant of LiP isozyme H8 increased from 38.4 % at pH
5 to 92.5% at pH 2.6. At pH 2.6, the observed product distribution
resulted from 65.5% β-O-4’ ether bond cleavage, 27.0% Ca-C1
carbon bond cleavage.
• Quantum calculations confirmed protonation of aliphatic hydroxyl
groups under extremely acidic conditions resulted in lower
energetic barriers for bond-cleavages.
• Lignin protonation implies catalysis of lignin depolymerization will
be more efficient at low pH, independent of the catalyst.
Pham L., Deng K., Northen T. R., Singer S. W., Adams P. D., Simmons B. A., Sale K. L. (2021) “Experimental and theoretical insights
into the effects of pH on catalysis of bond-cleavage by the lignin peroxidase isozyme H8 from Phanerochaete chrysosporium.”
Biotechnology for biofuels, 14(1), 108. doi: 10.1186/s13068-021-01953-7
Experimental and theoretical insights into the effects of pH
on catalysis of bond-cleavage by the lignin peroxidase
isozyme H8 from Phanerochaete chrysosporium
pH dependence of lignin peroxidase from
Phanerochaete chrysosporium
Proposed mechanism for b-O-4’ ether bond cleavage from
protonated Ca-OH, GGE cationic radical

7. Papers Enabled by JBEI Technologies

8. Leveling the cost and carbon footprint of circular
polymers that are chemically recycled to monomer
Background
• Mechanical recycling of polymers downgrades them such that they are
unusable after a few cycles
• Chemical recycling to monomer offers a means to recover the
embodied chemical feedstocks for remanufacturing
• Designing novel polymers that can be made with bio-based materials
can reduce the energy footprint of chemical recycling to virgin-quality
monomers
Approach
• We use systems analysis to quantify the costs and life-cycle carbon
footprints of virgin and chemically recycled polydiketoenamines
(PDKs), next-generation polymers that depolymerize under ambient
conditions in strong acid
• By developing process simulations and exploring long term shifts in
system-wide costs and emissions, we can establish targets for making
PDKs competitive with conventional plastics
Outcomes and Impacts
• We find that the cost of producing virgin PDK resin using unoptimized
processes is ~30-fold higher than recycling them
• The cost of recycled PDK resin ($1.5 kg−1
) is on par with PET and
HDPE, and below that of polyurethanes
• Virgin resin production is carbon intensive (86 kg CO2e kg−1
), while
chemical recycling emits only 2 kg CO2e kg−1
• This cost and emissions disparity provides a strong incentive to
recover and recycle future polymer waste
• Future adjustments in the chemistry and a shift toward greater bio-
derived content will be required to reduce the GHG footprint and cost
of virgin PDK
Vora et al. (2021) Science Advances, doi: 10.1126/sciadv.abf0187
Figure 2. Minimum selling price and life-cycle GHG
footprint for different conventional and PDK scenarios.
Figure 1. System boundary for chemically recyclable PDK

9. Further engineering of R. toruloides for the production
of terpenes from lignocellulosic biomass
Background
• Terpenes encompass a wide variety of molecules with a diverse
range of commercial applications, including biofuels
• Efficient production of terpenes from cheap carbon sources, such
as lignocellulose, is essential for them to be economical
• This study focuses on optimizing terpene production in a
conversion host capable of converting a wide range of renewable
carbon, Rhodosporidium toruloides
Approach
• To improve terpene production, strains producing the biofuel
precursor bisabolene was subjected to multi-omic analysis, and the
data generated was leveraged to identify specific enzymes in the
mevalonate pathway for optimization
Outcomes and Impacts
• Proteomic, metabolomic, and transcripomic analysis were
conducted on several bisabolene-producing strains and several
mevalonate enzymes were identified for optimization, including
HMG-CoA reductase, mevalonate kinase, phosphomevalonate
kinase
• The optimization was conducted for two terpenes, 1,8-cineole and
bisabolene, and titers were improved for both terpenes to 1.4 g/L
and 2.6 g/L, respectively
Kirby et al. (2021) Biotechnology for Biofuels, doi: 10.1186/s13068-021-01950-w