The document describes research that identified the first enzyme capable of producing toluene through a biochemical reaction. Key findings include: - Researchers successfully identified the enzyme phenylacetate decarboxylase (PhdB) and its activating enzyme (PhdA) that enable the production of toluene from microbial communities. - This is the first reported enzyme for toluene biosynthesis and expands the catalytic abilities of glycyl radical enzymes. - The discovery will allow the first biochemical synthesis of renewable toluene from lignocellulosic biomass rather than petroleum, offering potential for biofuel production.

1. Discovery of enzymes for toluene synthesis from
anoxic microbial communities
Outcomes and Impacts
• Successfully identified the toluene-producing enzyme,
phenylacetate decarboxylase (PhdB), and its cognate activating
enzyme (PhdA) from a pool of more than 300,000 candidates.
• From a fundamental biochemical perspective, this is the first
reported toluene biosynthesis enzyme, and its discovery expands
the known catalytic range of glycyl radical enzymes (only seven
reaction types had been characterized previously).
• From a biofuels perspective, this discovery will enable first-time
biochemical synthesis of a renewable aromatic hydrocarbon from
renewable resources, such as lignocellulosic biomass, rather than
from petroleum.
Beller et al. (2018) Nature Chemical Biology, doi: 10.1038/s41589-018-0017-4
Background
• Toluene is a widely used octane booster in gasoline that has a global market of 29 million tons per year.
• From the perspective of energy sustainability and security, it would be desirable to offset the huge volume of petroleum-
derived toluene with microbially produced toluene made from a renewable resource, such as lignocellulosic biomass.
• Microbial toluene biosynthesis was reported in anoxic lake sediments more than three decades ago, however, the identity
of the enzyme catalyzing this biochemically challenging reaction has been a mystery for decades.
Approach
• Cultivation of anoxic, toluene-producing microbial communities
from lake sediment and sewage sludge.
• Activity-guided fractionation of proteins in microbial community
cell lysate to narrow down the list of candidates for toluene
biosynthesis.
• Use metagenomics and metaproteomics to identify candidate
gene/protein sequences.
• In vitro testing of the heterologously expressed and purified
candidate enzymes for toluene biosynthesis.
Lake in Berkeley, California, that was one source of the
toluene-producing enzyme (phenylacetate decarboxylase,
PhdB) discovered in this JBEI study. Toluene-producing
cultures that derived from anoxic sewage sludge contained a
nearly identical version of this enzyme (with more than 90%
sequence identity). The phenylacetate decarboxylase reaction
is shown. (Photo credit: Chickmarkley via Wikipedia
Commons/CC-BY-SA-3.0).

2. Annotation of the Corymbia terpene synthase gene
family shows broad conservation but dynamic
evolution of physical clusters relative to Eucalyptus
Outcomes and Impacts
• Identified 102 putative functional TPS genes in CCV, which is similar to
the numbers found in Eucalyptus grandis (113) and E. globulus (106).
• The number of TPS genes in all three eucalypts studied is notably high
compared to other plants.
• Orthology between TPS genes in E grandis and E. globulus was
common, with 60% of genes found in orthologous pairs, but only 9% of
TPS genes in CCV were orthologous with pairs from the other eucalypts.
• Both the proportional representation of subfamilies and the syntenic
physical position of gene clusters indicated a high degree of conservation
in the TPS gene family between CCV and E. grandis.
• Increased knowledge of TPS genes across multiple plant species will
enable their targeted use for the production of specific biofuels and
bioproducts.
Butler et al. (2018) Heredity, doi: 10.1038/s41437-018-0058-1
Background
• Terpenes are economically and ecologically important phytochemicals,
and their synthesis is controlled by the terpene synthase (TPS) gene
family, which is highly diversified throughout the plant kingdom.
• The recent assembly of two Corymbia citriodora subsp. variegata (CCV)
genomes presents an opportunity to examine the conservation of TPS’s
across more divergent eucalypt lineages.
Approach
• Annotated the terpene synthase gene family in the CCV reference
genome and compared it to other plants (Vitis, Populus, Arabidopsis).
• Focused on the comparison of Corymbia with E. grandis and E.
globulus.
Comparison of copy number and genomic location of TPS
physical clusters between E. grandis (Egr) and C. citriodora
subsp. variegata (CCV). Chromosomes are scaled by physical
size. Locus names show the number of TPS genes and the
subfamily they belong to.

3. Functional genomics of lipid metabolism in the
oleaginous yeast Rhodosporidium toruloides
Outcomes and Impacts
• Successfully demonstrated the application of RB-TDNAseq to the study
of lipid metabolism in an oleaginous yeast.
• Identified 150 genes with significant roles in lipid accumulation, notably
genes involved in signaling cascades (28 genes), gene expression (15
genes), protein modification or trafficking (15 genes), ubiquitination or
proteolysis (9 genes), autophagy (9 genes), and amino acid synthesis (8
genes).
• Identified a large set of genes from a wide array of subcellular functions
and compartments that impact lipid catabolism and accumulation.
• The principles learned from exploring lipid metabolism and storage
across diverse eukaryotes can inform biotechnological innovations for
the production of biofuels and bioproducts.
Coradetti et al. (2018) eLife, doi: 10.7554/eLife.32110
Background
• Oleaginous yeasts are capable of producing significant amounts of
intracellular lipids that may be suitable as biofuels and bioproducts.
• One of these yeasts, R. toruloides, can form large lipid droplets under a
variety of conditions and is an attractive platform to study conserved
aspects of the cellular biology of these important organelles across
diverse eukaryotes.
• To realize the biosynthetic potential of R. toruloides, a better
understanding of the unique aspects of its biosynthetic pathways, gene
regulation and cellular biology is required.
Approach
• Construction of a randomly barcoded, random insertion library in R.
toruloides by ATMT and its application for functional genomics using RB-
TDNAseq.
• Study lipid metabolism in R. toruloides under a wide range of conditions
and stresses that are known to elicit lipid production.
(A) DIC microscopy of R. toruloides grown in low nitrogen
media for 40 hours and stained with BODIPY 493/503 to
label lipid droplets. (B) Two strategies to enrich populations
for high or low TAG content cells. Buoyant density
separation on sucrose gradients. Lipid accumulated cells are
loaded on to a linear sucrose gradient and centrifuged.

4. Depiction of how engineered yeast that produce
monoterpenes could be used to replace
conventional hopping methods and processes.
Industrial yeast engineered for the production of
primary flavor determinants in hopped beer
Outcomes and Impacts
• Successfully controlled the production of linalool and geraniol during
ethanol productionthat exhibited significantly higher hop flavor/aroma
than the parental control.
• Similar monoterpene concentrations were observed between different
batches, demonstrating the consistent performance of the engineered
strain.
• Results demonstrate that monoterpenes derived from yeast
biosynthesis during fermentation give rise to hop flavor/aroma in
finished beer and that biosynthesis provides greater consistency than
traditional hopping.
Denby et al. (2018) Nature Communications, doi: 10.1038/s41467-018-03293-x
Background
• Hops are an expensive ingredient for breweries to source, and total
domestic sales have tripled over the past 10 years.
• Farming of hops requires a large amount of natural resources: ~100
billion L of water is required for annual irrigation of domestic hops.
• The production of the terpenes primarily associated with the flavor of
hops, linalool and geraniol, using engineered yeast would address these
issues and could be integrated directly into the brewing process.
Approach
• Monoterpene synthases encoding for linalool and geraniol were identified,
synthesized and expressed in S. cerevisiae.
• Engineered these yeast strains to be capable of producing monoterpenes
during beer fermentation to eliminate exongenous delivery of these
compounds.
• Focused on tuning the expression of key genes in a biosynthetic pathway
to simultaneously make precise concentrations of multiple flavor
molecules.
Plot of linalool and geraniol production of engineered yeast
strains compared to concentrations found in commercial beers,
plotted in log10 space.

5. Background
• Fungi and plant primary cell walls both possess an
integrity signaling system that detects and feeds
back damage.
• A similar system had been proposed for the plant
secondary cell wall.
• If this existed, the hypothesis was that it would limit
biomass engineering.
Approach
• We performed transcriptomics on the developing
stems of Arabidopsis mutants which had altered
xylan structures (the second most abundant cell
wall polysaccharide after cellulose (Fig 1A, B)
Transcriptomic analysis of xylan mutants does
not support the existence of a secondary cell
wall integrity system in Arabidopsis
Faria-Blanc et al. (2018) Frontiers in Plant Science, doi.org/10.3389/fpls.2018.00384
Figure 1. (A) Images of mutant plants used in this study. Scale bar =
20 mm. (B) Diagram representing the effects of these mutations on
xylan structure. Acetylation and the reducing end oligosaccharide on
the xylan molecule is not shown for clarity. Pentagons = xylose,
hexagons = GlcA, circles = methyl groups. (C) Number of differentially
expressed genes in each genotype compared to WT (fold change <
0.5 or > 2.0, p-value < 0.05).
Outcomes and Impacts
• Our data revealed remarkably few changes to the
transcriptome. We hypothesize that this is because cells
undergoing secondary cell wall thickening have entered
a committed program leading to cell death, and
therefore a secondary cell wall integrity system would
have limited impact.
• The absence of transcriptomic responses to secondary
cell wall alterations may facilitate engineering of the
secondary cell wall of plants.

6. Integrated analysis of isopentenyl pyrophosphate (IPP)
toxicity in isoprenoid-producing Escherichia coli
Background
• Isopentenyl pyrophosphate (IPP) toxicity presents a challenge in
engineered microbial systems since its formation is unavoidable in
terpene biosynthesis.
• However, there are several issues to using E. coli as an industrial
biofuel producing host, including susceptibility to phage contamination,
inefficiency in growing on certain desirable carbon sources (especially
sucrose), and low recombination capacity of DNA into its
chromosome.
Approach
• We developed an experimental platform to study IPP toxicity in
isoprenol-producing Escherichia coli.
• We first characterize the physiological response to IPP accumulation,
demonstrating that elevated IPP levels are linked to growth inhibition,
reduced cell viability, and plasmid instability.
• Using multi-omics data, we demonstrated that endogenous E. coli
metabolism is globally impacted by IPP accumulation.
Outcomes and Impacts
• We showed that IPP toxicity slows nutrient uptake, decreases ATP
levels, and perturbs nucleotide metabolism. It also selects for pathway
“breakage”, using proteomics to identify a reduction in phospho-
mevalonate kinase (PMK) as a probable recovery mechanism.
• We observed the extracellular accumulation of IPP and present
preliminary evidence that IPP can be transported by E. coli.
• We discovered that IPP accumulation leads to the formation of ApppI,
a nucleotide analog of IPP that may contribute to observed toxicity
phenotypes.
• These findings might be broadly relevant for the study of isoprenoid
biosynthesis.
George et al. (2018) Metabol Eng, doi: 10.1016/j.ymben.2018.03.004)

7. Background
• There is great potential to use S. cerevisiae for the
production of unique secondary metabolites by introducing
many of the metabolic production routes that have evolved
in plants, fungi or bacteria
• S. cerevisiae lacks a number of short-chain acyl-CoA
metabolites required in the biosynthesis of many
biologically and economically important compounds.
Approach
• Introduced biosynthetic metabolic pathways to five
different acyl-CoA esters into S. cerevisiae: isovaleryl-
CoA, n-butyryl-CoA, n-hexanoyl-CoA, propionyl-CoA,
and methylmalonyl-CoA.
Engineered production of short-chain acyl-
coenzyme A esters in Saccharomyces cerevisiae
Krink-Koutsoubelis et al. (2018) ACS Syn Bio, doi: 10.1021/acssynbio.7b00466
Production levels of propionyl-CoA (p-CoA) and methylmalonyl-
CoA (mm-CoA) production via the various biosynthesis routes.
(A) Biosynthetic schemes from malonyl-CoA or propionate (B)
Intracellular propionyl-CoA concentrations in the 3HP production
strain BJ5465 pNK30 (purple) and the PrpE production strain
BJ5465 pNK36 (green).
Outcomes and Impacts
• Metabolically engineered the budding yeast S. cerevisiae
to produce propionyl-CoA, butyryl-CoA, isovaleryl- CoA,
hexanoyl-CoA and methylmalonyl-CoA.
• Evaluated multiple routes toward propionyl-CoA and
methylmalonyl-CoA biosynthesis and established a
feeding free approach for the production of these acyl-
CoA esters in vivo.
• These engineered strains can serve as a platform for the
production of value added chemicals, ranging from
alcohols, acids, biopolymers and (un)natural fatty acids
and polyketides.
Scheme of gene cassettes encoding pathway enzymes and
their respective acyl-CoA products.

8. Background
• Valerian root extracts have been used by European and
Asian cultures for millennia for their anxiolytic and sedative
properties, and the the compound responsible for valerian's
well known activities is the sesquiterpenoid valerenic acid.
• The biosynthetic pathway for valerenic acid has not been
fully identified, and attempts at identifying the full
biosynthetic pathway for producing valerenic acid or
reconstituting this activity in a heterologous host have failed.
Approach
• Mined the V. officinalis transcriptome for genes with
homology to A. annua dehydrogenases and found an
alcohol dehydrogenase and an aldehyde dehydrogenase
VoADH1 and VoALDH1, respectively.
• Phylogenetic and expression analyses were necessary to
identify a valerenadiene oxidase, VoCYP71DJ1.
De novo synthesis of the sedative valerenic
acid in Saccharomyces cerevisiae
Wong et al. (2018) Metab Eng, doi: 10.1016/j.ymben.2018.03.005
(A) Important valerenic acid derivatives and related compounds.
Valerenic acid, hydroxyvalerenic acid and acetoxyvalerenic acid
are the three most abundant valerenic acids in V. officinalis.
Valerenic acid and valerena-4,7(11)-diene are reported to have
sedative effects, while acetoxyvalerenic acid has antagonistic
activities. (B) P450 activity in Asteraceae, Apiaceae and
Caprifoliaceae sesquiterpenoid drug pathways.
Outcomes and Impacts
• Engineered a yeast chassis for the production of the
valerenic acid precursor valerenadiene at a titer of ~140
mg/L.
• Used phylogenetic and expression analysis to identify a
root-upregulated V. officinalis valerenadiene oxidase, Vo-
CYP71DJ1, and integrated dehydrogenases to produce a
yeast strain capable of generating valerenic acid at 4
mg/L.

9. Background
• Lignin valorization is critical for economic viability of future
biorefineries but is hindered due to the challenges of
engineered biochassis such as the slow kinetics, aromatics
toxicity, and cost.
• Robust engineered microbes or microbial consortia that can
effectively depolymerize lignin are highly desired.
Approach
• Synthetic pathway based on vanillin degradation (LigV and
LigM) from the aromatic-catabolizing bacterium
Sphingomonas paucimobilis SKY-6 and a protocatechuate
decarboxylase (aroY) was reconstructed in an engineered
E. coli strain to covert a lignin-derived aromatic vanillin to
catechol.
• Placed pathway under control of the ADH7 promoter that
responds to the presence of vanillin to establish an
autoregulatory system.
Toward engineering E. coli with an
autoregulatory system for lignin valorization
Wu et al. (2018) Proc Nat Acad Sci, doi: 10.1073/pnas.1720129115
Schematic presentation of an autoregulatory microbial
system for valorizing lignin-derived aromatics into value-
added chemicals. A catechol biosynthesis pathway
coexpressed with an aromatic transporter CouP under the
induction of vanillin-inducible promoter ADH7. The
transporter (blue) represents other unknown aromatics
transporters.
Outcomes and Impacts
• Strain coexpressing pathway genes with transporter CouP
produced about 40% more catechol than the strain that did not
coexpress the transporter CouP.
• Demonstrated a smart, autoinducible, and efficient system for
vanillin bioconversion by utilizing the vanillin-inducible
promoter ADH7.

10. Background
• Interactions between plant roots and soil microorganisms
are critical for plant fitness in natural environments.
• It is not clear how the interaction between root exudate
chemistry and microbial substrate preferences combines to
influence rhizosphere community assembly and succession.
Approach
• Integrate information from comparative genomics and a
recently described exometabolomics approach to explore
the metabolic potential of soil bacteria, the composition of
root exudates produced by an annual grass through its
developmental stages and the substrate uptake preferences
of isolated soil bacteria representing groups that display
distinct successional responses to growing plant roots.
Dynamic root exudate chemistry and microbial
substrate preferences drive patterns in
rhizosphere microbial community assembly
Zhalnina et al. (2018) Nat Microbiol, doi: 10.1038/s41564-018-0129-3
Distributions of select traits in the genomes of soil
bacterial isolates classified into two groups based on the
response to plant root growth. a, The minimum
generation times predicted from genome sequences. b,
Genome size of isolates. c, Extracellular enzymes for
plant polymer degradation. d, Monomer transporters. All
gene frequencies were adjusted for differences in
genome size.
Outcomes and Impacts
• There is a preference by rhizosphere bacteria for consumption
of aromatic organic acids exuded by plants.
• Results show that programmed developmental processes in
plants result in dynamic patterns of the chemical composition
of root exudates.
• Chemical succession in the rhizosphere interacts with
microbial metabolite substrate preferences that can be
predicted from genome sequences.

11. A transgene design for enhancing oil content in
Arabidopsis and Camelina seeds
Background
• Increasing the oil yield is a major objective for oilseed crop
improvement. Oil biosynthesis and accumulation are influenced by
multiple genes involved in embryo and seed development.
• The Leafy Cotyledon1 (LEC1) is a master regulator of embryo
development that also enhances the expression of genes involved in
fatty acid biosynthesis.
Approach
• We speculated that an Artificial Positive Feedback Loop approach
could be used to increase oil yield by targeted overexpression of a
master regulating transcription factor for oil biosynthesis, using a
downstream promoter for a gene in the oil biosynthesis pathway.
• To verify the effect of such a combination on seed oil content, we
made constructs with maize (Zea mays) ZmLEC1driven by serine
carboxypeptidase-like (SCPL17) and acyl carrier protein (ACP5)
promoters for expression in transgenic Arabidopsis
thaliana and Camelina sativa.
Outcomes and Impacts
• Agrobacterium-mediated transformation successfully generated
Arabidopsis and Camelina lines that overexpressed ZmLEC1 under
the control of a seed-specific promoter.
• Overexpression of ZmLEC1 increased the oil content in mature
seeds by more than 20% in Arabidopsis and 26% in Camelina.
• The findings suggested that LEC1, driven by a downstream seed-
specific promoter, can be used to increase oil production in
Arabidopsis and Camelina, and could be a promising target for
increasing oil yield in oilseed crops.
Zhu et al. (2018) Biotechnol Biofuels 11:46. https://doi.org/10.1186/s13068-018-1049-4
Seed oil in selected lines of Arabidopsis (a) and Camelina (b). Increase
in the best lines (95% confidence interval) was 18-24% and 18-37%,
respectively.
WT CsAL3 CsSL2 CsSL5CsAL3 WT
a ***
***
34
36
38
40
42
edoilcontent(%)
b
*** ***
30
32
34
36
38
40
42
WT AtSL1 AtSL4 AtSL5
Seedoilcontent(%)
a ***
***
28
30
32
34
36
38
40
42
WT CsAL1 CsAL5 CsSL1 CsSL2
Seedoilcontent(%)
b
*** ***
30
32
34
36
38
40
42
WT AtSL1 AtSL4 AtSL5
Seedoilcontent(%)
The engineered plants are indistinguishable from wild type. This
photo shows four Camelina lines engineered with pACP5:ZmLEC1
(AL) or pSCPL17:ZmLEC1 (SL).
pSCP17:GUS
pACP5:GUS
Several promoters were tested. The two selected promoters were
confirmed by promoter:GUS analysis to have high expression in
seeds and embryos. pSCP17 is seed specific while pACP5 leads
to some expression also in vegetative tissues.