The document discusses optimization of the mevalonate pathway and fed-batch fermentation for isoprenol production in E. coli. Key steps included: 1) Optimizing pathway intermediates to provide optimal levels for isoprenol production. 2) Conducting fed-batch fermentations in a bioreactor, which improved isoprenol titer to 10.8 g/L, the highest reported. 3) Eliminating acetate production and adding a solvent overlay further improved titer and productivity in fed-batch fermentations.

1. Molecular characteristics of plant UDP-
arabinopyranose mutases
Background
• L-arabinofuranose is a ubiquitous component of the cell wall and
various natural products in plants, where it is synthesized from
cytosolic UDP-arabinopyranose. The biosynthetic machinery
long remained enigmatic in terms of responsible enzymes and
subcellular localization.
• With the discovery of UDP-arabinopyranose mutase in plant
cytosol, the demonstration of its role in cell-wall arabinose
incorporation, and the identification of UDP-arabinofuranose
transporters in the Golgi membrane, it is clear that the cytosolic
UDP-arabinopyranose mutases are the key enzymes converting
UDP-arabinopyranose to UDP-arabinofuranose for cell wall and
natural product biosynthesis.
• In contrast to the solid evidence pertaining to UDP-
arabinopyranose mutase function in vivo, the molecular features,
including enzymatic mechanism and oligomeric state, remain
unknown.
Outcomes and Impacts
• Here, we review the UDP-arabinopyranose mutase and
reversibly glycosylated polypeptide literature together, to
summarize and systemize reported molecular characteristics
and relations to other proteins.
• Arabinofuranose is a major component of grass biomass, and is
generally more difficult to convert to biofuels and useful
bioproducts than hexoses. Understanding the biosynthesis of
arabinofuranose provides targets for improving biomass
composition.
Saqib et al. (2019) Glycobiology, doi: 10.1093/glycob/cwz067
Schematic of UDP-Arabinose mutase (UAM) in the
plant cell. The catalyzed reaction is shown above a
hypothetical model (in stipled enclosure) of UAM
autoglycosylation, oligomerization, and membrane
association. UAfT = UDP-arabinofuranose
transporter. Grey spheres = Class 1 UAM. White
spheres = Class 2 UAM. Small blue sphere =
glucose. Green star = arabinofuranose. Orange line =
xylan. Yellow line = pectin

2. Complete genome sequence of agrobacterium
33MFTa1.1 isolated from Thlaspi arvense roots
Langley et al. (2019) Microbiology Resource Announcements, doi: 10.1128/MRA.00432-19
Image of flowering Thlaspi arvense
Background
• This report describes the complete genome sequence of
Agrobacterium sp. 33MFTa1.1 that will facilitate plant-
microbe interaction studies at JBEI
• Agrobacterium sp. strain 33MFTa1.1 was isolated for
functional from the Thlaspi arvense root-associated
microbiome
Approach
• Bacteria were streaked onto LB plates, single colonies
were amplified, and an aliquot was used for 16S V1 and
V4 PCR
• Whole genome sequencing was performed using a
combination of Oxford Nanopore long-read sequencing on
the MinION Mk1B and Illumina paired-end 300-bp read
sequencing for quality
Outcomes and impacts
• The circular chromosome annotation predicts 2,654
protein-coding genes, 63 pseudogenes, 2 rRNA operons,
and 40 tRNAs, with canonical anticodon triplets that base
pair with codons for amino acids
• The linear chromosome annotation predicts 1,800 protein-
coding genes, 69 pseudogenes, 2 rRNA operons, and 14
tRNAs
• We identified 41 gene clusters of interest for further
research at JBEI

3. Insights into the mechanism of phenylacetate
decarboxylase (PhdB), a toluene producing glycyl radical enzyme
Outcomes and Impacts
• The DFT results indicated that all three mechanisms are
thermodynamically challenging, reflecting the remarkable
demands on PhdB for catalysis of this reaction.
• Along with other data, evidence that PhdB was able to bind
α,α-difluorophenylacetate but was unable to catalyze its
decarboxylation supported the enzyme’s abstraction of a
methylene H atom (green mechanism at left).
• An improved understanding of the mechanism of PhdB will
inform efforts at protein engineering to optimize the enzyme,
paving the way to more efficient, first-time green
biosynthesis of toluene, a widely used octane booster in
gasoline that has a global market of 29 million tons per year.
Rodrigues et al. (2019) ChemBioChem doi: 10.1002/cbic.201900560
Background
• We recently reported the discovery of phenylacetate decarboxylase (PhdB), representing one of only ten glycyl-radical-
enzyme reaction types known, and a promising biotechnological tool for first-time biochemical synthesis of toluene from
renewable resources, such as cellulosic biomass.
Approach
• We used experimental and computational data to evaluate
the plausibility of three candidate PhdB mechanisms (see
figure at right), involving either attack at the phenylacetate
methylene carbon (green) or carboxyl group [protonated
(blue) vs. deprotonated (red)].
• In vitro experimental data included assays with F-labeled
phenylacetate, kinetic studies, and tests with site-directed
PhdB mutants; computational data involved estimation of
reaction energetics using density functional theory (DFT).

4. Optimization of the IPP-bypass mevalonate pathway and
fed-batch fermentation for the production of isoprenol
in Escherichia coli
Kang et al. (2019) Metabolic Engineering, doi.org/10.1016/j.ymben.2019.09.003
Background
• Isoprenol is a drop-in biofuel and precursor
for commodity chemicals. Production has
been optimized in small-scale batch
fermentations using IPP-bypass pathway
but improvements in yield and productivity
are necessary to reach commercial success
Approach
• The IPP-bypass mevalonate pathway (Fig.
1) was optimized to provide optimal levels of
the pathway intermediates (Fig. 2)
• Fed-batch fermentations were carried out in
a 2-L bioreactor
Outcomes and impacts
• Isoprenol titer reached 3.7 g/L in batch
cultures, which represents 44% of the
theoretical yield
• Optimization of the fed-batch fermentation
conditions (Fig. 3) resulted in 10.8 g/L of
isoprenol, the highest titer ever reported for
this compound.
Fig 1. The IPP-bypass mevalonate
pathway was optimized for isoprenol
production. Acetate production was
eliminated
Fig 2. Different HMGR, HMGS and
MK variants were tested with
engineered PMD mutants
Fig 3. Titer was improved in
fed-batch fermentations
after elimination of acetate
and addition of a solvent
overlay
Isoprenol titer
Isoprenol(g/L)

5. Distinct functional roles for hopanoid composition
in the chemical tolerance of Zymomonas mobilis
Brenac et al. (2019) Mol. Microbiol., doi: 10.1111/mmi.14380
Background
• Hopanoids are a class of membrane lipids found in diverse
bacterial lineages, such as Z. mobilis, but their physiological
roles are not well understood
• The lack of genetic tools for manipulating hopanoid
composition in this bacterium has limited their further
functional analysis
Approach
• Collected a redundant set of 25 Tn5 mutants of the base
strain ZM4 covering annotated genes in the hopanoid
biosynthesis pathway
• Measured chromosome numbers per cell directly using a
qPCR-based approach
Outcomes and impacts
• Found that Z. mobilis cells were highly polyploid, containing
> 50 copies of two different genomic loci under our growth
conditions
• Hopanoids protect against several ethanol-driven phase
transitions in membrane structure, including lipid
interdigitation and bilayer dissolution conditions
• Demonstrated that both reduced hopanoid content and
modified hopanoid polar head group composition mediate
growth and survival in ethanol
The hopanoid composition of Z. mobilis derived
liposomes mediates changes to bilayer structure
induced by ethanol. (A) Cartoon showing the sequential
phase transitions in membranes caused by increasing
ethanol concentration, (B) emission spectra of Laurdan
in liposomes reconstituted from extracted Z. mobilis, and
(C) quantification of membrane ordering, measured as
Laurdan GP, and its triphasic dependence on ethanol
concentration.

6. Genome sequence of Striga asiatica provides
insight into the evolution of plant parasitism
Background
• Parasitic plants in the genus Striga, commonly known
as witchweeds, pose a threat to agriculture worldwide
• An understanding of Striga parasite biology, which
could lead to agricultural solutions, has been
hampered by the lack of genome information
Approach
• Developed draft genome sequence of Striga asiatica
with 34,577 predicted protein-coding genes, which
reflects gene family contractions and expansions that
are consistent with a three-phase model of parasitic
plant genome evolution
Outcomes and Impacts
• Striga seeds germinate in response to host-derived
strigolactones (SLs) and then develop a specialized
penetration structure, the haustorium, to invade the
host root
• Found evidence for horizontal transfer of host genes
as well as retrotransposons, indicating gene flow to S.
asiatica from hosts
• Results provide valuable insights into the evolution of
parasitism and a key resource for the future
development of Striga control strategies
Yoshida et al. (2019) Curr Biol., doi: 10.1016/j.cub.2019.07.086
(A) Developmental stages used for the transcriptome analysis
of Striga, (B) expression profile of each transcript is
represented in PCA space, (C) heatmap of normalized gene
expression of each transcript, and (E-J) in situ hybridization on
haustorial sections of Striga at 1 day (E and F) and 7 days (G–J)
after rice infection.

7. Common principles and best practices for
engineering microbiomes
Background
• Microbial communities have seemingly limitless
capabilities, driving Earth’s biogeochemical cycles and
occupying every environmental niche
• However, knowledge gaps hinder their efficient use for
addressing urgent societal and environmental
challenges
Approach
• Structuring research and technology developments
around a design–build–test–learn (DBTL) cycle will
advance microbiome engineering and spur new
discoveries
Outcomes and Impacts
• In this review paper, we present key elements of an
iterative DBTL approach that can be implemented to
advance the rational engineering of microbiomes for
functions that benefit society
• We discuss how the DBTL cycle can be applied to
build model systems to establish basic principles of
microbial ecosystems and provide an outlook on the
frontiers of microbiome engineering
Lawson et al. (2019) Nature Reviews Microbiology, doi: 10.1038/s41579-019-0255-9
The design–build–test–learn (DBTL) cycle for microbiome
engineering. The key aspects and approaches of each phase of the
design–build–test–learn cycle are presented. The cycle starts with
a defined engineering objective that determines the design and
produces an engineered microbiome that performs the desired
function(s).

8. Robust characterization of two distinct glutarate
sensing transcription factors of Pseudomonas
putida l-lysine metabolism
Background
• A significant bottleneck in synthetic biology involves
screening large genetically encoded libraries for
desirable phenotypes
• Transcription factor-based biosensors can be leveraged
to screen thousands of genetic designs for optimal
biofuel and bioproduct generation by engineered
microbes
Approach
• Characterized two glutarate sensing transcription
factors (CsiR and GcdR) from Pseudomonas putida
• The genomic contexts of csiR homologues were
analyzed, and their DNA binding sites were
bioinformatically predicted
Outcomes and Impacts
• CsiR was shown to dissociate from DNA in vitro when
exogenous glutarate was added, confirming that it acts
as a genetic repressor
• Developed a novel mathematical approach to describe
the usable range of detection for genetically encoded
biosensors
Thompson et al. (2019) ACS Synthetic Biology, doi: 10.1021/acssynbio.9b00255
Performance of CsiR and GcdR biosensors in P. putida. (A) RFP
expression of either wild type P. putida or with the ability to
metabolize glutarate knocked out, (B) RFP expression of wild type,
ΔdavT, or Δglutarate strains, (C) Intracellular concentration of 5-
aminovalerate of wild type, ΔdavT, or Δglutarate strains of P.
putida harboring either the CsiR or GcdR biosensor when grown on
10 mM glucose and 10 mM 5-aminovalerate (5AVA).

9. One-pot bio-derived ionic liquid conversion
followed by hydrogenolysis reaction for biomass
valorization
Background
• The ionic liquid one-pot (IL-OP) process using [Ch][Lys]
has been shown to be very promising
• One critical aspect that needs to be addressed is the
conversion of the lignin generated
Approach
• Lignin-enriched residue from switchgrass and poplar
recovered after the IL-OP process was converted to low
molecular weight aromatic/phenolic compounds by
catalytic hydrogenolysis reactions
• Reaction products were recovered and their mass
balance, composition, and structure investigated using
GC-MS, FT-IR, and GPC
Outcomes and Impacts
• The depolymerization of residual grassy and woody
lignins from catalytic hydrogenolysis over metal
catalysts Pd/C and Ni(SO)4 achieved up to 35wt%
(initial lignin basis) yields of lignin monomers
• Insights produced will help inform the design of
biorefineries that process both polysaccharides and
lignins into biofuels and bioproducts
Carrozza et al. (2019) Bioresource Technology, doi: 10.1016/j.biortech.2019.122214
Effect on lignin solubilization degree as a function of catalyst
dosage in combination with reaction temperature (a) and pressure
(b) during hydrogenolysis of switchgrass (SG) and poplar (POP)
lignin residue derived from the IL one-pot process.