This document summarizes a research article that studied the transport of the antibiotic tetracycline (Tet) in Escherichia coli. The key findings are: 1. Tet is transported into E. coli cells by three transporters: TetA, TetB, and TetH. 2. TetA is a primary transporter that uses ATP hydrolysis to actively transport Tet into cells. TetB and TetH are secondary transporters that harness gradients to transport Tet. 3. Experiments showed TetA is the major transporter and is responsible for most Tet uptake. TetB and TetH play lesser but still significant roles in Tet transport. 4. Understanding the roles and interactions of these

1. The JBEI quantitative metabolic
modeling library (jQMM)
Birkel et al. (2017) “The JBEI quantitative metabolic modeling library (jQMM): a python library for
modeling microbial metabolism” BMC Bioinformatics, doi: 10.1186/s12859-017-1615-y
Background
• Modeling of microbial metabolism is a topic of growing
importance in biotechnology. Mathematical modeling helps
provide a mechanistic understanding for the studied process,
separating the main drivers from the circumstantial ones,
bounding the outcomes of experiments and guiding
engineering.
jQMM produces measurements of metabolic
fluxes for comprehensive genome-scale models
Approach taken for using proteomics data to improve biofuel yields
Approach
• The jQMM library presented here provides an open-source,
Python-based framework for modeling internal metabolic
fluxes and leveraging other -omics data for the scientific study
of cellular metabolism and bioengineering purposes.
Outcomes
• Firstly, it presents a complete toolbox for simultaneously
performing two different types of flux analysis that are typically
disjoint: Flux Balance Analysis and 13C Metabolic Flux Analysis.
• Moreover, it introduces the capability to use 13C labeling
experimental data to constrain comprehensive genome-scale
models through a technique called two-scale 13C Metabolic
Flux Analysis (2S-13C MFA).
• In addition, the library includes a demonstration of a method
that uses proteomics data to produce actionable insights to
increase biofuel production.
Significance
• jQMM will facilitate the design and metabolic engineering of
organisms for biofuels and other chemicals, as well as
investigations of cellular metabolism and leveraging -omics
data.

2. Lipid engineering reveals regulatory roles for
membrane fluidity in yeast flocculation and
oxygen-limited growth
Degreif et al. (2017) “Lipid engineering reveals regulatory roles for membrane fluidity in yeast
flocculation and oxygen-limited growth” Metab Eng, 41, 46-56. doi: 10.1016/j.ymben.2017.03.002
Background
• Yeast synthesize unsaturated lipids using the lipid desaturase OLE1
in order to maintain membrane fluidity
• OLE1 expression is regulated by membrane ordering sensors in the
ER, Spt23p and Mga2p; Ole1p activity is oxygen dependent
• Flocculation is the clumping of yeast cells together due to cell wall
interactions; it is an important phenotype in industrial fermentation
Approach
• OLE1 expression was titrated using the MET3 promoter and changed
in methionine concentration, generating a strain that allows to
functional characterization of lipid unsaturation in yeast
• Various aspects of cell physiology (growth, fermentation), membrane
properties (membrane fluidity), and gene expression (global RNA-Seq
and targetd qRT-PCR) were correlated as a function of changes in
lipid composition (unsaturation)
Outcomes
• Low lipid unsaturation triggers yeast to flocculate, which results from
the membrane fluidity sensor Spt23p being a strong regulator FLO1, a
mediator of yeast flocculation
• RNA-Seq reveals that Spt23p and Mga2p regulate hundreds of other
genes in yeast’s response to hypoxia
Significance
• Membrane fluidity in the ER regulates yeast flocculation during
fermentation via the effects on changing oxygen concentrations on
unsaturated lipid synthesis. A host of other genes involved in
metabolism and physiology are regulated similarly.
A B
D
250
Δflo1Δflo8
PMET3
-OLE1 grown in 250 µM met
Man Glc Gal Mal C18:1
shaking
medium
change
E
250
[met] (µM)
200 150 100 50 0
-
BY4742
-
PMET3
-OLE1
0.0
0
20
40
60
80
100
CoA-S
O
CoA-S
9
10
O NADH + H+
NAD+
O2
2 H2
O
OLE1
14 15 16 17 18
[met]retention time (min)
C14:1
C16:1 C16:0
C18:1 C18:0
C14:0
(myristoleic acid) (myristic acid)
(palmitoleic acid) (palmitic acid)
(oleic acid) (stearic acid)
10 mM
320 µM
160 µM
10 µM
%acylchainsunsaturated
DC
A
B
D
p.
250
[met] (µM)
200 150 100 50 0
C
A
C
13xmyc
N
C
High
UFA
Low
UFA
p90p90
FLO1
OLE1met
ER membrane
myc
Spt23p
Repression of the lipid desaturase OLE1 (top) using the MET3
promoter causes yeast to flocculate with increasing methionine
concentrations (bottom), reflecting a decrease in unsaturated
lipid content. Mechanistic model for this process involves
activation of the ER membrane ordering sensor Spt23p.

3. Isopentenol(mg/L)
0
300
600
900
1200
Growth Rate (/h)
0.0 0.2 0.5 0.7 0.9
R² = 0.7226
475
WT
0.4
WT
Figure 6
AtoB HMGS HMGR MK R74HIDI MtIPK
K22M
WT
S208E
Figure 2b
R74H
WT
K22M
S208ESlower = less
Faster = more
2.4-fold Titer
improvementtion activity. At such high concentrations of MVAP in vivo, the rate
enhancements on kcat/ KM or kcat observed in vitro for select mutants
would not necessarily be observed. Considering the importance of the
native mevalonate pathways for isoprenoid metabolism, the activity of
PMDsc might betightly regulated by MVAPconcentrations in vivo since
a low KI would significantly decrease the rate of decarboxylation as
excess MVAP accumulates. This directs MVAP flux through the
phosphomevalonate kinase (PMK) enzyme to generate MVAPP, the
preferred native substrate for PMDsc, rather than increasing futile
decarboxylation of MVAP to IP.
WT and four mutants, R74G, V230E, R74G-R147K-M212Q and
R74H-R147K-M212Q, werespecifically selected for substrate inhibition
study, where activity wasmeasured in wide range of MVAPconcentra-
tions up to 100 mM (Fig. 7). These variants representatively spanned a
wide range of isopentenol titers (8 – 1080 mg/ L), and their substrate
inhibition behavior was characterized by determining each mutant's KI
(Table 3). Interestingly, two higher isopentenol producing mutants
(R74G and R74H-R147K-M212Q) exhibited significantly higher KI's
(110 mM and 80 mM, respectively) than those with lower isopentenol
producers (WT, 18 mM; V230E, 10 mM; and R74G-R147K-M212Q,
11 mM) (Fig. 7). A correlation emerged between growth rates (and/ or
titers) and PMD activity when an observed turnover number was
calculated at 100 mM MVAP using the analytical expression for
noncompetitive substrate inhibition, kobs (Supplementary Fig. S7). This
turnover number (kobs) depends upon each mutant's kcat, KM, and KI,
perhapsyielding amoreaccurate assessment of theturnover conditions
in vivo (Kang et al., 2016). Therelationship between growth and kobs of
these mutants suggests that the screening platform directly reports on
the extent of substrate inhibition in PMDsc mutants (Fig. 7 and
Supplementary Fig. S7). This sensitivity of an in vivo screening
platform to a mutant's KI modulation speaks to its power in analyzing
the robustness of heterologously expressed mevalonate pathways with-
in overexpressing bacterial strains.
3. Conclusion
Evaluation of enzyme li
a screening method. In this
because it does not require
designs with the desirabl
essential for growth of the
study, wedesigned agrowt
decarboxylation activity to
achieve thisgoal, the grow
decarboxylation rate of M
formation of IP is ultimate
MVA pathway (Fig. 1A). C
mutagenesis generated two
which were tested by our
growth-based screening pla
that significantly increased
L. Correlation of growth ra
PMD mutants confirmed
platform, and kinetics stud
of this screening platform b
to the target enzymeactivit
Alteration of PMD and IPK
inhibition strength of fosm
with more flexibility, pot
isopentenol production via
4. Materials and method
4.1. Plasmids and Strains
All plasmids and str
Supplementary Table S1.
and PMD variants are show
4.2. Development of thePM
For the screening platf
HMGR, HMGS, MK, Idi a
constructed by adding Idi
for isopentenol production
while HMGR, HMGS, and
archaeal IP kinases from
(MTH) and Thermoplasma a
plasmids, pET15b-MTH a
Poulter, 2010), using p
IPKTHA-F-BglII and IPKTH
coding apotential IPkinase
two primersEcIPK-BglII-Fa
Wild type PMDsc and PMD
plasmid under control of a
based plasmid under contr
et al., 2011). Mutant libra
Table 3
Analysis of Ki and kobs for select mutants. N.D.: not detected.
kcat, sec− 1
KM, mM Ki, mM kobs, sec− 1
WT 0.15 2.3 18 0.02 (0.01)
R74G 0.14 3.4 110 0.07 (0.04)
V230E 0.07 0.8 10 0.006 (0.004)
R74G-R147K-M212Q 0.22 0.5 11 0.022 (0.008)
R74H-R147K-M212Q 0.16 0.43 80 0.07 (0.03)
Fig. 7. Determination of the noncompetitive substrate inhibition constant, KI, for select
PMDsc mutants: WT (circles); R74G(squares); V230E (upward triangles); R74G: R147K:
M212Q (downward triangles); R74H: R147K: M212Q (diamonds). All activities reported
are normalized relative to each mutant's kcat derived from fits to the noncompetitive
substrate inhibition expression. Parameters were derived from three technical replicates
(n = 3).
A. Kang et al.
132
Higher KI
High-throughput enzyme screening platform for the
IPP-bypass mevalonate pathway for isopentenol
production
Kang et al. (2017) "High-throughput enzyme screening platform for the IPP-bypass mevalonate
pathway for isopentenol production" Metab Eng. 41:125-134, doi:10.1016/j.ymben.2017.03.010
Background
• Isopentenol (or isoprenol, 3-methyl-3-buten-1-ol) is a drop-in biofuel and a precursor for commodity chemicals such as isoprene. To
overcome limitations of the original mevalonate pathway for isopentenol production such as high ATP demand and IPP toxicity, we
developed an “IPP-bypass” isopentenol pathway using the promiscuous activity of a mevalonate diphosphate decarboxylase
(PMD). However, the promiscuous PMD activity toward non-native substrate was still limiting and it is important to improve the
enzyme activity to apply this new pathway for high titer, rate, yield production.
Approach
• We developed a high-throughput screening platform that correlated promiscuous PMD activity toward MVAP with E. coli cell
growth. By inhibiting all IPP production from the endogenous MEP pathway, we made the heterologous pathway which depends on
promiscuous PMD activity the only pathway to produce IPP which is required for the E. coli growth.
Outcomes and Significance
• Design of growth-linked screening platform to improve promiscuous activity of PMD.
• Identification of PMD mutants that improve the conversion of MVAP to IP.
• 2.4-fold improvement of isoprenol titer via the IPP-bypass pathway with PMD mutants.
• Kinetic study of PMD mutants to elucidate modes of action for the titer improvement.

4. Impact of lignin polymer backbone esters
on ionic liquid pretreatment of poplar
Kim et al. (2017) “Impact of lignin polymer backbone esters on ionic liquid pretreatment
of poplar” Biotechnol Biofuels, 10(1), 101. doi, 10.1186/s13068-017-0784-2
Background
• Biomass pretreatment remains an essential step in lignocellulosic biofuel
production, largely to facilitate the efficient removal of lignin and increase
enzyme accessibility to the polysaccharides
• There have been significant efforts in planta to reduce lignin content or
modify its composition to overcome the inherent recalcitrance
Approach
• In this work, transgenic poplar lines in which monolignol ferulate conjugates
were synthesized during cell wall development to introduce, during
lignification, readily cleavable ester linkages into the lignin polymer
backbone (i.e., “Zip lignin” produced by John Ralph at GLBRC) were
pretreated with different ionic liquids (ILs).
Outcomes
• The strategic introduction of ester bonds into the lignin backbone resulted in
increased pretreatment efficiency and released more carbohydrates with
lower energy input.
• After pretreatment with any of three different ILs, the transgenic poplars,
especially those with relatively higher amounts of incorporated monolignol
ferulate conjugates, yielding up to 23% higher sugar levels compared to
wild-type plants.
Significance
• This work clearly demonstrate that the introduction of ester linkages into the
lignin polymer backbone decreases biomass recalcitrance in poplar has the
potential to reduce the energy and/or amount of IL required for effective
pretreatment.
• This result could enable the development of an economically viable and
sustainable biorefinery process

5. Structure of aryl O-demethylase offers insights into
its tyrosine-dependent catalytic mechanism
Outcomes
• Solved the X-ray crystal structure of LigM at 1.81-Å resolution
(Fig 1) and showed it has a novel protein fold.
• Characterize aryl O-demethylation reaction as having an
ordered sequential mechanism.
• Computationally determined the positions of the substrates and
showed Tyr247 acts a general acid in the reaction mechanism.
Kohler et al. (2017) “Structure of aryl O-demethylase offers molecular insight into a catalytic tyrosine-dependent
mechanism” Proc. Natl Acad. Sci. USA, 114(16), E3205–E3214. http://doi.org/10.1073/pnas.1619263114
Background
• Some strains of soil and marine bacteria have
evolved intricate metabolic pathways for
metabolizing environmentally-derived aromatics.
• Many of these metabolic pathways go through
intermediates such as vanillate.
• Demethylation of vanillate is essential for
downstream assimilation in the TCA cycle.
• LigM from Sphingomonas paucimobilis is a novel
demethylase biochemical assays.
Significance
• First structural and mechanistic characterization of single-domain aryl
demethylase using a novel tyrosine-dependent mechanism.
• Insights from this work will inform synthetic biology approaches to
convert underutilized aromatics into higher value compounds.
Approach
• We studied the structure – function relationship of
LigM using X-ray crystallography (@ALS),
molecular simulations and enzymology (@JBEI).
Figure 1. The 1.81 Å X-ray crystal structure of LigM reveals a novel
demethylase fold and well-conserved folate-binding architecture. A) Overall
structure of LigM showing both vanillate and tetrahydrofolate in the active sit
where –CH3 is transferred from vanillate to tetrahydrofolate during the reaction.
B) Zoomed in view highlighting the the tyrosine (Tyr247) dependent mechanism.
Figure 2. LigM utilizes an ordered, sequential kinetic
mechanism. The steady-state kinetics for LigM O-
demethylation of vanillate were determined by varying the
concentration of one substrate while the concentration of
the other substrate was fixed at different concentrations.

6. Transport of the essential cell wall precursor
UDP-arabinofuranose into the Golgi of plants
Rautengarten et al. (2017) “The elaborate route for UDP-arabinose delivery into the Golgi
of plants” Proc. Natl Acad. Sci. USA 114, 4261–4266, doi: 10.1073/pnas.1701894114.
Background
• Arabinose is a key component of plant wall polymers and glycoproteins.
The majority of Ara found in glycans occurs as a furanose ring (Araf), while
its precursor has a pyranose ring configuration (UDP-Arap).
• The biosynthesis of UDP-Arap originates from UDP-xylose in the Golgi
lumen, subsequently UDP-Arap is transported out of the lumen into the
cytosol where it is converted by a mutase into UDP-Araf.
a) Plants overexpressing UDP-Araf transporters
showed clear developmental and growth defects,
and b) The amount of Araf in total cell wall material
from plants overexpressing UDP-Araf transporters
demonstrated increase total arabinose content, thus
indicating that the phenotype could be related to the
amount of arabinose in the cell wall.
In vitro assay demonstrating the transport
of UDP-Araf by candidate transporters.
Approach
• Given the partitioned biosynthesis of UDP-Ara species, it was envisioned
that a transporter capable of transporting UDP-Araf into the Golgi
apparatus should exist.
• Employing the state-of-the-art transporter assay developed at JBEI, we
screened the entire family of Arabidopsis nucleotide sugar transporters
using UDP-Araf as a substrate.
Outcomes
• We were able to identify a small family of four transporters with the ability
to efficiently transport UDP-Araf under in vitro conditions.
• By manipulating the expression levels of the transporters, we were able to
demonstrate that plants with elevated expression can have increased Araf
content in cell wall polymers which are accompanied by developmental
defects.
Significance
• The identification of four UDP-Araf transporters has confirmed the
circuitous route of this important cell wall substrate from the Golgi to the
cytosol and back into the Golgi.

7. Treatment of lignite and thermal coal with low cost
amino acid based ionic liquid-water mixtures
To et al. (2017) "Treatment of lignite and thermal coal with low cost amino acid based
ionic liquid-water mixtures" Fuel, 202, 296-306, https://doi.org/10.1016/j.fuel.2017.04.051.
Background
• Coal remains a significant source of energy worldwide, and
efficient pre-processing of coal can significantly enhance
efficiency.
• The objective of this study is to explore the use of cheaper ionic
liquids; made from simple acid-base reactions between two
naturally occurring chemicals, choline and amino acids; for the
pretreatment of lignite coal.
5x Microscopic profile of lignite and thermal coal soaked in
[Ch][Lys] on a heated stage at 70OC. Darkening of the solution and
swelling of the particles can be observed.
Approach
• Two types of coal were investigated in this study, a lignite coal
with particle size 20 mesh (841 µm), and a thermal bituminous
coal with particle size 75-120 µm.
• Treatment of the two types of coal in the ionic liquids were
carried out at a 1:5 weight/volume ratio i.e. 0.1 g of coal in 0.5
mL of ionic liquids, at 70o C with stirring for 3 hours.
Outcomes
• The treatment of lignite using [Ch][ARG], [Ch][GLY] and
[Ch][PHE], reduces the size of the lignite particles 3-5x.
• The removal of oxygenated functional groups is a desirable
outcome, since oxygen reduces the efficiency of converting coal
to fuels/chemicals.
Significance
• Treatment of coal by ionic liquids reduces the decomposition
temperature of coals, the size of the coal particles, and removes
oxygenated functional groups, all of which are beneficial to
downstream combustion of coal.

8. Preliminary crystallographic analysis and in vitro
characterization of the nonmutase UAM2 from
Oryza sativa
Outcomes
• Preliminary crystallographic analysis we performed as well as analysis of the
oligomeric state in crystal and solution. UAM2 crystallizes readily but forms
crystals of limited quality.
• We show by size exclusion chromatography that the nonmutase is monomeric
in solution, and employ limited proteolysis to show DTT-enhanced proteolytic
digestion, indicating the existence of intramolecular disulphide bridges that can
protect the protein from proteolysis.
Welner et al. (2017). "X-ray diffraction analysis and in vitro characterization of the UAM2 protein from Oryza
sativa" Acta Crystallogr F Struct Biol Commun, 73(Pt 4), 241-245, doi: 10.1107/S2053230X17004587
Background
• The maintenance of UDP-activated sugars in plant cells is important for the
synthesis of complex carbohydrates including cellulose and hemicelluloses.
• The existence of seemingly non-enzymatic proteins in complexes
interconverting UDP-arabinopyranose and UDP-arabinofuranose in the plant
cytosol remains an enigma.
Significance
• A better understanding of the underlying molecular mechanism and pathways
involved in the maintenance of activated sugar pools in plant cells will help in
the engineering of bioenergy feedstocks
Approach
• To shed light on their function, we have undertaken crystallographic and
functional studies of the nonmutase UAM2 from Oryza sativa.
Crystals of OsUAM2. The largest
dimension is approximately 100 µm.
Size exclusion chromatogram of
OsUAM2 (blue), overlaid with
molecular weight standards (red)

9. Leveraging microbial biosynthetic pathways
for the generation of ‘drop-in’ biofuels
Background
• As the majority of petroleum consumption in the United States consists of
gasoline (47%), diesel fuel and heating oil (21%), and jet fuel (8%), ‘drop-
in’ biofuels that replace these petrochemical sources are particularly
attractive.
• With our increased understanding of biosynthetic logic of metabolic
pathways, we discuss the unique advantages of fatty acid, terpene, and
polyketide synthases for the production of bio-based gasoline, diesel and
jet fuel.
Production of biofuels in E. coli from three metabolic pathways
Maximum possible theoretical yield and highest titer given for each
compound based on the substrate glucose under anaerobic conditions.
Approach
• Diverse fuel energy needs of the global economy may be best
served by diversifying the metabolic pathways we choose to
develop biofuel substitutes.
Outcomes
• Fatty acid synthesis is currently the most mature pathway for
bioproduction of straight-chain hydrocarbons of short to medium
chain length that compose gasoline and diesel.
• Exquisite control over product formation by polyketide synthases
could generate customized products with desired combustion
properties as biofuel substitutes.
• Monoterpenes (C10) and sesquiterpenes (C15), produced from
isoprenoid synthases, are identified as diesel and jet fuel substitutes.
Significance
• Fatty acid synthases currently produce the highest titers, isoprenoid
synthases result have higher energy density fuels, and the
customizability of polyketides could allow the production of tailored
biofuel products with desired physical properties.
Pathway Compounds
Mass yield %
(g/ghexose)
Highest
reported titre
Fatty Acid
Synthesis
Alkanes (C13-C17) 30.7 – 30.8% 300 mg /L [11]
Alkanes (C9-C14) 30.5 – 30.7% 580 mg /L [16]
Propane 29.4 % 32 mg /L [20]
Isoprene Synthesis
Bisabolene 32.4 % 5.2 g / L [33]
Farnesene 32.4 % 1.1 g/L [36]
Pinene 32.4 % 32 mg/L [38]
Limonene 32.4 % 700 mg/L [46]
Polyketide
Synthesis
Multi-methyl-branched esters 27.2 – 27.6 % 98 mg/L [51]
Pentadecane 30.8 % 140 mg/L [52]
Zargar et al. (2017) “Leveraging microbial biosynthetic pathways for the generation of ‘drop-
in’ biofuels” Curr Opin Biotechnol, 45, 156-163, doi: 10.1016/j.copbio.2017.03.00

10. NH
O
n
OH
O
H2N
n
ORF26
n=1-3
H2N
O
OH
ORF26
E. coli
H2N
O
NH2
H2N
O
OH
NH2
DavB
DavA
NH
O
H2N
O
OH
NH2
NH
O
Application of an acyl-CoA ligase from
Streptomyces aizunensis for lactam biosynthesis
Zhang et al. (2017) “Application of an acyl-CoA ligase from Streptomyces aizunensis
for lactam biosynthesis” ACS Synth Biol. doi: 10.1021/acssynbio.6b00372
Background
• -caprolactam and -valerolactam are important commodity chemicals
used in the manufacture of nylons, with millions of tons produced
annually.
• Biological production of these highly valued chemicals has been
limited due to a lack of enzymes that cyclize ω-amino fatty acid
precursors to corresponding lactams under ambient conditions.
• In this study, we demonstrated production of these chemicals using
ORF26, an acyl-CoA ligase involved in the biosynthesis of ECO-
02301 in Streptomyces aizunensis (Fig. 1)
Fig 1. Lactam chemistry of ORF26 and the
valerolactam biosynthesis from lysine
Fig 2. Production of valerolactam and
caprolactam from precursor feeding
Approach
• To apply ORF26 for renewable chemical production, ORF26 was
overexpressed in E. coli, and both valerolactam and caprolactam
were formed in vivo by feeding their respective precursors, 5-
aminovaleric acid (5-AVA) and 6-aminocaproic acid (6-ACA) (Fig. 2)
• To achieve renewable production of valerolactam, we introduced a
two-enzyme pathway into E. coli that converts lysine to 5-AVA.
Outcomes
• High concentrations of lysine (e.g., 10 g/L) resulted in ~5 fold increase
in valerolactam production compared to non-supplemented cultures.
Significance
• The enzyme ORF26 enables five-membered, six-membered and even
seven-membered ring formation at mild temperatures, resulting in the
production of important industrial lactams such as valerolactam and
caprolactam via fermentation
0
0.5
1
1.5
2
2.5
pET28-Empty pET28-ORF26 pET28-CaiC
Caprolactam(mg/L)
24h
48h
72h
0
50
100
150
200
250
pET28-Empty pET28-ORF26 pET28-CaiC
Valerolactam(mg/L)
24h
48h
72h
H2N
O
OH
H2N
O
OH ORF26
or
CaiC
E. coli
NH
O
NH
O O
OH
H2N
NH
O
NH
O
O
OH
H2N
E. coli
A. B.
152.7 ± 5.4
48.6 ± 1.2
2.02 ± 0.08
ORF26
or
CaiC
188.8 ± 7.5
N.D. N.D.