JBEI Highlights June 2016

1. Improving Synthetic Biology
Communication: Recommended Practices
Outcomes
• ACS Synthetic Biology Registry (https://acs-registry.jbei.org) established and incorporated into the “Web of Registries”
• Authors incentivized and enabled to depict genetic designs and fully disclose complete sequences following SBOL standards
Significance
• New recommendations for authors and the availability of the new ACS Synthetic Biology Registry
add value to the manuscript publications process for authors, reviewers, editors, and the broader
scientific community alike
Hillson, NJ. et al. (2016) “Improving Synthetic Biology Communication: Recommended Practices for Visual Depiction
and Digital Submission of Genetic Designs”. ACS Synth. Biol., 5 (6), pp 449–451. DOI: 10.1021/acssynbio.6b00146
Background
• Communication about research and development is
made more effective by the adoption of community
standards
• For synthetic biology, research articles should
consistently depict genetic designs and fully disclose
the complete sequences of all the constructs they
describe
Approach
• Create a Registry for the journal ACS Synthetic
Biology, leveraging JBEI’s ICE repository platform
• ICE supports Synthetic Biology Open Language
(SBOL) data-exchange (SBOL 2) and genetic design
visualization (SBOL Visual) community standards
• ACS Synthetic Biology now recommends that authors
deposit strains, sequences, parts, and seeds into its
Registry as part of the manuscript submission process

2. A Simple and Inexpensive Strategy for
Reproducible Analysis of Aqueous Lignin
Outcomes
• The assay generated unique reactivity profiles that was used distinguished between suspensions of alkali lignin, ionic liquid
derived lignin (Eucalyptus) and Klason lignin (switchgrass).
• The assay revealed the sequestration of water-soluble high and low molecular weight moieties within insoluble lignin
aggregates that often leads to erroneous interpretation of data from biological lignin degradation studies
• A 96-well format of the assay was also used to rapidly monitor consumption of syringic acid by Sphingobium sp. SYK-6.
Significance
• The simplicity and reproducibility of the assay makes it an inexpensive and versatile tool for routine
qualitative and semi-quantitative comparative analysis aqueous lignin samples.
Joshua, C.J. et al. (2016). “Ferricyanide-based analysis of aqueous lignin suspension revealed
sequestration of water-soluble lignin moieties.” RSC Advances. DOI: 10.1039/C6RA04443C
Background
• Lignin is a complex amphiphilic plant
cell wall derived phenolic polymer
with vast potential as a source of
high-value renewable products.
• Lignin amphiphilic moieties interact to
form heterogeneous insoluble
aggregates that are often difficult to
characterize.
• Developing reproducible analytical
strategies for lignin analysis is critical
for its valorization.
Approach
• We developed simple ferricyanide
and size exclusion chromatography
based technique for rapid analysis of
aqueous lignin samples.

3. Impact of Engineered Lignin Composition on Biomass
Recalcitrance and Ionic Liquid Pretreatment Efficiency
Outcomes
• Cleavage of β-O-4 linkages in the H-lignin dominant mutant was
greater than those in the G-lignin dominant mutants.
• Glycome profiling revealed that altering the structure and composition
of lignin components results in overall changes in the structural
architecture of the cell walls, which in turn leads to changes in the
extractability of wall polymers.
Shi, J. et al. (2016). "Impact of engineered lignin composition on biomass recalcitrance
and ionic liquid pretreatment efficiency". Green Chem. DOI: 10.1039/c6gc01193d
Background
• Collaboration between researchers at JBEI, BESC, and the EFRC
C3Bio focused on understanding how changes in lignin composition
impact ionic liquid pretreatment efficiency.
• Used a combination of approaches to characterize the structural
and compositional features of wild-type Arabidopsis and mutants
with distinct lignin monomer compositions: fah1-2 (Guaiacyl, G-
lignin dominant), C4H-F5H (Syringyl, S-lignin dominant), COMT1
(G/5-hydroxy G-lignin dominant), and a newly developed med5a
med5b ref8 (p-hydroxyphenyl, H-lignin dominant) mutant.
Approach
• Coupled traditional cell wall and hydrolysate characterization
techniques with glycome profiling and DFT modeling to develop a
fundamental understanding of how lignin engineering impacts
pretreatment efficiency using 1-ethyl-3-methylimidaozlium acetate.
Significance
• This study provides insights into the role of lignin monomer composition
on the enzymatic digestibility of biomass and the effect of lignin
modification on cell wall architecture and biomass pretreatment
performance.
Glucose yields before (a,c) and after (b,d) IL pretreatment at 3
(a,c) and 10 (b,d) mg enzyme/g biomass loading
Changes in lignin bond moieties before and after IL pretreatment

4. Insights into Polyketide Biosynthesis gained from
Repurposing Antibiotic-producing Polyketide
Synthases to Fuels and Chemicals
Outcomes
• We have repurposed PKSs to produce a number of short-chain
molecules that could have applications as fuels or chemicals.
• We have uncovered a number of expanded substrate
specificities and requirements of various PKS domains not
previously reported and determined an unexpected difference in
the order of enzymatic reactions within a module.
• We were able to efficiently change the stereochemistry of side
chains in selected PKS products.
Yuzawa, S. et al., (2016) “Insights into polyketide biosynthesis gained from repurposing antibiotic-
producing polyketide synthases to fuels and chemicals” J Antibiot (Tokyo) DOI: 10.1038/ja.2016.64.
Background
• Complex polyketides comprise a large number of
natural products that have broad application in
medicine and agriculture.
• They are produced in bacteria and fungi from enzyme
complexes named type I modular polyketide synthases
(PKSs) that contain discrete enzymatic domains
organized into modules.
• The modular nature of PKSs has enabled a multitude
of efforts to “repurpose” the PKS genes to produce
novel compounds in a predicted manner.
Significance
• These results greatly enhance the mechanistic
understanding of PKS and pave the way for exploitation
of PKS as a platform to produce fuels and chemicals.
-Lipomycin
LipNrps, LipMt etc.
(B)
LipPks1
Domain insertion (TE)
AT
LipPks1+TE
ACP KS AT KR ACP TE
R OH
OOH
R
HO
O
S
R
O
S
O OO
OH
OH
OH
N
O
OH
OH
O
O OO
OH
OH
OH
N
O
OH
OH
OH3C
21-Methyl--lipomycin
LipPks2 (M2, M3） LipPks3 (M4, M5）
KR KSDHAT
LipPks4 (M6, M7)
ATAT
LipPks1 (M1）
ACP KS AT KR ACP KS AT DH KR ACP KS AT DH KR KS
(A)
S
O
O
R
HO
S
O
R
HO
R
HO
O
S
R
HO
O
S
R
HO
O
S
R
HO
O
S
R
HO
O
S
R
O
S
R =
~~
~~
ACP ACPKRACP
R =
~~
~~
~~
~~
~~
~~
kcat/KM (M-1 s-1) = 70 23 304 237 38 4
Starter acyl-CoA = Propionyl n-Butyryl Isobutyryl 2-Methylbutyryl Isovaleryl Pivaloyl
KS AT DH KR ACP KS AT DH KR ACP
AT ACP KS AT KR ACP
BorA3 (M2, M3)
AT
BorA1
KS AT KS AT KR ACP KS AT DH KR ACP
(A)
(B)
BorA2 (M1)
ACP KR ACP
S
O
H
HO2C
H
HO2C
S
O
HO
H
HO2C
HO
S
O
H
HO2C
HO
S
O
BorA1
AT
BorA1
KS AT
KR-swapped BorA2+TE
ACP KR ACP TE
Domain swapping (KR)
Domain insertion (DH, ER, KR, TE)
ER
O
HO
O
OH
S
O
O
HO
O
HO
O
S
DH
S
O
O
HO
S
O
HO
O
HO
Borrelidin
BorA4-BorA6
BorI, BorJ
O
O
CO2H
NC
OH
OH
DH
BorA2 (M1)
AT KS ATACP KR ACP

5. Feedstocks for aviation biofuels.
Chuck, Singh et al. (2016). "Biofuels for Aviation, Chapter 2
(pgs. 17-34), Elsevier ISBN: 978-0-12-804568-8.
Background
• Collaboration between researchers at the University of Bath-UK,
Plymouth Marine Lab-UK and JBEI-USA to understand the cost,
availability and sustainability of feedstocks to successfully produce
aviation biofuels.
• There are numerous routes to aviation fuels and as such it is likely
that multiple feedstocks will be used globally to produce future
aviation biofuels.
• The chapter provides a general overview of all the major feedstocks
available, where they are regionally produced, the challenges
inherent in increasing production to necessary levels and key
forecasts for bioenergy use ahead to 2050.
Significance
• While the properties of the fuel itself is key to the eventual
replacement of fossil resources, it is more likely that the availability
and logistical consideration around the feedstock will be the key
determinant in what fuels are produced and where they are
situated.
• While a large investment must still be made in research
development and infrastructure to make aviation biofuels a reality,
there is probably sufficient feedstock to produce aviation biofuels if
the demand for road transport biofuels is reduced by significant
technological advancements in electric and battery technology.
• It is also clear that no one feedstock will supply the entire aviation
sector, and a range of technologies to develop aviation biofuels
from lipids, lignocellulose, marine resources will need to be
developed concurrently to create aviation biofuels.
• Prediction on whether a paradigm shift due to genetic engineering
or the development of 3rd generation, non-land based biomass, will
alter the biofuel market is difficult with current data.
Urban food waste produced as percentage of continent
Low and high estimates for the level of biomass available for
bioenergy in 2050
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
Fisher et al 2001
Yamamoto 2001
Wolf et al 2003
Hoogwijk et al 2005
Field et al 2008
Hoogwijk et al 2009
Smeets et al 2007
WBGU 2009
Van Vuuren et al 2009
Beringer 2011
Searle 2015
IEA 2 °C scenario
GEA Efficiency
IPCC complied…
ECOFYS / WWF…
Greenpeace energy…
Potenital global annual bioenergy contibution
(EJ)
Low scenario
High scenario
Africa
10%
Europe
16%
Americas
23%
Asia
51%
Total UFW is approx. 600 million tonnes