JBEI Research Highlights - September 2017

1. Outcomes and Significance
• We discovered the enzyme that catalyzes the oxidative cyclization of prodigiosin to form cycloprodigiosin. This membrane
protein is dependent on iron, and requires a tetrahydroptern as reducing cofactor
• The enzyme is very closely related (functionally and sequence-wise) to an enzyme important in human health: Alkylglycerol
Monooxygenase (AGMO) - this is a notoriously difficult enzyme to work with, so a bacterial homolog presents a great
opportunity to simplify studies on AGMO, and AGMO researchers are already requesting our strains for this purpose
• This newly discovered enzyme is unrelated to the enzyme responsible for cyclizing prodiginines in Streptomyces, which
means that the pathways evolved independently
de Rond et al. (2017) “Oxidative cyclization of prodigiosin by an alkylglycerol
monooxygenase-like enzyme,” Nat. Chem. Biol., DOI: 10.1038/nchembio.24714
Background
• To be able to biomanufacture a wide variety of products, we need to
expand the range of characterized enzymatic transformations (i.e. our
“biocatalytic toolkit”). Enzymes catalyzing C–H activation reactions
are particularly valuable additions to the toolkit
• The bacterium Pseudoalteromonas rubra produces cycloprodigiosin,
an interesting bioactive molecule
Approach
• We harnessed JBEI’s unique resources – shotgun proteomics and the
Diva PCR service – to design and generate constructs capable of
disrupting genes in P. rubra, a non-model organism
• We disrupted a key gene in the pathway to verify its function, and
found that a neighboring gene was responsible for the cyclization of
prodigiosin to form cycloprodigiosin
• Some basic properties of the enzyme were measured in vitro in
inverted membrane vesicles of E. coli heterologously expressing it
Prodiginines are bright pink, making it easy to identify P.
rubra mutants in which prodigiosin biosynthesis was altered.
The ease of measuring this phenotype made it possible for
two UC-Berkley undergraduates to contribute significantly to
the project.
The enzyme we discovered catalyzes a type of reaction
notoriously difficult to do with traditional synthetic organic
chemistry: C–H activation followed by C–C bond formation.
Oxidative Cyclization of Prodigiosin by an
Alkylglycerol Monooxygenase-like Enzyme

2. Survey of Lignin-Structure Changes and
Depolymerization during Ionic Liquid
Pretreatment
Outcomes
• Triethylammonium hydrogensulfate ([TEA][HSO4]) results in the maximum decrease in β-aryl ether bonds, but a minimum
decrease in molecular weight
• 1-ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]) yields a smaller decrease in the β-aryl ether content and the maximum
decrease in molecular weight
• Cholinium lysinate ([Ch][Lys]) shows an intermediate result, with moderate depolymerization and recondensation observed
Datta et al. (2017) "Survey of Lignin-Structure Changes and Depolymerization during Ionic
Liquid Pretreatment,” ACS Sust. Chem. & Eng., DOI: 10.1021/acssuschemeng.7b02123
Background
• Understanding the chemical changes in lignin
structure during ionic liquid (IL) pretreatment
process is not only needed for understanding
and overcoming biomass recalcitrance during IL
pretreatment but is also necessary for designing
new routes for lignin valorization
Significance
• The insight gained on lignin structure changes and possible depolymerized products during IL pretreatment process will help
future lignin valorization efforts in a potential IL-based lignocellulosic biorefinery
Monomer yields (g/kg) obtained as a function of pretreatment
condition, IL used, and type of monomer released
Approach
• Changes in the lignin structure due to three IL
pretreatment processes were monitored using
1H−13C heteronuclear single quantum
coherence (HSQC) nuclear magnetic resonance
(NMR) spectrometry, 31P NMR, elemental
analysis, and gel permeation chromatography
(GPC) supported by an EMSL user project
0
0.2
0.4
0.6
0.8
1
1.2
1.4
[C2C1Im][OAc] [Ch][Lys] [TEA][HSO4]
140$C$/$1h$
Guaiacol
Vanillin
Acetovanillone
Guaiacylacetone
0
0.5
1
1.5
2
2.5
3
[C2C1Im][OAc] [Ch][Lys] [TEA][HSO4]
160$C$/$1h$
Guaiacol
Vanillin
Acetovanillone
Guaiacylacetone
0
0.5
1
1.5
2
2.5
3
3.5
[C2C1Im][OAc] [Ch][Lys] [TEA][HSO4]
160$C$/$2h$
Guaiacol
Vanillin
Acetovanillone
Guaiacylacetone
0
0.5
1
1.5
2
2.5
3
3.5
4
[C2C1Im][OAc] [Ch][Lys] [TEA][HSO4]
160$C$/$3h$
Guaiacol
Vanillin
Acetovanillone
Guaiacylacetone

3. Life-Cycle GHG and Water Intensity of
Cellulosic Biofuel Production Using
[Ch][Lys] Ionic Liquid Pretreatment
Outcomes
• We found that the current iHG process achieves an ~45%
reduction in GHG-intensity relative to gasoline on a per-
MJ of fuel output basis (using ethanol)
• Moving from water-wash to iHG does not signifcantly
change water use, but the iHG-projected reaches parity
with the water consumption for dilute-acid biorefineries
and reduces GHG emissions by ~75% relative to gasoline
1) Integrated high-gravity configuration simulated. This is the first study
to examine the water and greenhouse gas implications of using ionic liquid
pretreatment in cellulosic biorefineries, and we found that the iHG process
represents a major step forward for both cost and sustainability.
Neupane et al. (2017) "Life-Cycle Greenhouse Gas-and Water-Intensity of Cellulosic Biofuel Production Using
Cholinium Lysinate Ionic Liquid Pretreatment,” ACS Sust. Chem. & Eng., DOI: 10.1021/acssuschemeng.7b02116
Background
• Ionic liquids (ILs) for pretreatment has been shown to
offer several advantages over other pretreatment
processes, including high delignification, production of a
clean lignin stream, reduced processing time for
enzymatic hydrolysis, high surface area in the recovered
biomass, and higher sugar yields at low enzyme
loadings
Significance
• Completed the first-ever LCA for IL-based biorefineries,
gauged progress to-date in GHG reductions and
identified importance of protic ILs
2) Net greenhouse gas emissions per MJ of fuel produced. In the water-
wash configuration, on-site natural gas demands for distillation of water to separate
and recovery ionic liquids results in unacceptably high GHG emissions. Moving to an
iHG configuration eliminates that need, and moving to a protic IL allows for the facility
be operate as a net-exporter of electricity.
Approach
• We simulated the conventional water-wash process,
integrated high gravity process (iHG), and a projected
integrated high gravity process (iHG-projected) using a
hypothetical protic IL in SuperPro and our life-cycle
assessment model to determine net GHG and water use

4. Base-catalyzed Depolymerization of
Lignin for Microbial Conversion
Outcomes
• A low-temperature BCD can generate partially-depolymerized lignin streams that are
compatible with microbial conversion
• Several organisms were demonstrated to convert the depolymerized lignin, including
Corynebacterium glutamicum, Pseudomonas putida and Rhodotorula mucilaginosa.
• Muconic acid was produced from a BCD lignin liquor by engineered P. putida
Scheme of the steps applied to obtain the
lignin-enriched solid substrate DMR-EH
and the BCD lignin liquor for further
biological upgrading. Measured values are
indicated by a, calculated values are
indicated by b.
Rodriguez et al. (2017) “Base-Catalyzed Depolymerization of Solid Lignin-Rich Streams Enables
Microbial Conversion,” ACS Sust. Chem. and Eng., DOI: 10.1021/acssuschemeng.7b01818
Background
• Lignin valorization can improve the economic viability of lignocellulosic biorefineries
• Because of the heterogeneity and recalcitrance of lignin, its transformation to value-
added products remains a considerable technical challenge
• Microorganisms with the natural ability to metabolize lignin-derived compounds offer
a possibility for conversion into biomass and bioproducts
• This work is a collaboration with NREL through a BETO AOP
Significance
• This study highlights the potential for a mild lignin depolymerization process to
enable microbial valorization of lignin and enhance biorefinery economics
Approach
• The base-catalyzed depolymerization (BCD) of a solid lignin-rich substrate was
examined under a range of temperatures at two base concentrations
• A microbial screen was used to assess the biocompatibility of the resultant BCD
liquors and to select organisms that can grow rapidly on this substrate
• The yields of monomeric aromatic compounds after the BCD and the changes in
lignin concentration and molecular weight distribution after microbial growth were
monitored

5. Evolution of the 3-Hydroxypropionate
Bicycle and Recent Transfer of Anoxygenic
Photosynthesis into the Chloroflexi
Outcomes
• Analysis shows that the phototrophic Chloroflexales clade
evolved much later than previously thought, with the
appearance of both anoxygenic photosynthesis and the 3-
hydroxypropionate (3HP) bicycle within the phylum
sometime near the end of Proterozoic time
• Findings demonstrate the utility of molecular estimates on
the divergence times of bacterial phyla to years—to test
hypotheses that bear on the age and role of important
microbial metabolisms
Summary of key divergence events from cross-
calibrated molecular clock analyses
Shih et al. (2017) "Evolution of the 3-hydroxypropionate bicycle and recent transfer of anoxygenic
photosynthesis into the Chloroflexi,” Proc Natl Acad Sci USA, DOI: 10.1073/pnas.1710798114
Background
• It is widely thought that anoxygenic photosynthesis
preceded the development of oxygenic photosynthesis and
the rise of atmospheric oxygen
• Several phototrophic bacterial clades are thought to have
evolved before oxygenic photosynthesis emerged,
including the Chloroflexi
Significance
• There is a strong possibility that phototrophy may have
originally evolved in an undiscovered or extinct stem group
lineage
Evolutionary relationships show that critical enzymes involved in the 3HP
bicycle were horizontally transferred from other taxa into the phototrophic
Chloroflexales order. (A) Phylogeny of the key 3HP enzyme, propionyl-CoA
synthase, illustrating the derived placement of the Chloroflexales (green
branches). The many basal taxa from non-Chloroflexi species indicate that the
Chloroflexales appear to have acquired this gene via HGT, as it is not found in
any other species within the Chloroflexi phylum. The acetyl-CoA synthetase
family was used as the outgroup. (B) Phylogeny of malyl-CoA lyase (MCL) also
shows a derived position and points to an HGT origin within the Chloroflexales
Approach
• Collaboration between LBNL and Cal Tech
• Used a combination of comparative genomics and
molecular clock analyses to compare to estimate key
divergences within the phylum and when they occurred

6. Effect of Ionic Liquid Pretreatment on
the Porosity of Pine
Outcomes
• Porosity increases after IL pretreatment at a biomass
concentration of 5 wt%, and keeps increasing with
increasing biomass concentration until 20 wt%
• With biomass concentrations increasing from 5 to 15
wt%, pretreated pine samples show a transition from
cellulose I to II allomorphs
(a) XRD data of pretreated pine samples before and after extraction with
petroleum ether and acetone; (b) XRD data pretreated pine samples at 130 C;
(c) sugar conversion after 72 h hydrolysis by DNS method.
Yuan et al. (2017) "Effect of Ionic Liquid Pretreatment on the Porosity of Pine: Insights from Small-
Angle Neutron Scattering, Nitrogen Adsorption Analysis, and X-ray Diffraction,” Energy & Fuels,
DOI: 10.1021/acs.energyfuels.7b01567
Background
• Biomass pretreatment can alter lignocellulose in many
ways. One of the most important is thought to be
increasing the porosity of the lignocellulose, thereby
increasing the accessibility of enzymes to the
polysaccharides present.
• Small angle neutron scattering is a powerful analytical
technique for measuring porosity, yet it is not widely
used currently.
Significance
• The changes in porosity and cellulose crystalline structures suggest that IL molecules interacted with biomass more efficiently
at higher biomass concentrations, which is attributed to the presence of resin acid in pine
• These findings provide a deeper understanding of how ILs interact with lignocellulose and indicate that ILs are effective at high
biomass loading levels, which is a significant factor in the costs of biomass conversion
Approach
• Changes in porosity of pine samples as a function of
ionic liquid (IL) pretreatment conditions were studied
using both SANS and nitrogen adsorption
measurements at the HFIR at ORNL.