JBEI Highlights June 2020

1. An Arduino based Automatic Pressure Evaluation
System (A-APES) to quantify growth of non-model
anaerobes in culture
Background
• Current techniques to monitor growth of anaerobic gut
fungi require time consuming manual pressure
measurements.
• High resolution data is time intensive to acquire and
typically limited to ~3 measurements per day.
• Goal: automate the measurement process so that higher
resolution growth rate data can be used to investigate gut
fungal growth dynamics.
Wilken et al. (2020) AIChE J., doi:10.1002/aic.16540
Instantaneous growth rate of Neocallimastix lanati on corn
stover (lignocellulose) varies over time.
Approach
• Arduino is an open-source and accessible electronic
design framework that is increasingly used to build
custom lab automation devices.
• We constructed a modular device using off-the-shelf
components (incl. an Arduino microcontroller) that can
continuously monitor pressure in a sealed vessel as well
as vent it at programmable intervals/conditions.
Outcomes and Impacts
• Design and construction guide of a low cost, modular and
easily extendable automated system is available online.
• Highest resolution growth curve of an anaerobic gut
fungus to date reveals that growth rate varies over time,
i.e. the gut fungi do not exhibit a classic exponential
growth phase.
• Pressure accumulation and hydrogen inhibition are
unlikely to inhibit growth.
Automatic vs. manual system growth monitoring the growth of
anaerobic gut fungi
Measurements
taken every minute
Growth rate varies likely due to
differentially metabolized breakout
products of lignocellulose

2. Visualizing pectin polymer-polymer entanglement
produced by interfacial water movement
Background
• Pectin polymers have bioadhesive properties that make them
interesting for applications e.g. in wound healing, drug delivery
and as mesothelial sealants. However, the physico-chemical
properties are not fully understood, and a more predictive
understanding of the structure-function relationships is required.
• In this report, we investigated the physical conditions for creating
pectin polymer-polymer (homopolymer) entanglement.
Approach
• The role of water movement in creating pectin entanglement was
investigated by placing water droplets between two glass phase
films and compressing the films at variable probe velocities.
• Adhesion strength and polymer entanglement were determined
as a function of time and compression strength.
• This work was a collaboration with Harvard Medical School,
Center for Bioenergy Innovation (CBI) and two German
universities.
Outcomes and Impacts
• Slow probe velocity (0.5 mm/sec) demonstrated no evidence of
stranding or polymer entanglement.
• In contrast, fast probe velocity (5 mm/sec) resulted in 1) an
increase in peak adhesion strength, 2) a progressive debonding
curve, and 3) increased work of cohesion (p < .001).
• Strong adhesion developed rapidly.
• We conclude that water movement can supply the motive force
for the rapid chain entanglement between pectin films.
• The findings are important for developing pectin films and
protocols for use as mesothelial sealants.
• Pectin is a biomass component and its use in biomedical
applications could increase the value of bioenergy crops.
Pierce et al. (2020) Carbohydrate Polymers, doi: 10.1016/j.carbpol.2020.116618
Schematic of the adhesion test. The probe moves down at a
set velocity until a set force is encountered and is then held at
that force. After a set time the probe is moved up at 0.2
mm/sec and adhesion measured.
Adhesion develops rapidly with at least 75% of maximum
observed after 20 sec. Entanglement is evidenced when
adhesion is not instantaneously relaxed but persists as the
probe moves away (arrow).

3. Do cell wall esters facilitate forest response to
climate?
Background
• Stresses associated with environmental change impact plant
growth, development and mortality and ecological succession.
• Carbon stocks and biosphere-atmosphere fluxes of CO2, water and
VOCs (volatile organic compounds including methanol and acetic
acid) are dramatically changing in response to climate factors.
• Understanding what drives the forest response to climate change is
important for predicting future terrestrial ecosystems responses.
• Little is known about the function of cell wall esterification in trees,
but pectin methylation is suggested to be important for gas
exchange via stomatal control and xylan acetylation is required for
proper xylem functioning.
• Cell wall esters are also a target for engineering improved biomass
for biofuels and renewable chemicals as they can impact
downstream fermentation.
Dewhirst et al. (2020) Trends in Plant Science, doi: 10.1016/j.tplants.2020.05.011
Integration of cell wall esters with atmospheric
emissions and primary carbon metabolism
Outcomes and Impacts
• In this review, we suggest that cell wall methyl and O-acetyl
esters play an important role mediating responses to
environmental change, linking modifications in cell wall structure
and function with atmospheric fluxes of methanol, acetic acid,
CO2 and water.
• In addition to being released into the atmosphere, methanol and
acetate derived from cell wall esters has the potential to be re-
integrated into central carbon metabolism via photorespiration.
These cell wall derived compounds could represent an alternative
carbon source and play a role in thermal tolerance.
• We highlight the need for further interdisciplinary research linking
cell wall biochemistry and metabolism with plant physiology and
biosphere-atmosphere gas exchange.
Climate change related forest dynamics associated
with cell wall-derived methanol and acetic acid

4. Purification and characterization of a native lytic
polysaccharide monooxygenase from Thermoascus
aurantiacus
Background
• Lytic polysaccharide monooxygenases (LPMOs) have
emerged as key proteins for depolymerization of cellulose.
These copper-containing enzymes oxidize C-1 and/or C-4
bonds in cellulose, promoting increased hydrolysis of the
oxidized cellulose chains.
• The LPMO from Thermoascus aurantiacus, a thermophilic
ascomycete fungus, has been extensively studied and has
served as a model LPMO.
Fritsche et al. (2020) Biotechnology Letters, doi: 10.1007/s10529-020-02942-w
Approach
• LPMOs are usually expressed in heterologous host, here a
method was developed to purify the LPMO from T. aurantiacus
filtrates along with a cellobiohydrolase and endoglucanase.
• Also, the effect of the LPMO in T. aurantiacus filtrates was
measured by comparing saccharifications with CTec2, which
has an LPMO in a commercial cellulase mixture.
Outcomes and Impacts
• The activity of the purified LPMO was measured with a
colorimetric assay that established the Topt of the native LPMO
at 60℃.
• Quantification of the amounts of cellulases present in the T.
aurantiacus culture filtrate established that the LPMO was the
most abundant protein in the culture supernatants.
• The importance of the LPMO to activity of the mixture compared
to CTec2 was demonstrated by saccharifications with Avicel and
acid-pretreated corn stover.
Purified cellulases from T. aurantiacus
Effect of ascorbate supplementation
on glucose release
Avicel Acid-pretreated
corn stover

5. An iron (II) dependent oxygenase performs the last
missing step of plant lysine catabolism
Background
• Lysine is an essential amino acid, and due to its low abundance in
cereals and legumes it is produced on a scale of one million tons a
year to supplement global food supply needs.
• Engineered plants with increased lysine could be an integral part of
a holistic supply chain for bioenergy crops with multiple end uses.
• Despite intensive study, plant lysine catabolism beyond the 2-
oxoadipate (2OA) intermediate remains unvalidated.
Approach
• Recently JBEI described a missing step in the D-lysine catabolism of
Pseudomonas putida in which 2OA is converted to D-2-
hydroxyglutarate (2HG) via hydroxyglutarate synthase (HglS), a
DUF1338 family protein.
• We obtained the crystal structure of HglS to 1.1 Å resolution in
substrate-free form and in complex with 2OA and use this
information to characterize and engineer variants in planta.
Outcomes and Impacts
• We propose a successive decarboxylation and intramolecular
hydroxylation mechanism forming 2HG in a Fe(II)- and O2-
dependent manner.
• An Arabidopsis thaliana HglS homolog coexpresses with known
lysine catabolism enzymes, and mutants show phenotypes
consistent with disrupted lysine catabolism.
• Results suggest DUF1338-containing enzymes catalyze the same
biochemical reaction, exerting the same physiological function
across bacteria and eukaryotes.
• This knowledge can serve as a guide in engineering plants with
increased lysine content.
Thompson et al. (2020) Nature Communications, doi:10.1038/s41467-020-16815-3
Ribbon diagram of the HglS crystal structure. The left image
shows the active site entrance, containing the metal cofactor
(red sphere) and metal-coordinating residues (orange) located
in the central β-sheet protein domain. A 180°
rotation on the right shows the overall protein structure.
Overlay of holo (green) and substrate-bound (cyan) structures
of HglS displaying the enzyme active site and the interaction
of Val402 and Ser403 with the 2-oxoadipate substrate.

6. Investigation of indigoidine synthetase reveals a
conserved active-site base residue of nonribosomal
peptide synthetase oxidases
Pang et al. (2020) J Am Chem Soc., doi:10.1021/jacs.0c04328
Mechanistic insight into NRPS Ox domains. (a) The key
active-site residue, Tyr 665, of the BpsA Ox domain (yellow) was
determined by structural alignment with McbC (gray), a RiPP
dehydrogenase. The generality of the key Tyr residue was
confirmed by (b) multiple sequence alignment of known NRPS Ox
domains and (c) biochemical assays of EpoB-Ox, the Ox domain in
the PKS− NRPS assembly line for epothilone biosynthesis. (d)
Proposed E2-like mechanism for NRPS Ox domains.
Background
• Nonribosomal peptide synthetases (NRPSs) and some polyketide
synthases (PKSs) are modular enzymes that synthesize secondary
metabolites that can be valuable bioproducts.
• The NRPS oxidase (Ox) domain usually oxidizes thiazoline, derived
from L-cysteine by the cyclization (Cy) domain, to thiazole on the
growing intermediate.
• The lack of a mecha nistic understanding of the NRPS Ox domains,
especially for the defining biochemical active-site residue, has
hindered its engineering.
Approach
• To establish a suitable system for this study, we chose BpsA, the
indigoidine synthetase from Streptomyces lavendulae, which is
readily expressed in several hosts, and reconstituted its activity in
vitro.
• Screened different variants to determine activity, mechanism and
specificity of BpsA.
Outcomes and Impacts
• Results show that the Ox domain utilizes an active-site base
residue, tyrosine 665, to deprotonate a protein-bound L-glutaminyl
residue.
• The revealed mechanism of NRPS Ox domains echoes the unified
paradigm for azole biosynthesis in RiPPs and NRPS peptides.
• These findings not only resolve the biosynthetic pathway mediated
by indigoidine synthetase but enabled mechanistic insight into NRPS
Ox domains to be used in future pathway engineering efforts.

7. Construction of a novel dual-inducible duet-
expression system for gene (over)expression in
Pseudomonas putida
Background
• Pseudomonas putida is a widely used host for metabolic
engineering and synthetic biology. However, the use of P.
putida could be expanded by designing new plasmid vectors
for the expression of heterologous proteins.
Gauttam et al. (2020) Plasmid, doi: 10.1016/j.plasmid.2020.102514
Approach
• To widen the scope of expression vectors for gene co-
expression studies, a previously established dual-inducible
expression vector, pRG_Duet2, developed for Corynebacterium
glutamicum has been modified for use in P. putida.
• This expression vector, named pRGPDuo2, harbors two origins
of replication, ColE1 for replication in E. coli and pRO1600 for
replication in P. putida.
Outcomes and Impacts
• Two multiple cloning sites (MCS1 and MCS2) in pRGPDuo2 are
individually controlled by inducible promoters Ptac or PtetR/tetA.
• Functional validation of pRGPDuo2 was confirmed by the co-
expression of genes for the fluorescent proteins superfolder
green fluorescent protein (sfGFP), and red fluorescent protein
(RFP).
• The expression vector pRGPDuo2 is an attractive addition to the
existing repertoire of expression plasmids for expression
profiling and adds to the tools available for P. putida metabolic
engineering.
Plasmid map of duet vector
RFP and sfGFP activity of duet vectors