Research summaries for papers published by the Joint BioEnergy Institute in December 2021

1. Diversifying isoprenoid platforms via atypical carbon
substrates and non-model microorganisms
Background
• Isoprenoid production has been largely successful in E. coli and S.
cerevisiae with metabolic engineering of the mevalonate (MVA) and
methylerythritol phosphate (MEP) pathways coupled with the
expression of heterologous terpene synthases.
• Conventional microbial chassis pose several major obstacles to
successful commercialization including the affordability of sugar
substrates at scale, precursor flux limitations, and intermediate
feedback-inhibition.
Approach
• Recent studies have challenged typical isoprenoid paradigms by
expanding the boundaries of terpene biosynthesis and using non-
model organisms including those metabolizing C1 substrates.
• We highlights the advances in isoprenoid biosynthesis with specific
focus on the synergy between model and non-model organisms
that may elevate the commercial viability of isoprenoid platforms by
addressing the dichotomy between high titer production and
inexpensive substrates.
Outcomes and Impacts
• Atypical carbon sources and non-model organisms harbor
metabolic advantages that could be harnessed to reduce substrate
costs and the associated emissions of bioproduction.
• Co-substrate utilization by certain organisms as in the case of R.
toruloides and P. putida has the potential to unlock lignocellulosic
biomass and many methylotrophs could tap into inexpensive and
highly abundant substrates.
• Systems engineering strategies are of particular interest for C1
metabolism. The translation of successful whole systems
engineering strategies from E. coli and S. cerevisiae to non-model
organisms will prove useful in further optimization.
Carruthers and Lee (2021) Frontiers Microbiology, doi: 10.3389/fmicb.2021.791089
Figure 1. A depiction of isoprenoid synthesis through the core 6
enzyme MVA and 7 enzyme MEP pathways. Also depicted are
the newly discovered archaeal branches from the MVA
pathway. The thermoarchaeal-type branch begins with
mevalonic acid whereas the archaeal and haloarchaeal-type
branches stem from MVAP.

2. Managing soil organic carbon for climate change
mitigation and food security
Background
• Soil organic carbon (SOC) determines the sustainability and
resilience of agroecosystems under changing climate
• Soil functions, such as climate change mitigation and food
production, are regulated by different properties of individual
SOC pools
Approach
• We review the literature and provide empirical evidence on the
functions of different SOC fractions, and their management
strategies to mitigate climate change impacts and ensure food
security
Outcomes and Impacts
• A new input of organic carbon is partially decomposed by soil
microbes and released back into the atmosphere, while some
C can be stored in the soil for decades to millennia. The
residence time of SOC is mainly determined by biochemical
recalcitrance, chemical stabilization, and physical protection.
• Conservation tillage sequestered more SOC and total nitrogen
in the soil than conventional tillage.
• Agroforestry and organic amendments increase crop
production through supplying more nutrients by increasing the
dissolved organic matter and free particulate organic matter
pools.
• Biochar showed the most favorable effect on soil C
sequestration and pore formation with its inherent porous
structure and recalcitrance, as well as its interaction with soil
particles and aggregates.
Kim et al. (2021). Managing Soil Organic Carbon for Climate Change Mitigation and Food
Security, doi: 10.1201/9781003243090-2.
Figure 1. Relations of soil organic carbon pools with soil
functions
Figure 2. Response ratios of soil organic amendments to no
addition for soil organic carbon (SOC), total nitrogen (TN), total
soil porosity (Porosity), and crop yield (Yield)

3. Lignin p-Hydroxybenzoate Decoration Regulates
Gravitropic Response of Poplar
Background
• Angiosperm woody species display gravitropic response in respect to
gravity and produce tension wood.
• Despite the long history of recognition of p-hydroxybenzoate
decoration in cell wall lignin, our understandings on its biological
significance remain largely elusive.
• This study explores physiological effects of lignin p-
hydroxybenzoylation on gravitropic response of poplar.
Approach
• Lignin p-hydroxybenzoate deficient and hyperaccumulating poplar
transgenic lines were treated with mechanical bending and/or
gravistimulation.
• The autotropism and gravitropism behaviors of the treated plantlets
were observed and recorded.
• The expression of secondary cell wall biosynthetic genes and the
alteration in cell wall composition were determined in the generated
tension wood, opposite wood and normal wood of the plants.
Outcomes and Impacts
• Mechanical bending or gravistimulation significantly enhances the
expression of PHBMT1 and the accumulation of p-hydroxybenzoates
in tension wood.
• Paradoxically, hyperaccumulation of p-hydroxybenzoates mitigates
gravitropism and/or enhances autotropism of the plants. Thus, lignin
p-hydroxybenzoylation negatively coordinates with the action of
tension wood cellulose fibers to control poplar wood deformation and
plant growth.
Zhao et al. (2021) Front Plant Sci 12, 755576, doi: 10.3389/fpls.2021.755576
The larger secant bending angle and more obvious
upward curving at the basal internodes were observed in
the PHBMT1 knockout plants, where the lignin-bound
pBAs are eliminated; by contrast, when pBAs were
hyper-accumulated in the stem cell walls of the PHBMT1
OE plants, smaller secant bending angle exhibited and
the stem upward curving occurred at the apical
internodes. The PHBMT1-mediated monolignol p-
hydroxybenzoylation and the subsequent accumulation
of lignin-bound pBAs play observable roles in the
regulation of poplar gravitropic response.
WT g1-8 g1-9 OE1 OE2
WT g1-8 g1-9 OE1 OE2
0.0
0.5
1.0
1.5
2.0
Top
length/
bottom
length
**
*
WT g1-8 g1-9 OE1 OE2
0.0
0.5
1.0
1.5
2.0
Relative
secant
bending
angle
**
*

4. Prediction of Solubility Parameters for Lignin and Ionic
Liquids Using Multi-resolution Simulation Approaches
Background
• The solubility parameters of a molecular species is a vital
feature that evaluates polarity and quantifies the ‘like-seeks-
like' principle.
• The performance of Hansen solubility parameters (HSP) has
previously been demonstrated for lignin solubility using different
solvent systems.
Approach
• The objective of this study is to evaluate the Hansen solubility
parameters of lignin, ILs, and DESs using multi-resolution
simulation approaches.
Outcomes and Impacts
• The SPs of lignin were obtained in the range of 23‒27 MPa1/2
,
which is close to the polymeric lignin solubility parameters.
• The correlation between experimental lignin dissolution in ILs
and DESs and predicted REDs of lignin had shown an
excellent agreement.
• The SPs of ILs namely [Ch][Lys], [Ch][Oct], and [Emim][Lys]
were predicted to be ~26 MPa1/2, which is close to lignin’s SPs
and resulted in increased biomass delignification.
• The molecular dynamics simulated SPs were validated by both
the COSMO-RS model and experimental investigations, with
the results showing a close agreement between predicted and
experimental solubility parameters.
• The predicted solubility parameters were applicable for
hardwood and grassy-type biomass, whereas the softwood
biomass has a different set of SPs for lignin
Mohan et al. (2021) Green Chemistry, https://doi.org/10.1039/D1GC03798F
Biomass
Type
Ionic Liquid
Lignin
removal (%)
RED Reference
Kraft lignin [Ch][For] 28.3 1.04
Hou et al.
(2015)
[Ch][Ace] 31 0.83
[Ch][But] 32.5 0.59
[Ch][Hex] 37.2 0.41
[Ch][Oct] 39.5 0.28
Sorghum
(grass)
[Ch][Ace] 45 0.83
Yao et al.
(2021)
[Ch][Oct] 51.86 0.28
[Ch][Lys] 77.41 0.34
Switchgrass
(grass)
[Emim][Ace] 16.5, 48.9 0.91
Sun et al.
(2014)
[Ch][Ace] 17.0, 50.2 0.83
[Ch][Lys] 69.3, 85.1 0.34
[Emim][Lys] 80.3, 86.6 0.28
Switchgrass
(grass)
[Ch][Lys] 74 0.34
Dutta et al.
(2018)
Eucalyptus
(hardwood)
[Ch][Lys] 70 0.34
Dutta et al.
(2018)
Pine
(softwood)
[Ch][Lys] 20 -
Dutta et al.
(2018)
Table : Correlation between RED values of solvent (ILs)−lignin
interaction and experimental lignin solubility or biomass
delignification

5. Genomics Characterization of an Engineered
Corynebacterium glutamicum in Bioreactor Cultivation
Under Ionic Liquid Stress
Background
• Ionic liquids (ILs) are potent reagents that can extract sugars from
renewable carbon streams for downstream bioconversion processes.
One emerging ionic liquid with favorable properties is cholinium lysinate
([Ch][Lys]).
• C. glutamicum shows a subtle growth defect at high concentrations of
[Ch][Lys] which suggested potential changes to its physiology in
response; a fundamental understanding of this microbe’s cellular
response to [Ch][Lys] stirred tank bioreactors during bioproduction could
inform future production strategies.
Approach
• We generated a new PacBio genome assembly of an engineered
isoprenol producing C. glutamicum strain BRC-JBEI 1.1.2 using whole-
genome sequencing.
• Differential gene expression profiling during isoprenol production in a
fed-batch bioreactor revealed the cellular response in the presence and
absence of [Ch][Lys].
Outcomes and Impacts
• This analysis identified genetic differences in C. glutamicum BRC-JBEI
1.1.2 isolate compared to the reference C. glutamicum genome.
• Transcriptomics analysis implicated ectP, a BCCT family transporter
similar to the E. coli betT and P. putida betT-III, as a transporter for
[Ch][Lys]; ectP was overexpressed in the presence of [Ch][Lys].
• This transcriptomics analysis of an isoprenol producing C. glutamicum
strain proposes several actionable targets including gltA, brnF for future
DBTL cycles that may lead to improved isoprenol production.
Banerjee et al. (2021) Front. Bioeng. Biotechnol., doi: 10.3389/fbioe.2021.766674
(A) Glucose and organic acids profiles in the 2-L stirred tank
bioreactor cultivation in the presence of 50 mM [Ch][Lys].
(B) Venn diagrams indicating the number of upregulated (left)
and downregulated (right) DEGs in response to [Ch][Lys]. (C)
Differential transcript profiles of C. glutamicum BRC-JBEI 1.1.2
under three discrete conditions, during scale transition (ST),
bioreactor cultivation in the absence of IL (BR) and presence of
IL (IL).
A B
C

6. Comparative genomics reveals a novel gene whose
product is involved in cyclization of a polyketide
Background
• Fungal genome sequencing has identified a tremendous
diversity of secondary metabolite gene clusters encoding a
huge breadth of biochemical reactions
• This study is the first functional study of a snoaL domain
cyclase (otaY) that is essential for the production of a
fungal polyketide, ochratoxin A, produced by Aspergillus
carbonarius
Approach
• A gene deletion approach was utilized to show the
essentiality of a polyketide cyclase for production of a
known polyketide
Outcomes and Impacts
• The cyclization process of the heterocyclic structure of
OTβ, in the first steps of OTA biosynthesis pathway had not
previously been associated with an enzyme
• Complete deletion of otaY gene demonstrated the inability
of ΔotaY mutant to synthesize the OTA molecule
• Defining the biochemical reactions performed by individual
enzymes in secondary metabolite biosynthetic pathways is
critical for growing the enzymatic “parts list” that may be
used to create novel pathways and compounds
Ferrara et al, Toxins 2021, 13(12), 892; https://doi.org/10.3390/toxins13120892
UPLC-FLD chromatograms of OTA standard
solution (1018 ng/g) (blue trace), extract of A.
carbonarius wild-type strain ITEM 5010 (6467
ng/g) (black trace), and extract of A.
carbonarius ∆otaY deletion mutant strain
AC2021 (red trace).