Research summaries for papers published by the Joint BioEnergy Institute in March 2022

1. Office of Biological and Environmental Research
Scale-up of the ionic liquid based biomass conversion
processes
Background/Objective
• Significant progress has been made with ionic liquid (IL) pretreatment due to their unique solvation properties, disrupting the
intermolecular forces that hold biomass biopolymers together and superior performance compared to other types of pretreatment
processes.
• One aspect that remains to be addressed is the scale-up of these ionic liquid-based processes
Approach
• This review paper discusses the different combinations of biomass
feedstocks and type of ionic liquid and their performance as a function
of scale
Results
• ILs are a promising pretreatment technology with advantages including
feedstock agnostic, efficient sugar conversion, fast kinetics, and
consolidated process configuration.
Significance/Impacts
• These tailored solvents have the potential to be a game changing
technology for the bioenergy enterprise.
Papa G., Simmons B.A., Sun N. (2022) “Scale-Up of the Ionic Liquid-Based Biomass Conversion Processes.” In: Zhang S. (eds) Encyclopedia of Ionic Liquids. Springer, Singapore. doi: 10.1007/978-981-10-6739-6_49-1

2. Office of Biological and Environmental Research
Applications of targeted proteomics in metabolic
engineering: advances and opportunities
Background/Objective
• Optimization of metabolically engineered organisms requires good
understanding of producing balanced level of pathway proteins.
• Targeted proteomics via selected-reaction monitoring (SRM) has been
increasingly used in metabolic engineering research to detect and quantify
sets of proteins with high selectivity and multiplexity.
Approach
• In this review, we present recent applications of targeted proteomics in
metabolic engineering research and highlight several successful studies of
targeted proteomics in production of high value chemicals.
• We also discuss challenges and limitations of current targeted proteomics
and map opportunities for future research.
Results
• Targeted proteomics has been a useful tool in metabolic engineering to
bring a more predictable and rational engineering of biology.
Significance/Impacts
• Integration of high quality and accuracy protein expression data input from targeted proteomics and other omics tools to machine learning
will become an avenue of interest in many metabolic engineering studies.
Yunus I.S., Lee, T.S. (2022) Current Opinion in Biotechnology. doi: 10.1016/j.copbio.2022.102709

3. Office of Biological and Environmental Research
Scalable and automated CRISPR-based strain
engineering using droplet microfluidics
Background/Objective
• CRISPR-MAGE allows one to make many simultaneous changes to an
organism’s chromosome across a population of cells, but this method is difficult
to use for high-throughput efforts.
• Droplet microfluidics has become an attractive option for performing such large
scale synthetic biology experiments.
Approach
• We developed a droplet microfluidic system that miniaturizes and automates
CRISPR-MAGE. The system can carry out up to 100 genetic modification
reactions in parallel.
Results
• The system proved to be effective in two test cases: (1) to successfully disrupt
the function of an enzyme found in E. coli, and (2) to modify E. coli to produce
indigoidine, a sustainable alternative to traditional indigo.
Significance/Impacts
• This is a scalable platform for producing large numbers of engineered strains,
which are needed to optimize genetic pathways and perform predictable
bioengineering.
Iwai, K., et al. (2022) Microsyst Nanoeng, doi: 10.1038/s41378-022-00357-3
CRISPR-MAGE steps. The ones inside the box are performed on the chip. Cells are removed from the chip
after the recovery step for induction and plating. b The microfluidic chip in a 3D printed holder (left), the
electrode pattern (right), a top-view of an individual well, and a side-view schematic of a well. The chip is
designed to contain 100 discrete reaction chambers with individually addressable electrodes for multiplexed
CRISPR-MAGE recombineering, and its 384-well format design can be interfaced with lab automation
equipment. c Droplets containing plasmids and cells are dispensed into each chamber through the inlet port,
mixed by electrowetting, and electroporated by applying a voltage pulse

4. Office of Biological and Environmental Research
Continental United States may lose 1.8 petagrams of soil organic
carbon under climate change by 2100
Background/Objective
• High-resolution information on soils’ vulnerability to climate-induced soil organic
carbon (SOC) loss can enable environmental scientists, land managers, and policy
makers to develop targeted mitigation strategies
Approach
• We used recent SOC field observations, environmental factors, and an ensemble
machine learning (ML) approach to estimate baseline SOC stocks in surface soils
across the continental United States at 100-m spatial resolution, and decadal changes
under the projected climate scenarios of CMIP6 earth system models (ESMs).
Results
• Baseline SOC projections from ML approaches captured more than 50% of
variability in SOC observations, whereas ESMs represented only 6–16% of
observed SOC variability (Figure 1).
• Both ML and ESM predictions agree on the direction of SOC change (net emissions
or sequestration) across 46–51% of continental US land area (Figure 2).
Significance/Impacts
• ML estimates showed a mean total loss of 1.8 Pg C from US surface soils under
the high-emission scenario by 2100, whereas ESMs showed no significant
change in SOC stocks with wide variation among ESMs.
Gautam et al. (2022) Global Ecology & Biogeography, https://doi.org/10.1111/geb.13489
Figure 1
Figure 2

5. Office of Biological and Environmental Research
Lignin synthesis and bioengineering approaches toward
lignin modification
Background/Objective
• Lignin plays important roles in plants, but often represents
hurdles to the utilization of cellulosic biomass for bioenergy.
• Understanding lignin synthesis is a prerequisite to
manipulate its content and composition in crops.
Approach
• The recent literature on lignin biosynthesis and engineering
has been reviewed.
Results
• With the advance of synthetic biology, it has become feasible to
fine-tune lignin content and composition by tailoring gene
expression, exploiting enzyme post-translational modifications,
altering enzyme cofactors, and rerouting lignin metabolic
precursors.
Significance/Impacts
• Novel bioengineering strategies can be implemented in bioenergy
crops to facilitate biomass conversion into bioproducts.
Liu C.J., Eudes A. (2022) “Lignin synthesis and bioengineering approaches toward lignin modification”
Book Chapter in Advances in Botanical Research, Lignin and hydroxycinnamic acids: biosynthesis and the buildup of the
cell wall. doi: 10.1016/bs.abr.2022.02.002
Figure 1: Current view of the lignin
biosynthetic pathway. At least thirteen
enzymes have been involved in
monolignol synthesis.
Figure 2: Chemical structures of lignin acyl groups (pCA: p-coumarate; HBA:
4-hydroxybenzoate) and potential alternative cleavable lignin monomers.

6. Office of Biological and Environmental Research
Nitrogen metabolism in Pseudomonas putida: functional
analysis using RB-TnSeq
Background/Objective
• Pseudomonas putida is an attractive host for the valorization of biomass hydrolysates
• Many nitrogenous target bioproducts, such as lactams and amino acids, can serve as a
source of nitrogen for P. putida
• To efficiently engineer the host, an extensive knowledge of its metabolism is required
Approach
• Using pooled mutant fitness assays, this study identifies genes involved in the
assimilation of 52 different nitrogen containing compounds. Further validation
experiments include biochemical enzyme characterization, biosensor development, and
mutant growth assays
Results
• We provide evidence for known nitrogen assimilation pathways and their
regulatory systems, and assign functions to genes whose exact roles in nitrogen
metabolism were not previously known
Significance/Impacts
• The presented functional genomics data is contextualized within the literature spanning
decades of research about nitrogen metabolisms.
• This work underlines the utility of functional genomics in the context of metabolic
engineering
Schmidt and Pearson et al. (2022) Applied and Environmental Microbiology doi: 10.1128/aem.02430-21
Overview of RB-TnSeq and data processing workflow.
Competitive growth assays using a pooled and barcoded
transposon mutant library are conducted under different nitrogen
conditions. Fitness values are calculated from barcode frequencies
and clustered using the manifold learning method t-SNE