JBEI highlights November 2019

1. Genomic and proteomic biases inform metabolic
engineering strategies for anaerobic fungi
Background
• Anaerobic gut fungi (AGF) of phylum Neocallimastiglomycota are
powerful degraders of lignocellulose.
• The genomes of AGF are notoriously repeat rich, which delayed
acquisition of high quality AGF genomes until the advent of long
read sequencing.
• The unusual composition of AGF genomes has hindered
interpretation of genomic information and heterologous expression
of AGF enzymes. Only ~30% of AGF genes are annotatable and
few proteins have been successfully heterologously expressed.
• This study provides strategies for engineering AGF that account for
the atypical compositions of AGF genomes and proteomes.
Approach
• We carried out analyses that compared AGF genomes and
predicted AGF proteomes to other available fungal genomes.
• Our analyses seek to inform engineering efforts including direct
genetic editing, effective generation of genetic variants, and
heterologous expression of valuable AGF enzymes.
Outcomes and Impacts
• The AT richness of AGF genomes make common gene editing
approaches challenging, but we suggest that recent advances in
alternative T-rich recognition sites provide a path forward.
• Non-standard codon usage and an abundance of homopolymeric
amino acid runs in AGF enzymes currently present challenges for
heterologous expression. To address this, we provide an AGF
codon usage table and we implicate homopolymeric amino acid
runs in glycosylation of valuable AGF enzymes.
Wilken et al. (2019) ACS Metabolic Engineering Communications, doi: 10.1016/j.mec.2019.e00107
Among all fungi, the predicted proteomes of AGF are most similar to those of
Cryptomycota, Glomeromycota, and Microsporidia. In contrast to these
neighbor phyla, predicted AGF proteomes are highly enriched in asparagine.
Anaerobic gut fungi have the most GC depleted coding genomes of all fungi,
complicating efforts to engineer AGF and their enzymes.

2. Function-driven single-cell genomics uncovers
cellulose-degrading bacteria from the rare biosphere
Background
• Assigning a functional role to a microorganism has historically
relied on cultivation of isolates or detection of environmental
genome-based biomarkers using a posteriori knowledge of
function. However, the emerging field of function-driven single-cell
genomics aims to expand this paradigm by identifying and
capturing individual microbes based on their in situ functions or
traits.
Approach
• We developed and benchmarked a function-driven single-cell
screen for cellulase activity in uncultivated microbes, which we
applied to a microbial community inhabiting the Great Boiling
Spring (GBS) Geothermal Field, northwest Nevada.
Outcomes and Impacts
• Single bacterial cells that bound to the fluorescent cellulose probe
were sorted and characterized by 16S rRNA and single cell
sequencing.
• Putative cellulases were identified by bioinformatics and screened
using an in vitro assay and NIMS detection.
• Along with well-characterized phyla, divergent cellulases encoded
in the genome of a representative of a recently described
candidate phylum from the rare biosphere, Goldbacteria, were
identified and characterized.
• We expect that this function-driven single-cell approach can be
extended to a broad range of substrates, linking microbial
taxonomy directly to in situ function..
Doud et al. (2019) ISME J, doi:10.1038/s41396-019-0557-y

3. Evaluating protic ionic liquid for woody biomass
one-pot pretreatment + saccharification, followed
by Rhodosporidium toruloides cultivation
Background
• One-pot ionic liquid-based processes are a promising
bioproduction strategy to reduce water consumption (removing the
washing of biomass), reducing costs and minimizing the presence
of inhibitory compounds.
Approach
• We evaluated the one-pot ionic liquid pretreatment +
saccharification configuration as a scheme for the deconstruction
and conversion of two different woody feedstocks, eucalyptus, and
pine.
• A comparative study of protic ionic liquids 2-
hydroxylethylammonium acetate-based ionic liquids, bis-2-
hydroxyethyl ammonium acetate ([2-HEA][OAc]), and cholinium
lysinate ([Ch][Lys]) for pretreatment of eucalyptus and pine was
conducted.
Outcomes and Impacts
• Protic ionic liquids were more effective in eucalyptus than in pine
pretreatment and less toxic than cholinium lysinate at
concentrations ≤10 % w/w.
• 2-hydroxylethylammonium acetate yielded the highest digestibility,
of up to 75% in eucalyptus.
• This work is a starting point for further studies aimed at increasing
cellulose digestibility in a one-pot configuration in the presence of
protic ILs.
Rigual et al. (2019) ACS Sustainable Chem. Eng., doi:10.1021/acssuschemeng.9b04451
Effect of pH adjustment on glucose and xylose
consumption over the course of growth and production
using R. toruloides in the presence of IL [2-HEA][OAc].

4. Omics-driven identification and elimination of
valerolactam catabolism in Pseudomonas putida
KT2440 for increased product titer
Background
• Pseudomonas putida is a promising host for metabolic engineering
due to its diverse catabolic range allowing for the valorization of
lignin derivatives.
• P. putida is also able to degrade or catabolize multiple lactams,
important precursors to polymers such as nylon. This catabolism
dramatically impacts titers in engineered strains.
Approach
• We utilized a combination of Random-Barcode Transposon
Sequencing (RB-TnSeq), as well as shotgun proteomics to identify
enzymes that hydrolyze lactams in P. putida. We then used this
information to engineer more productive strains of lactam
producing strains of P. putida.
Outcomes and Impacts
• RB-TnSeq validated that valerolactam is metabolized via the L-
lysine catabolic pathway.
• Shotgun proteomics identified that OplBA is likely responsible for
lactam hydrolysis.
• Knocking out oplBA in P. putida prevented the bacterium from
growing on valerolactam, as well as preventing caprolactam
hydrolysis in vivo.
• Knocking out oplBA and other loci in P. putida increased
valerolactam titers from 0 mg/L to ~90 mg/L after 48-hour
fermentations with added L-lysine.
Thompson et al. (2019) Metab Eng Commun. doi: 10.1016/j.mec.2019.e00098
Using a combination of RB-TnSeq and shotgun proteomics a
lactam hydrolase was identified in P. putida (A). By eliminating
the hydrolase and other pathways that compete for precursors
valerolactam titer was significantly increased (B).

5. Genome sequence of the model rice variety
KitaakeX
Background
• Rice (Oryza sativa) provides food for more than half of the world’s
population and also serves as a model for studies of biofuel crops such
as sorghum and switchgrass
• The Kitaake cultivar (ssp. japonica), which originated at the northern
limit of rice cultivation in Hokkaido, Japan, has emerged as a model for
rice research. It has a rapid life cycle (9 weeks seed to seed) and is
easy to transform and propagate
• KitaakeX, a Kitaake variety carrying the Xa21 immune receptor gene
Jain et al. (2019) BMC Genomics, doi:10.1186/s12864-019-6262-4
Genome wide analysis of KitaakeX genome and its
comparison with other rice varieties
Approach
• To obtain a high-quality, de novo genome assembly, we sequenced the
KitaakeX genome using a strategy that combines short-read and long-read
sequencing. Sequencing reads were collected using Illumina, 10x
Genomics, PACBIO, and Sanger platforms at the Joint Genome Institute
(JGI) and the HudsonAlpha Institute
Outcomes
• The assembled sequence contains 377.6 Mb, consisting of 33 scaffolds
with a contig N50 of 1.4 Mb, covering a total of 99.67% of assembled
bases in chromosomes
• We predicted 35,594 protein-coding genes in the KitaakeX genome
representing 31.5% genic space of the assembled genome size.
• We found 331,335 variations between KitaakeX and Nipponbare and
nearly 10 times as many (2,785,991) variations between KitaakeX and
Zhenshan97
Significance
• The high quality, de novo assembly of the KitaakeX genome will serve as a
useful reference genome for rice and will accelerate functional genomics
studies of rice and other monocotyledonous species such as sorghum.
The early flowering rice variety KitaakeX

6. Stochastic economic and environmental
footprints of biodiesel production
from Jatropha curcas Linnaeus in Nepal
Background
• Despite some initial failures, the required growing conditions for
commercial success of Jatropha have now identified, which lead to a
renewed commercial interest and necessitate a new and updated
analysis.
• This study considers a novel set of essential parameters specifics to
geography, climate, soil conditions, and irrigation to determine
commercially feasible land for Jatropha farming and documented a
system level economic and environmental impacts analyses.
Approach
• We developed stochastic process models and quantified selling price
and carbon footprint.
• We determined suite of avenues to address the current global failure
of Jatropha-based biodiesel plant.
Outcomes and Impacts
• A seed yield of >3.9 t/ha and a high oil content Jatropha variety (oil
yield of >50 wt%) are required to achieve the selling price of biodiesel
close to the current local price of the conventional diesel of $1/L.
• Including the impacts from direct and indirect land use changes, the
carbon footprint could reach below the conventional fuel equivalent by
achieving a very high seed yield (>5 t/ha), using only marginal lands,
and encouraging aggressive afforestation.
• Results indicate the pathways and sensitivities for developing policies
to enable the production of sustainable biodiesel from Jatropha.
Baral et al. (2019) Renew. Sustain. Energy Rev., doi: 10.1016/j.rser.2019.109619
Minimum selling price of biodiesel
Greenhouse gas emissions
including direct land use changes