Development of a High Throughput Platform for Screening Glycoside Hydrolases based on Oxime-NIMS Background• Development of high performance, process compatible,

glycoside hydrolases enzymes (GHs) are critical to costeffective biomass to biofuels and/or bioproducts processes.

• High throughput assays are critical to the cocktaildevelopment process given the diversity of plant feedstocksand potential GH enzymes which are predicted to have150,000 unique domain arrangements.

Approach and Outcomes• JBEI has pioneered approach integrating oxime based

bioconjugate chemistries, acoustic printing andnanostructure-initiator mass spectrometry (Oxime-NIMS) forquantitative kinetic analysis glycan resulting from GHreactions including with heterogenous biomass substrates.

• Defined a standard panel of twelve substrates spanning thetypes of glycosidic linkages found in plant cell walls areincluded in the experimental workflow.

• Integrated this approach with automatic biomass handling,enzyme reactions with oxime-NIMS analysis.

• To test the application of this platform and substrate panel, wecollaborated with GLBRC to study the reactivity of threeengineered cellulases and their synergy of combinationacross a range of reaction conditions and enzymeconcentrations.

Significance• large-scale screening using the standardized platform and substrates will generate critical datasets

to enable direct comparison of enzyme activities for cocktail design.

Deng et al. (2015) "Development of a High Throughput Platform for Screening Glycoside Hydrolases Based on Oxime‐NIMS”, Frontiers in Bioengineering and Biotechnology, DOI: 10.3389/fbioe.2015.00153

Standardized substrates list

Workflow of oxime‐NIMS automation

Precursor-Directed Combinatorial Biosynthesis of Cinnamoyl, Dihydrocinnamoyl, and Benzoyl Anthranilates in Saccharomyces cerevisiaeBackground• Biological synthesis of pharmaceuticals and biochemicals

offers an environmentally friendly alternative toconventional chemical synthesis.

• Cinnamoyl, dihydrocinnamoyl, and benzoyl anthranilatesare natural metabolites which possess beneficial activitiesfor human health.

• By exploiting the substrate flexibility of 4-coumarate-CoA ligase (4CL) and hydroxycinnamoyl/benzoyl-CoA:anthranilate N-hydroxycinnamoyl/benzoyltransferase(HCBT), we achieved rapid biosynthesis of more than 160 cinnamoyl, dihydrocinnamoyl, and benzoyl anthranilates in yeast upon feeding with both natural and non-natural cinnamates, dihydrocinnamates, benzoates, and anthranilates.

Approach and Outcomes• 4CL and HCBT were co-expressed in yeast

Saccharomyces cerevisiae.• Analysis of the culture medium of the recombinant 4CL-

HCBT yeast using LC-MS demonstrated the synthesis ofa wide variety of cinnamoyl, dihydrocinnamoyl, andbenzoyl anthranilates.

Significance• The substrate promiscuity of 4CL and HCBT can be exploited for

the production of novel pharmaceuticals in yeast and for the optimization of plant biomass for co-products.

Eudes, A., Teixeira Benites, V., Wang, G., Baidoo, E. E., Lee, T. S., Keasling, J. D., & Loque, D. (2015). "Precursor‐Directed Combinatorial Biosynthesis of Cinnamoyl, Dihydrocinnamoyl, and Benzoyl Anthranilates in Saccharomyces cerevisiae". PLoS One, 10(10), e0138972. 

HCBT (enzyme 2)

4CL(enzyme 1) +

CinnamatesBenzoates

Dihydrocinnamates(supplied to the culture medium)

Cinnamoyl-CoAsBenzoyl-CoAs

Dihydrocinnamoyl-CoAs

Anthranilates(supplied to the culture medium)

Cinnamoyl-anthranilates Benzoyl-anthranilates Dihydrocinnamoyl-anthranilates

Series of valuable metabolites produced in Saccharomyces cerevisiae(R = H, OH, OCH3, CH3, CF3, OCHF2, Br, Cl, or F)

Lignin-modifying processes in therhizosphere of arid land grasses.

Outcomes• We identified 127 unique putative genes with potential biomass-degrading activity.• After further elongation, we found five extended assembly products potentially

representing novel lignin-modifying enzymes; incuding a Basidiomycetes Class II lignin peroxidase, two laccases, a ferroxidase, and a copper-dependent LPMO.

Background• We conducted a metatranscriptomic study of

a high lignin-turnover environment to identify potentially novel lignolysis genes.

Approach• In collaboration with the Joint Genomics

Institute, we developed a bioinformatics strategy for identifying potentially novel lignolysis genes.

• This strategy 1) assembles nearly complete genes from metatranscriptomic reads, 2) searches the translated assembled genes against a database of lignolysis proteins, 3) places the potentially lignolytic gene matches on gene trees to determine novelty, and 4) uses a sequence-threading approach to model the protein structure of potentially novel genes and determine their feasibility.

Significance• Mining natural habitats with high

lignin turnover can provide insights that can be leveraged for rational design of new molecules to overcome current technological bottlenecks.

Hudson, C. M. et al. (2015) “"Lignin‐modifying processes in the rhizosphere ofarid land grasses”, Environ Microbiol, doi: 10.1111/1462‐2920.13020

Converting Sugars to Biofuels: Ethanol and Beyond

Kang, A.; Lee, T.S. (2015) “Converting Sugars to Biofuels: Ethanol and Beyond”, Bioengineering, 2, 184‐203.

Background• To date, the most significant sources of

biofuels are starch- or sugarcane-based ethanol. However, the ultimate goal of biofuel production is to produce fuels from lignocellulosic biomass (LCB)-derived sugars with optimal fuel properties and compatibility with the existing fuel distribution infrastructure

Significance and perspectives• In this review, we provided a brief summary of

recent efforts on cellulosic ethanol production and an overview of current metabolic engineering efforts to improve the productivity and the yield of several key advanced biofuel molecules including fatty acids-derived fuels and isoprenoid-derived fuels.

• Strategies used in these metabolic engineering efforts were discussed and summarized as follows: (1) identification of better enzymes; (2) flux control of intermediates and precursors; (3) elimination of competing pathways; (4) redox balance and cofactor regeneration; (5) bypassing regulatory mechanisms; and (6) improvement in host sugar utilization and robustness

• Most recent progress in advanced biofuels production from LCB-derived sugars or hydrolysates were highlighted.

Host strains and pathways

BiofuelsSugarsBiomass

Identification and resolution of microdiversity through metagenomic sequencing of parallel consortiaBackground• Although metagenomic sequencing has enabled the de novo reconstruction of some

microbial genomes from environmental communities, microdiversity (closely-related organisms exhibit different metabolic activities and occupy distinct niches) confounds current genome reconstruction techniques.

• The metagenomes of parallel microbial consortia, defined as consortia cultivated under the same conditions from the same natural community with overlapping species composition, was sequenced to identify the microdiversities among the community members.

Outcomes• 17 and 20 near-complete genomes were reconstructed using MaxBin 2.0 from the

parallel consortia by taking advantage of the abundance differences between the two microbial communities.

• The genomes of Halomonas, Rhodobacteraceae, and Rhizobiales populations were reconstructed. The comparison within each population suggests differences in functional potential that may result in distinct roles in the community.

• Experiments on two closely related Halomonas spp., shows that only one of the species can grow using glucuronate as sole carbon source while the other cannot, confirming the observation in microdiversity within this population.

Significance• Parallel consortia allows microdiversity comparison among closely related species.• The recovery of genomes with different functional potential can be served as a great

source for future applications.

Nelson et al. (2015) “Identification and resolution of microdiversity through metagenomic sequencing of parallel consortia”, Applied and Environmental Microbiology, doi: 10.1128/AEM.02274‐15.

Phylogenetic trees of the reconstructed genomes relative to model organisms.

MaxBin 2.0: an automated binning algorithm to recover genomes from multiple metagenomic datasetsBackground• Recovering population genomes from metagenomic samples remains a

difficult task since sequences of all microbial populations are sampled simultaneously; however it is a critical procedure to understand the functional potential of the uncultivated microbes in natural and engineered ecosystems.

• Automating the binning process provides a high-throughput method to recover microbial genomes from metagenomes.

Outcomes• MaxBin 2.0, the next generation of the MaxBin algorithm that recovers

genomes from co-assembly of multiple metagenomic samples.• In comparison to other binning algorithms that utilize multiple

metagenomic datasets, MaxBin 2.0 has the highest accuracy in recovering genomes from simulated metagenomes.

• The ability of MaxBin 2.0 to measure the coverage levels of the genome bins also allows comparisons of the genome-resolved microbial community composition across multiple samples.

Significance• JBEI has developed and deployed MaxBin 2.0, a very accurate genome recovery software, to better analyze underlying

microbial populations in various natural or man-made environments.• Comparisons of the microbial populations among different environments can be easily achieved using MaxBin 2.0, which not

only recovers microbial genomes but also estimates their abundances in different environment.

MaxBin 2.0 is the most accurate tool amongst all genome recovery tools.

Wu et al. (2015) “MaxBin 2.0: an automated binning algorithm to recover genomes from multiple metagenomic datasets”, Bioinformatics, doi: 10.1093/bioinformatics/btv638.