Plastid-produced interorgannellar stress signal MEcPPpotentiates induction of the unfolded proteinresponse in endoplasmic reticulum

Outcomes• Elevated levels of MEcPP induce UPR genes.• MEcPP produced in the chloroplast acts as an interorgannellar communication signal that transmits information to prime the UPR machinery,

ensuring the cell is able to handle ER stress resulting from adverse conditions.

Schematic model depicting alternative routes by which MEcPP potentiates induction of selected UPR genes.

Background• Eukaryotic cellular homeostasis in response

to intracellular and environmental stimulirequires a coordinated interorgannellarcommunication network.

• Consequently, endoplasmic reticulum (ER)homeostasis is perturbed by unfolded proteinresponse (UPR) genes, which monitor ERprotein-folding capacity and communicate theER status to gene expression programs thatup-regulate genes encoding components ofthe protein folding machinery or the ER-associated degradation system.

• In this study, methyl erythritolcyclodiphosphate (MEcPP), a novel stress-specific retrograde signaling metabolite (1), isshown to potentiate the induction of the UPRin ER.

Significance• These findings provide a link between a plastidial retrograde signal and transcriptional reprogramming of ER genes

critical for readjustment of protein-folding capacity in stressed cell.

Xiao, et al. (2015). “Retrograde signaling by the plastidial metabolite MEcPP regulates expression of nuclear stress-response genes.” Cell, 149:1525-1535.

Approach• To demonstrate, using a combination of

transcriptomics, proteomics and metaboliteprofiling approaches, that mutant plants(ceh1) with high endogenous MEcPP levelsdisplay increased transcript and proteinlevels for a subset of core UPR genes.

Isopentenol fermentation from pretreated mixed feedstocks

Outcomes• It is shown that ionic liquid pretreatment liberates the most sugar during enzymatic saccharification when compared with

dilute sulfuric acid and soaking in aqueous ammonia pretreatment. • Isopentenol production from the IL pretreated biomass was significantly higher than those for SAA and DA treatments.

These results indicate that IL pretreatment with [C2mim][OAc] is a promising conversion technology for the conversion of mixed and pelleted feedstocks.

Shi, J. et al. (2015). “Impact of pretreatment technologies on saccharification and isopentenol fermentation of mixed lignocellulosic feedstocks.” BioEnergy Research, DOI: 10.1007/s12155-015-9588-z

Background• Mixing and pelleting of cellulosic

biomass provides an attractive solution for biorefineries due to improved homogeneity/availability, increased bulk and volumetric energy densities.

• In order to effectively convert the mixed feedstock, the pretreatment technology must be capable of efficiently processing a wide range of feedstocks.

Approach• We studied the impact of pretreatment

technologies on saccharification and isopentenol fermentation of mixed lignocellulosic feedstocks .

Significance• This study provides insights on developing biorefinery technologies that produce

advanced biofuels based on mixed feedstock streams.

Scale-up of Ionic Liquid Based Fractionation of Mixed Feedstocks

Outcomes• An integrated scale up process including pretreatment, homogenization, continuous washing/separation, and product

recovery was effectively developed to simplify feedstock handling, reduce IL inhibition and reduce water consumption, all of which can be further integrated with downstream enzyme hydrolysis and microbial fermentation for lignocellulosicbiorefinery.

Li, C. et al. (2015). “Scale-up of Ionic Liquid Based Fractionation of Single and Mixed Feedstocks”. BioEnergy Research, DOI 10.1007/s12155-015-9587-0

Background• Lignocellulosic biorefineries have

tonnage and throughput requirements that must be met year round and there is no single feedstock available in any given region that is capable of meeting the price and availability demands.

• IL pretreatment can enables fractionation of a wide range of feedstocks and produces high yields of fermentable sugars suitable for biofuel production.

Significance• This scale-up evaluation demonstrates that IL pretreatment technology is feedstock

agnostic and can be effectively scaled to larger operations.

Overall glucan recovery from solid stream = 99.7%

Overall xylan recovery from solid stream = 62.8%

Overall lignin recovery from solid stream = 59.9%

Overall[C2C1Im][OAc] balance = 99.5%  

 

140ºC, 1h, atmosphere pressure

10% solid loading

Biomass/IL slurry

6 kg

Ionic Liquid Pretreatment

Precipitation &Washing

Solids

Liquid

600.0 g dry weight

216.3 g glucan 88.7 g xylan 11.3 g arabinan 8.0 g galactan 181.7 g lignin 12.5 g ash 81.5 g ND

Pretreated solids

421.7 g dry weight 216.0 g glucan 55.7 g xylan 6.8 g arabinan 2.2 g galactan 108.9 g lignin 5.1 g ash 27.0 g ND 0.9 g [C2C1Im][OAc]

178.3 g biomass in IL for recovery 0.3 g glucan 33.0 g xylan 4.5 g arabinan 5.8 g galactan 72.8 g lignin 7.4 g ND 5372.9 g [C2C1Im][OAc]

Mixed Feedstock (1:1) Switchgrass Eucalyptus

Water [C2C1Im][OAc]5400 g

Approach• This work demonstrates the scale-up of IL pretreatment of

single feedstocks (switchgrass and eucalyptus) and mixed feedstock (mixtures of these two) by 30-fold, relative to the bench scale (6L vs 0.2L) at 10% solid loading.

Fast forward genetics in filamentous fungi

Baker, S.E., Schackwitz, W., Lipzen, A., Martin, J., Haridas, S., LaButti, K., Grigoriev, I.V., Simmons, B.A., McCluskey, K. (2015). “Draft genome sequence of Neurospora crassa strain FGSC 73” Genome Announc., 3(2): pii:e00074-15.

Background• Neurospora crassa is a

filamentous fungus that is a model for induction of cellulaseand hemicellulose secretion

• It is also a model genetic system with several hundred “anonymous” mutant strains

Approach• Resequence Neurospora

mutant strains in order to associate mutant phenotypes with genotypes

• Part of a larger “fast forward genetics” program within the JBEI Fungal Biotechnology to associate phenotypes with genotypes

Outcomes• Resequencing accelerates the association of classical genetic phenotypes with genotypes • Develop a pipeline for gene discovered based on mutagenesis and screening for phenotypes

Significance• FGSC 73 is the second reference sequence for Neurospora crassa and was used to generate a

new set of gene models

Development of an orthogonal fatty acid biosynthesis system in E. coli for oleochemical production

Outcomes• We cloned and solubly expressed several type I FAS enzymes including those from C. glutamicum and M. tuberculosis• We demonstrated the in vivo activity of several type I FASs and used C. glutamicum FAS to produce fatty alcohols and

methyl ketones.

Haushalter, R. W. et al. (2015). "Development of an orthogonal fatty acid biosynthesis system in E. coli for oleochemical production". Metab Eng, DOI: 10.1016/j.ymben.2015.04.003

Background• E. coli’s native fatty acid synthase

(FAS) has been used for production of fuels and chemicals, but is subject to regulation that limits production

• Actinobacterial possess type I FAS enzymes that directly produce acyl-CoAs and are not transcriptionallyregulated in engineered E. coli.

Approach• Cloned and expressed type I FAS

enzymes from actinobacteria and investigated their capacity for oleochemical production

Significance• This is the first example of a heterologous FAS pathway in E. coli, and will be

further optimized to improve renewable production of oleochemicals.

Computational protein design enables a novel one-carbon assimilation pathway

Outcomes• JBEI robotics were used to screen several thousand variants of the computationally designed Formolase for improved

kinetics, several improved variants were found

Siegel, J.B. et al. (2015). “Computational protein design enables a novel one-carbon assimilation pathway” Proc. Natl. Acad. Sci.,112: 3704-3709.

Background• Novel strategies are needed

to address current challenges in energy storage and carbon sequestration.

• One approach is to engineer biological systems to convert one-carbon compounds into multicarbon molecules such as fuels and other high value chemicals

Approach• Utilize computational design

to design a novel carbon assimilation pathway

• Utilize JBEI robotics to improve computationally designed enzymes via a colorimetric screen

Significance• By combining a computationally designed and robotically-evolved enzyme with

several naturally occurring enzymes, a new carbon fixation pathway, the formolasepathway, which assimilates one-carbon units via formate was created.

Formic Acid Formyl-CoA Formaldehyde DHA DHAP

CO2

FDH

NADHH+

NAD+

HSCoAATP

AMPPPi

NADHH+

NAD+

HSCoA

ACS ACDH FLS DHAK

ATP ADP

INPUT OUTPUT

1. Reducing Equivalents 2. Carbon Activation and Reduction 3. Carbon Bond Formation 4. Metabolic Assimilation

Formolase Pathway

Robotic Screening Workflow