Next-generation Ammonia Pretreatment Enhances Cellulosic Biofuel Production

da Costa Sousa et al. (2016) “Next-generation ammonia pretreatment enhances cellulosic biofuel production”. Energy & Environmental Science (2016) [DOI: 10.1039/C5EE03051J]

Objective• To improve the deconstruction of cellulosic fuels using an

ammonia-based pretreatment.

Approach • Collaboration between GLBRC, JBEI, and BESC: this new

pretreatment was developed entirely within the BER family.• A new liquid ammonia pretreatment called Extractive Ammonia

(EA) simultaneously converts native crystalline cellulose to highly digestible material while selectively extracting up to ~45% of the lignin from the biomass (Fig. a).

Result/Impacts• EA-treated corn stover gave higher fermentable sugars yield

compared to an older ammonia treatment with 60% lower enzyme loading (Fig. b).

• EA preserves extracted lignin, offering the potential for lignin valorization with fuel and chemical co-products.

• Single-stage EA fractionation achieves high biofuel yields comparable to results obtained with Ionic Liquid pretreatment.

• Sugars derived from EA pretreatment are readily fermentable due to removal of most inhibitors.

a. Biomass delignification and b. impact glucan conversion of EA process

Exploiting the Substrate Promiscuity of Hydroxycinnamoyl-CoA:Shikimate Hydroxycinnamoyl Transferase (HCT) to Reduce Lignin.

Outcomes• Protocatechuate (PCA) was identified as a competitive inhibitor of HCT in vitro: It binds to the HCT catalytic site.• Arabidopsis plants overproducing PCA have 30% less lignin and show improved biomass saccharification (+ 25%).

Eudes A, et al. (2016). Exploiting the substrate promiscuity of hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyl transferase to reduce lignin. Plant Cell Physiology DOI: 10.1093/pcp/pcw016

Background• Lignin is an essential cell wall

polymer but confers recalcitrance to plant biomass.

• Novel approaches to reduce lignin in bioenergy crops without affecting biomass yield are desired for economical production of second-generation biofuels

Approach• Identify competitive inhibitors

of HCT, a key enzyme of the lignin biosynthetic pathway.

• Overproduce such inhibitors in Arabidopsis to reduce lignin synthesis in specific tissues and improve biomass saccharification.

Significance• Expression in plants of ubiC / pobA to overproduce PCA is a novel strategy to reduce lignin and reduce overall biofuel cost.• Crops expressing ubiC / pobA under the control of tissue-specific promoters are being developed.

1) Step catalyzed by HCT in the lignin biosynthetic pathway

3) A two-gene strategy for protocatechuate overproduction reduces lignin content and improves biomass saccharification in Arabidopsis

2) Protocatechuate (PCA) inhibits HCT activity in vitro

- 10 20 30 40 50 60 70 80 90

100

HCT

activ

ity (%

of c

ontr

ol w

ithou

t PCA

)

HCT activity reduced in the presence of PCA

3) Protocatechuate binds the HCT active site

PCA x25

LIGNIN -30%

WT

pobA-1

pobA-2

ubiC-1

ubiC-2

ubiC-pobA-1

ubiC-pobA-2

ubiC-pobA-3

02468

101214161820

Ligni

n co

nten

t (%

bio

mas

s)

WT

pobA-1

pobA-2

ubiC-1

ubiC-2

ubic-pobA-1

ubic-pobA-2

ubic-pobA-3

0

200

400

600

800

nmol

g-1

FW

COOH

OH

O

O

OH

COOH

OH

COOH

OH

OH

ubiC pobA

chorismate HBA PCA

0 24 48 72 96

0

50

100

150

200

WTubiC-pobA-1 ubiC-pobA-2ubiC-pobA-3Su

gars

(µg

mg-1

bio

mas

s)

Time (h)

SACCHARIFICATION +25%

PCAHBA

phenylalanine

p-coumaroyl-CoA

p-coumaroyl shikimate

feruloyl-CoA

H-unit

G-unit

HCT shikimate

coniferaldehyde

sinapaldehydeS-unit

A Droplet Microfluidic Platform for Automating Genetic Engineering

Outcomes• Duration and temperatures of the microfluidic heat-shock procedures

were optimized to yield transformation efficiencies comparable to those obtained by benchtop methods with a throughput up to 6 droplets/min.

• Continuous replenishment of oil to the culture chamber provides a fresh supply of oxygen to the cells for long-term ( 5 days) cell ∼culture.

• The microfluidic format permitted significant reduction in consumption (100-fold) of expensive reagents such as DNA and enzymes compared to the benchtop method.

1) Standard heat-shock steps are reproduced in droplets

Multiple fluidic handling operations are performed on the microfluidic chip; reagent partitioning, merging, mixing and thermocycling. Device schematic depicts transformation steps corresponding to benchtop procudures. Microfluidic system employs electrodes to manipulate aqueous droplets and peltier modules for temperature control.

Gach et al. (2016) “A Droplet Microfluidic Platform for Automating Genetic Engineering”, ACS Synth Biol., doi, 10.1021/acssynbio.6b00011

Background• The basic steps of genetic engineering involve

the mixing of plasmids and competent cells, transformation, outgrowth, addition of selection marker, culture and expression.

• Performing many of these operations manually or with robotics can be fairly expensive with respect to time, materials and reagents.

2) Cells transformed and cultured on-chip to express fluorescent proteinsThe platform was validated by transforming various plasmids into Escherichia coli, Saccharomyces cerevisiae and Aspergillus niger.Large scale sample processing demonstrated by performing 100 separate transformations in droplets.

Approach• A temperature-controlled hybrid microfluidic

platform was developed to implement heat-shock transformation, antibiotic selection, culture and assay in discrete droplets.

Significance• The proposed platform offers potential for automation of molecular

biology experiments significantly reducing cost, time and variability while improving throughput.

Investigation of Proposed Ladderane Biosynthetic Genes from Anammox Bacteria by Heterologous Expression in E. coli

Outcomes• Dozens of constructs were produced and tested in

this study. Although the ladderane biosynthetic pathway was not identified in this extremely challenging project, important data was generated (e.g., on a novel putative desaturase) and all constructs and data were made available to the scientific community for further studies.

Javidpour et al. (2016) “Investigation of proposed ladderane biosynthetic genes from anammox bacteria by heterologous expression in E. coli, ” PLOS ONE, doi: 10.1371/journal.pone.0151087

Background• Ladderanes are highly energetic compounds uniquely

produced by anammox (anaerobic ammonia-oxidizing) bacteria that could serve as high-energy-density jet fuel components (~46% higher than typical jet fuel). Their biosynthetic pathway is unknown.

Significance• This was the first experimental effort to identify

ladderane biosynthetic genes and provides the scientific community with considerable synthetic biology resources to further explore these potential renewable jet fuel components.

Approach• 34 candidate ladderane biosynthesis genes from an

anammox bacterium were synthesized by the Joint Genome Institute, assembled into synthetic operons, and systematically tested for function by heterologous expression in E. coli under anaerobic conditions. More focused studies were performed with some gene candidates.

Structure of a C20 [5]-ladderane fatty acid

Candidate ladderane biosynthesis genes from the anammox bacterium Kuenenia stuttgartiensis and synthetic operons constructed for this study to assess function.

Secondary Metabolism for Isoprenoid-based Biofuels

Kang, A., & Lee, T. S. (2016). “Chapter 2 - Secondary Metabolism for Isoprenoid-based Biofuels. Biotechnology for Biofuel Production and Optimization” (pp. 35-71), 1st Edition. Amsterdam, Elsevier. 

Background• Isoprenoids are most abundant and diverse in plants, but

it is not economically feasible to extract isoprenoids from plants in general. Therefore, microbes, particularly Escherichia coli and yeast, have been exploited as hosts for isoprenoids biosynthesis.

Approach• In this book chapter, we present current progress in

isoprenoid-based biofuel production with emphasis on secondary metabolism pathways for isoprenoid biosynthesis.

Outcomes• First, two distinct biosynthetic pathways for production of

the universal precursors of isoprenoids are discussed: the mevalonate (MVA) pathway, archaeal MVA pathway, and the methylerythritol phosphate (MEP) pathway. Second, biochemical properties of the relevant enzyme reactions are briefly explored: regulation of the enzyme activity and stoichiometry of two pathways. Lastly, the book chapter discusses the development of isoprenoid pathways in microbial hosts and the recent progress of isoprenoid-based biofuel production and engineering strategies to optimize the isoprenoid pathway.

Significance• Understanding of the isoprenoid biosynthetic pathways

present in various species will facilitate engineering the isoprenoid biosynthetic pathways in microbial systems.

1) Isoprenoid natural products

2) Biological pathways for Isoprenoids

End-to-end Automated Microfluidic Platform for Synthetic Biology

Outcomes• Microfluidic platform capabilities for multiple automated DNA assembly methods; Escherichia coli and Saccharomyces

cerevisiae transformation; and automated control of cellular growth, gene expression induction, and proteogenic and metabolic output analysis

• New method and software for Isothermal Hierarchical DNA Construction

Significance• Demonstration of the microfluidic platform’s potential to provide end-to-end solutions for synthetic

biology research, from design to functional analysis

Linshiz, G. et al. (2016). “End-to-end automated microfluidic platform for synthetic biology: from design to functional analysis.” Journal of Biological Engineering 10:3 DOI: 10.1186/s13036-016-0024-5

Background• Synthetic biology aims to engineer

biological systems for desired behaviors

• The construction of these systems can be complex, often requiring genetic reprogramming, extensive de novo DNA synthesis, and functional screening

Approach• Develop a programmable,

multipurpose microfluidic platform and associated software

• Apply the platform to major steps of the synthetic biology research cycle: design, construction, testing, and analysis

ATP Citrate Lyase Mediated Cytosolic Acetyl-CoA Biosynthesis Increases Mevalonate Production in Saccharomyces cerevisiae

Outcomes• Combining the two modifications increases accumulation

of mevalonate pathway intermediates, and that both modifications are required to substantially increase production.

• Replacing the native ERG12 (mevalonate kinase) promoter maximized accumulation of mevalonate.

Rodriguez, S., et al. (2016). "ATP citrate lyase mediated cytosolic acetyl-CoA biosynthesis increases mevalonate production in Saccharomyces cerevisiae". Microb Cell Fact, 15(1), 48. doi:10.1186/s12934-016-0447-1

Background• To date, it has been difficult to engineer S.

cerevisiae to continuously convert sugars present in biomass-based feedstocks to acetyl-CoA derived products due to intrinsic physiological constraints.

Significance• By combining the push/pull/block strategies, we significantly improved mevalonate production.• This strategy can be used to improve the efficiency with which industrial strains of S. cerevisiae

convert feedstocks to acetyl-CoA derived biofuels and renewable chemicals

Approach• We incorporated an alternative mode of acetyl-CoA

biosynthesis mediated by ATP citrate lyase (ACL) that may obviate such constraints.

• We employed a push/pull strategy to shunt citrate towards ACL by deletion of the mitochondrial NAD+-dependent isocitrate dehydrogenase (IDH1) and engineering higher flux through the mevalonate pathway. Time course of mevalonate production (a) and cellular squalene

concentrations (b) in engineered strains. Strains are: No symbols represent the unengineered parent strain BY4741; Empty squares represent BY4741 with MvaES genes expressed, JBEI 10569; Emtpy circles represent BY4741 with MvaES and ACLab genes expressed, JBEI 10690; Filled circles represent BY4741 with MvaES and ACLab genes expressed, and the IDH1 gene deleted, JBEI 10691; Crosses represent BY4741 with MvaES, ACLab, and the Y. lipolitica IDH genes expressed JBEI 10692. Strains were grown in low [(NH4)2SO4]

Rapid Discovery and Functional Characterization of Terpene Synthases from Four Endophytic Xylariaceae

Outcomes• Out of the 26 TPS’s profiled, 12 TPS’s were functionally

expressed in E. coli.• Majority of active TPS’s exhibited both monoterpene and

sesquiterpene synthase activity.

Wu, W., et al. (2016). "Rapid Discovery and Functional Characterization of Terpene Synthases from Four Endophytic Xylariaceae". PLoS One, 11(2), e0146983. doi, 10.1371/journal.pone.0146983

Background• Many endophytic fungi are known to produce a wide

spectrum of volatile organic compounds (VOCs) with potential applications in bioenergy, such as terpenes analogous to molecules in diesel fuels.

Significance• We discovered 12 novel and functional TPS’s from

endophytic fungi clustered into 4 homology groups.• These TPS’s have potential to be significant sources of

renewable diesel blendstocks, and may be useful for other applications in human health.

Approach• We discovered and characterized 26 terpene

synthases (TPSs) derived from four endophytic fungi.• Once identified, these TPS genes were were

expressed in an E. coli strain, and their product profiles were determined using Solid Phase Micro-Extraction (SPME) and GC-MS.

Compounds produced by terpene synthases studied

Effects of Arbuscular Mycorrhizal Fungi Colonization and Tree-herb Interaction on Legume Growth in Pb contaminated Soil

Outcomes• AMF inoculation of legume trees increased overall biomass yield of

inoculated plants compared to that of non-inoculated ones under Pb stress.• AMF impact on biomass yield could be from a direct interaction to retain Pb

in the underground part of the trees and prevent Pb toxicity and/or by influencing plant photosynthesis and nutrient acquisition.

Yang, YY. et al. (2016). “The roles of arbuscular mycorrhizal fungi (AMF) in phytoremediation and tree-herb interactions in Pb contaminated soil.” Sci Rep, 6, 20469. doi, 10.1038/srep20469

Background• Novel strategies are needed for growing plants

in heavy-metal contaminated soils found in some marginal lands.

Significance• This study demonstrated a strategy that combined microbial effects and planting patterns to

enhance biomass production of biofuel feedstock on contaminated marginal lands.

Approach• The effects of AMF colonization and the

presence of legume or grass herbs on the growth of a legume tree, Robinia pseudoacacia, in Pb contaminated soil were analyzed.

• The mechanism of increased biomass growth of legume trees were studied and discussed by measuring N, P, S, Mg and Pb concentrations in different organs.