Understanding limonene toxicity in E. coli

Outcomes• A mutation in alkyl hydroperoxidase

allowed significantly improved growth in the presence of limonene

• This led to the hypothesis that limonene forms a toxic hydroperoxide, which was verifiedby several methods

Background• Limonene, a promising biofuel

candidate, was previously shown to be highly toxic to E. coli

Approach• We allowed E. coli to evolve

tolerance towards limonene and sequenced the evolved strain, which was highly limonene-resistant

Significance• Laboratory evolution is a powerful method to uncover tolerance phenotypes• We obtained insight into the mechanism of limonene toxicity and have a

strain that is highly tolerant to the toxic hydroperoxide, which forms spontaneously in aerobic conditions.

Spontaneous oxidation of limonene to limonene-hydroperoxide and detoxification by AhpCL177Q.

Chubukov, V., Mingardon, F., Schackwitz, W., Baidoo, E. E., Alonso‐Gutierrez, J., Hu, Q., Lee, T. S., Keasling, J. D., & Mukhopadhyay, A. (2015). "Acute limonene toxicity in Escherichia coli is caused by limonene‐hydroperoxide and alleviated by a point mutation in alkyl hydroperoxidase (AhpC)". Appl Environ Microbiol, 81(14), 4690‐4696. doi, 10.1128/AEM.01102‐15

Strains expressing AhpCL177Q are highly tolerant to limonene.

Limonene-hydroperoxide is the major toxic compound to E. coli. Non-oxidized limonene is relatively non-toxic to wild type E. coli.

The spontenousE. coli mutant was resequencedat the DOE Joint Genome Institute.

Metabolic engineering for the high-yield production of isoprenoid-based C5 alcohols in E. coli

Outcomes• 60% increase in the yield of 3-methyl-3-buten-1-ol by engineering the Shine-Dalgarno sequence of nudB2

• Achieved final titers of 2.23 g/L of 3-methyl-3-buten-1-ol (~70% of pathway-dependent theoretical yield)2

1George, et al., “Correlation Analysis of Targeted Proteins and Metabolites to Assess and Engineer Microbial Isopentenol Production.” Biotechnology and Bioengineering. 111(8):1648-1658 (2014).2George, et al., “Metabolic engineering for the high-yield production of isoprenoid-based C5 alcohols in E. coli.” Scientific Reports. doi:10.1038/srep11128 (2015).

Background• Branched five carbon (C5) alcohols

are attractive targets for microbial production due to their desirable fuel properties and importance as platform chemicals

• Optimization of Initially engineered two-plasmid system for 3-methyl-3-buten-1-ol has been performed1

Approach• NudB, a promiscuous phosphatase,

was identified as a likely pathway bottleneck by metabolite profiling, and RBS engineering on NudB was attempted.

• Further engineering on mevalonate kinase expression and C5 alcohol recovery was also attempted.

Significance• Engineered a heterologous isoprenoid pathway in E. coli for the high-yield

production of 3-methyl-3-buten-1-ol, 3-methyl-2-buten-1-ol, and 3-methyl-1-butanol

RBS engineering to improve 3-methyl-3-buten-1-ol titer and reduce IPP accumulation.

~70% theoretical yield and 2.2 g/L titer in shake flask w/ overlay

Engineering of plant cell walls for enhanced biofuel production

1Linshiz, et al., “PaR-PaR: Laboratory Automation System.” ACS Synth. Biol. 2:216-222 (2013).2Linshiz, et al., “PR-PR: Cross-Platform Laboratory System.” ACS Synth. Biol. Article ASAP (2014).

Background• In this publication, we provide an overview of current advances in

engineering of plants with improved properties as feedstocks for biofuels production.

• The review focused on the following targets:• Reducing lignin• Decreasing inhibitors such as acetate• Increasing hexose/pentose ratio• Increasing cell wall sugar content

Significance/perspective• Up to date review on the advances in cell wall

engineering. • Summary of many recent studies including several

from the BRCs.• Annotated reference list with 87 references to

mostly recent literature• Discussion of remaining challenges, especially trait

stacking and predicting plant performance under a range of environmental conditions.

Loque D, Scheller HV, Pauly M (2015) Engineering of plant cell walls for enhanced biofuel production. Curr Opin Plant Biol 25: 151‐161. 

An example of engineering to obtain high density and low lignin. In these plants, normal lignin was maintained in the vessels while low lignin was restricted to fiber cells. 

Biomass is composed of different polysaccharides and lignin. The detailed composition differs between different types of feedstock. 

An unusual xylan in Arabidopsis primary cell walls is synthesised by GUX3, IRX9L, IRX10L and IRX14

Background• The polysaccharide xylan is a major component of

biomass (2nd only to cellulose).• It is a hindrance to cellulose deconstruction (blocks

cellulase access); difficult to depolymerise.

Approach and Outcomes• We identified and characterized a novel xylan structure in

Arabidopsis. This xylan had glycosidic linkages resistantto standard xylan degrading hydrolases.

• This xylan also has an novel pattern of side-chainspacing, pointing to exquisite molecular control overglycan synthesis. This pattern has unknown function.

• Using a multi ‘omics approach, we identified candidateglycosyltransferase genes for its biosynthesis.

• We used reverse genetics to test and confirm thesepredictions in planta.

• We developed a high-throughput, non-radioactivemethods for characterizing glycosyltransferase activity invitro.

Significance• We characterized a novel xylan structure and identified the majority of the

genes responsible for its synthesis.

Mortimer, JC et al. (2015). An unusual xylan in Arabidopsis primary cell walls is synthesised by GUX3, IRX9L, IRX10L and IRX14. Plant Journal. doi, 10.1111/tpj.12898

Cellulose Xylan

Work led by Prof. Paul Dupree, University of Cambridge, UK.

Trends In Microbiology: engineering solvent tolerant microbes

The reviewI reviewed strain engineering, primarily as it pertains tobacterial solvent tolerance, and on the benefits andchallenges associated with expression of membrane‐localized transporters in improving solvent tolerance andproduction.

BackgroundDuring microbial production of solvent‐like compounds,such as advanced biofuels and bulk‐chemicals,accumulation of the final product can negatively impactthe cultivation of the host microbe and limit theproduction levels. Consequently, improving solvent‐tolerance is becoming an essential aspect of engineeringmicrobial production strains. Transporters specificallyhave emerged as a powerful category of proteins thatbestow tolerance and often improve production but aredifficult targets for cellular expression.

Mukhopadhyay, A. (2015). "Tolerance engineering in bacteria for the production of advanced biofuels and chemicals". Trends Microbiol. doi, 10.1016/j.tim.2015.04.008 

SRM-based approach to assess organelle profiles in plant samples

Outcomes• It is indeed possible to use the SRM approach (targeted mass spectrometry) to profile organelle abundance in a plant

sample• We also demonstrated that the approach can be used to assess organelle profiles in plant tissues e.g. the abundance of

plastid markers was significantly higher in photosynthetic tissue.

Parsons and Heazlewood (2015). Beyond the Western front: targeted proteomics and organelle abundance

profiling. Front. Plant Sci. 5, 301. doi: 10.3389/fpls.2015.00301

Background• The characterization of subcellular

organelles involved in cell wall biosynthesis requires high purity preparations for proper analyses.

• Although western blotting enables profiling of organelle purity and contamination, there is a limited availability of antibodies in plant science

Approach• Use targeted mass spectrometry

approaches to profile protein extracts to determine suitability of this method for organelle profiling.

Significance• We have developed a collection of SRM transitions or peptide targets that can be now be used

to easily and quickly assess plant organelle profiles in fractions associated with cell wall biosynthesis e.g. plasma membrane and Golgi apparatus.

Non‐photosynthetic tissue Photosynthetic tissue

SRM signal for plastid markers from mass spectrometry

1 2 3

1 2 3

Complex regulation of prolyl-4-hydroxylases impacts root hair expansion

1Linshiz, et al., “PaR-PaR: Laboratory Automation System.” ACS Synth. Biol. 2:216-222 (2013).2Linshiz, et al., “PR-PR: Cross-Platform Laboratory System.” ACS Synth. Biol. Article ASAP (2014).

Background• Prolyl-4-hyroxylase is an enzyme responsible for converting

proline to hydroxyproline. The Hydroxyproline residues aresubsequently glycosylated. Thus, this step is essential forformation of many, possibly all, glycans and polysacchariesin the plant cell wall.

• Plants have several P4H isoforms and their individual roleshave not been understood.

Approach and Outcomes• Mutants in three major P4H isoforms expressed in

Arabidopsis roots were studied.• P4H5 has an essential function different from P4H2 and

P4H13, which are partly redundant.• Protein-protein interactions were investigated by co-

localization, bimolecular fluorescence complementation(BiFC) and FRET (Förster resonance energy transfer).

• The P4H proteins form homodimers, and additionally formsheterodimes with P4H5

• P4H5 is localized in the Er when P4H5 is absent. Only in thepresence of P4H5 is the P4H2 protein targeted to Golgi.

Significance• Understanding P4H function is important since these enzymes are necessary for glycan formation

in plant cell walls. The conditional targeting of P4H2 is an important illustration of the role of protein-protein interactions in the localization of function fo Golgi resident proteins involved in cell wall formation. This is a vastly understudied area of research.Velasquez al. (2015). "Complex regulation of prolyl‐4‐hydroxylases impacts root hair expansion". MolPlant 8(5), 734‐746. 

Left: In roots, deficiency in P4H is seen in the absence or deficiency in root hairs. Below: Protein‐protein interactions were investigated with a range of methods. Results obtained with BiFC are shown.