Development of a native E. coli induction system for ionic liquid tolerance

Outcomes• Ionic liquid inducible promoters were identified and their performance for expression of EilA was evaluated• One of the ionic liquid inducible promoters performed significantly better than an IPTG indubicle expression system,

especially at higher concentrations of the ionic liquid

Frederix, et al., “Development of a native E. coli induction system for ionic liquid tolerance” PLOS One (2014).

Background• Overexpression of a

heterologous transporter EilAincreases tolerance to ionic liquids in E. coli

• Heterologous expression with traditional gene expression systems requires the use of expensive inducer molecules

• Heterologous expression of membrane proteins is often toxic

Approach• Develop a ionic liquid inducible

expression system for EilA using stress responsive promoters that were identified via microarray analysis

Significance• The methodology used can be easily translated into the development of gene expression

systems for tolerance mechanisms for other toxic compounds, in any host organism.

Outcomes• Identified a strain that was the highest amorphadiene producer at low carbon concentration (MBIS5)• Identified a strain that was the highest FPP producer, accumulating FPP levels that were toxic to the

cell and affected growth and production (MBIS2)• Identified a strain that was the highest amorphadiene producer when there is no limitation of the carbon

source (MBIS1). • Showed that RBS strength could dramatically alter the production of enzymes in a heterologous

metabolic pathway and, thus, the flux through that pathway.• Showed that the highest amorphadiene titer can be achieved through balanced expression of pathway

enzymes and, hence, minimal accumulation of pathway intermediates.SignificanceAs higher throughput cloning and screening methods become widely available, it will be possible to test many morecombinations of RBS strength for all the genes in the pathway to achieve optimal protein and metabolite balance andwe anticipate that these methods will become integral to pathway optimization.

Metabolic pathway optimization using ribosome binding site variants and combinatorial gene assembly

Background• A combinatorial approach was used to

choose the most appropriate RBSs forthe genes of the lower half of themevalonate pathway (mevalonate toamorphadiene) to improveamorphadiene production in E.coli

• The most appropriate RBS combinationand carbon source were chosen,enabling us to reduce the accumulationof toxic metabolic intermediates,improve growth, and improve theproduction of amorphadieneapproximately five-fold

Approach• 9 MBIS constructs were made using

combination of different RBS strengths5’ of MK, PMK, and PMD

• The constructs were tested at highercarbon concentration at the sign ofcarbon limitation

• Due to accumulation of high levels ofFPP by some strains, expression ofADS was increased to relieve FPPtoxicity and also increase amorphadieneproduction

Time (h)

1. 2.

4.

3.

Figures: 1) Lower mevalonate pathway constructs, 2 & 3) Specific amorphadiene production, 4) Intracellular FPP concentration

24 h 48 h

Tailored isoprenoid products by engineering geranylgeranyl reductase

Outcomes• Sequence variants created that display increased rates of H6GGPP production or are able to halt the extent of reduction at

H2GGPP and H4GGPP• The structures of these variants reveal the basis for their altered activities and also shed light onto the catalytic mechanism.

1Kung, et al., “Constructing Tailored Isoprenoid Products by Structure-Guided Modification of GeranylgeranylReductase”. Structure 22:1028-36 (2014).

Background• Enzymatic reduction of isoprenoid

double bonds is of considerable interest to synthetic biologists interested in the microbial production of isoprenoid drug or biofuel molecules.

• The enzyme geranylgeranyl reductase(GGR) catalyzes hydrogenation of carbon-carbon double bonds to produce the saturated alkyl chains.

Approach• We solved the crystal structures of

GGR from Sulfolobus acidocaldarius, including the structure of GGR bound to geranylgeranyl pyrophosphate (GGPP) and designed mutations the change activity.1

Significance• The degree of GGPP reduction can be customized enzymatically, a feature that is particularly

useful in synthetic biology.

Background• Nucleotide sugar transporters (NSTs) provide the activated sugars, which are predominantly made in the cytosol, for plant cell wall biosynthesis within the Golgi lumen• Plant biomass, composed of cell walls, can be used for production of advanced biofuels• The specific functions of these NSTs were largely unknown

Approach• Development of a novel liposome and mass spec-based tool for determination of transporter functions• Enzymatic synthesis of putative substrates that are not commercially available

Outcomes• To 24 out of 44 potential candidates a specific function could be assigned including the bifunctionalUDP-rhamnose/galactose transporters (URGTs)1

• The modulation of NST expression in plants affects cell wall composition e.g. increased galactose content1

Significance• The gained knowledge about NST functions largely contributes to our understanding of how lignocellulosicmaterials are made in plants and will accelerate the development of improved bioenergy crops

Nucleotide sugar transporters of Arabidopsis

Ectopic expression of URGT1 leads to increased galactan content in plants

1Rautengarten et al., “The Golgi localized bifunctional UDP-rhamnose/UDP-galactose transporter family of Arabidopsis.” Proceedings of the National Academy of Sciences, (2014).http://www.pnas.org/content/111/31/11563.full

LC-MS/MS analysis of nucleotide sugar transporter activities

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Fuc Rha Ara Gal Xyl GalA GlcA

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SN Col‐0

SN 35Spro:URGT1

Res Col‐0

Res 35Spro:URGT1

The Arabidopsis URGTs are localized within the Golgi membrane