Uppsala University, Sweden Department of Photochemistry and - - PowerPoint PPT Presentation

• Uppsala University, Sweden
• Department of Photochemistry and

Molecular Science

– Hydrogenproduction with Cyanobacteria

Supervisors and Instructors

• Wetlab:

– Dr. Thorsten Heidorn – Daniel Camsund, PhD student

• Modelling:

– Mats Walden, PhD student

• Institute for Cell and Molecular Biology at

Uppsala University

The Uppsala iGEM Team

• Wetlab:

– Anders Kristoffersson (Sweden) – Erik Florman (Sweden) – Karl Brune (Germany) – Ruiqing Ni (China)

• Modelling:

– Jonatan Halvardson (Sweden)

Nowadays Problems

• The world has a lot of problems:

– Climate Change – Water shortage – Food shortage – Energy – To be continued..

• Beer in Sweden is incredibly expensive..

Booze Bugs

• The aim of our project

– Produce alcohols, in particular ethanol & isobutanol. – Directly from sunlight – With cyanobacteria – Investigate ways to increase production of alcohols of Synechocystis sp PCC 6803

The Future

• Imagine if park areas could look like this

and produce green energy !?

PhotoBioReactor designed by Charles Lee www.BIOSarch.com

The issue of ethanol from agriculture

• Today 25% of the US grain harvest is

used as fuel.1

1 Scientific American May 2009, Could food shortages bring down civilization. Lester R

Brown

The issue of ethanol from agriculture

• 175 million people in India is living on food

produced from fossil water. 1

• >1 Billion people is hungry in the world today

Hungry People in the world today, Source: UN

1 Scientific American May 2009, Could food shortages bring down civilization. Lester R

Brown

The issue of ethanol from agriculture

• The 25 % of U.S. grain harvest devoted to

fuel, could potentially feed half a billion

• people. 1
• This leads to the conclusion that

agriculture based fuels can not substitute fossil fuels on a broad basis.

1 Scientific American May 2009, Could food shortages bring down civilization. Lester R

Brown

The issue of ethanol from agriculture

• The 25 % of U.S. grain harvest devoted to

fuel, could potentially feed half a billion

• people. 1
• So, what to do ?!

1 Scientific American May 2009, Could food shortages bring down civilization. Lester R

Brown

Non-fermentative “Algae” Fuel Production: Booze Bugs

• Potentially non-competitive with food

production.

– Deployable at locations not suitable for conventional agriculture. – Low usage of fresh water. – Adaption to use salt water possible

The ethanol project

• Introducing genes for

ethanol production.

– ADH2 & Pdc (Zymomonas mobilis)

• Redirecting flow of

substrate.

– Inhibiting the PDC complex, using an antisense RNA and a protein approach.

The Implementation

• Ethanol Constructs

Increasing substrate levels

• Inhibiting the PDC, pyruvate dehydrogenase

complex

– Antisense RNA

• A set of RNA’s complementary to the PDC subunit

mRNA’s. Aiming to disturb translation of the PDC subunits.

– PirAB

• Pirin like proteins, shown to inhibit the PDC1, and

inducing fermentative pathways 2. Aiming to disturb functionality of the PDC.

1 A cyanobacterial gene encoding an ortholog of Pirin is induced under stress conditions. Yukako Hiharaa, Masayuki

Muramatsua, Kinu Nakamuraa and Kintake Sonoikeb, FEBS Letters 574 (2004) 101–105

2 Pirin Regulates Pyruvate Catabolism by Interacting with the Pyruvate Dehydrogenase E1 Subunit and Modulating

Pyruvate Dehydrogenase Activity. Po-Chi Lai et al. Journal of Bacteriology, January 2007, p. 109-118, Vol. 189, No. 1

The Implementation

• Inhibiting the PDC

Why Ethanol & Butanol

• Butanol

+ Equivalent to standard gasoline. Plug & Play  – More toxic to Synechocystis compared to ethanol – Not a proven concept.

• Ethanol

+ Widely used as a fuel and chemical + Wt Synechocystis already quite tolerant against EtOH – Require infrastructure investments – Corrosive – More hazardous compared to gasoline – Low energy density

The Butanol project

• Existing pathways in the Registry

– Alberta iGEM Team 2007:

• Fermentative pathway for butanol production
• But: Fermentative pathway not optimal in

aerobic conditions.

• Pathway shown by Shota Atsumi, Taizo Hanai,

James C. Liao

• “Non-fermentative pathways for synthesis of branched-

chain higher alcohols as biofuels” (Nature Vol 451|3 January 2008)

The Butanol project

• Introduction of genes for isobutanol

production, kivd (lactococcus lactis) and ADH2 (bakers yeast).

The Implementation

• Isobutanol Construct

Modeling

• We want to describe

the system without knowing the enzyme concentration e0 or the death term kt.

• Assuming quasi

steady state and that the system can be described as a “one step” reaction.

Modeling

• Setting up

equations

– With some additional assumptions and some math…..

Modeling

• Leads to an expression, that

allows us to optimize the system in respect to the promoter activities.

For a complete deduction please visit

• ur iGEM wiki.

Problems

• Getting our hands on the kivd gene

– Trials with PCR on “filmjölk”, swedish yoghurt. – Lots of mails to different people! – Bingo! Bioneer synthesizes the gene 

Problems

• PirA and the PirAB construct

– Problems with PCR for PirA – Problems with assembly of the PirAB construct – And due to lack of time this approach was abandoned. 

Problems

• Synechocystis

– Not the standard lab bug – Require special parts – Doubling time about11h

• 3 weeks from transformation to

measurements

Results

• Evaluation in E.coli (constructs with

plLac)

– Why use a GC when we can play around with statistics? – Construction of a PLS model using spectrophotometric data. – Measure ethanol/butanol concentration using the PLS model.

Results

Results

• Clear systematic difference between

functional and control constructs.

Results

• Clear systematic difference between

functional and control constructs.

Results

• Clear systematic difference between

functional and control constructs.

Results

• Evaluation of antisense RNA in

synechocystis

– Synechocystis culutres carrying two of the antisense RNA constructs grows very slow (A1, B2), even in “copper free” medium. – The other nine show inconclusive growth rates for copper induced versus non induced.

Future Prospects

Algae bio fuels = Big business

• > 1 billion USD invested since 2007
• Synthetic Genomics,

– A Craig Venter business backed by ExxonMobil

• Sapphire Energy

– Backed by Bill Gates

Gold Rush for algae; Nature 461, 460-461 (2009)

Tack o Hej, leverpastej

• Thank you for listening