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 PDC 1 , 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 • Ethanol • Butanol + Widely used as a fuel + Equivalent to and chemical standard gasoline. Plug & Play  + Wt Synechocystis already quite tolerant – More toxic to against EtOH Synechocystis compared to ethanol – Require infrastructure investments – Not a proven concept. – 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 e 0 or the death term k t . • 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 our 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