Rethinking Carbon Fixation Avi Flamholz, Milo Lab Department of Plant Sciences Weizmann Institute of Science
Where does the carbon go? (in the biological world)
Carbon Fixation: A New Frontier For Systems Biology Requires a majority of land and fresh water used by humanity. A molecular process that affects the global climate. Major uncertainties about rates and limits. Field CB. Primary Production of the Biosphere: Integrating Terrestrial and Oceanic Components. Science. 1998
Carbon Fixation By The Numbers Agriculture accounts for 60-70% of human fresh water use covers ~40% of the land surface. Foley J a, Defries R, Asner GP, et al. Global consequences of land use. Science. 2005 Postel SL, Daily GC, Ehrlich PR. Human appropriation of renewable fresh water. Science. 1996
The Calvin-Benson Cycle Drives Biological Carbon Fixation Carries the largest flux in the biosphere. Assimilates ~100 billion tons of carbon per year and employs Rubisco, the most abundant enzyme in the world. Raines CA. Increasing photosynthetic carbon assimilation in C3 plants to improve crop yield. Plant physiology. 2010
Can we use these resources differently? Better?
We work on metabolic engineering and bacterial factories to address these problems
Can we improve the Calvin cycle? (for specific applications) Breugel's "The Fall of Icarus"
E. Coli as a Testbed for Carbon Fixation
What governs the efficiency of photosynthesis and carbon fixation? Constraints on metabolites, catalytic rates and pathway structures limit growth Noor et al, Mol. Cell 2010; Bar-Even et al, Biochemistry 2011; Bar-Even et al, PLOS CB 2011 Rubisco is already optimized Savir et al, PNAS 2010 Design principles in photosynthesis Milo, Photos. Res. 2009 Synthetic carbon fixation pathways for higher productivity Bar-Even et al, PNAS 2010 BioNumbers and equilibrator for quantitative analysis Phillips & Milo, PNAS, 2009; Moran, Phillips & Milo, Cell 2010; Milo et al., NAR 2010
Future Direction: "Benchmarking" Carbon Fixation Cycles Compare novel carbon fixation pathways to the Calvin cycle in E. coli.
Gigatons of CO2 are fixed annually - can we use it better? C
Methylotrophic Bacteria Grow on C1 Compounds Methane 2e- Formaldehyde Assimilation Pathways Methanol Methylotrophic organisms 2e- Formaldehyde 2e- Formic acid Carbon Fixation Pathways 2e- Autotrophic organisms CO2 Energy e- oxidation state
The Biotech Industry Runs on Sugar (Glucose Mostly)
Sugars are Tasty and Expensive Sugars are derived from edible parts of plants and prices are on the rise.
The Methanol Economy Methanol is a promising alternative fuel and can be used to make useful polymers. It is also easy to make chemically in various ways and cheaper than sugar. Methanol, USD 200-400 / MT Sugar, USD 700-800 / MT Olah G a. Beyond oil and gas: the methanol economy. Angewandte Chemie. 2005
Methanol Production Currently made from fossil fuels and natural gas, but could potentially be made from (inedible) plant biomass in various ways.
Let them eat methanol! C1
Methylotrophs in the Methanol Economy Natively methylotrophic organisms are not very useful for biotechnological applications: their metabolism is not wellunderstood and they are hard to manipulate genetically.
Our solution: engineer a methylotrophic E. coli C1
Adding the Ribulose Monophosphate Pathway to E. coli Requires only 3 exogenous enzymes
Balancing Protein Levels is Important Very difficult to predict the ideal enzyme levels for a pathway a priori.
Exploring Expression Space with Combinatorial Cloning We test thousands of enzyme levels by combinatorially modulating RBS.
It'll Never Work on the First Try Use laboratory evolution to persuade E. coli to switch metabolic modes. It helps that methanol and formaldehyde are toxic.
Closing Thoughts Carbon fixation uses limited resources. Providing food and fuel for a growing population is becoming difficult. Challenges: Use water and land resources more efficiently Fix more carbon Use more of the carbon we fix We can make biofuels and fine chemicals in bacteria: important what we make them from. Might require us to look outside the box and use all the tools available in nature.
Questions?