Engineering a synthetic enzyme pathway for the conversion of biomass to hydrogen

Biomass to Hydrogen

Phillip R. Smith1, Kunal K. Mehta2, and James R. Swartz1,2

1Department of Chemical Engineering, 2Department of Bioengineering Stanford University

Enzyme Pathway Cell-free Technology

Hydrogen Production Data Future Work: Kinetic Model

and Protein Evolution Conclusions

Proteins require different conditions for maturation/activation Fermentors would feed several reactors

• 75 million metric tons of H2 produced/year, from fossil fuels1,2 • 413 million metric tons of CO2 released/year (stoichiometric basis) • ∼1% of global CO2 emissions3,4

Goal: enzyme pathway to convert biomass sugars to hydrogen

FNR Ferredoxin-NADPH-

reductase

Fd Ferredoxin

H2ase [FeFe] Hydrogenase

Target Concentrations

Production of H2 in a cell-free system affords much greater control over the reaction environment than that available with in vivo hydrogen production platforms

Data Collection:

• Glucose is powering H2 production • The FNR/Fd/H2ase pathway is limiting

Zhang et al. (2007) PLoS ONE; Smith, Bingham, Swartz (2011). Int. J. Hyd. Energy

• We are building a kinetic model of the hydrogen-producing pathway to guide future engineering efforts

• To determine the relevant kinetic parameters of the enzymes, we are developing an assay to observe the performance of the enzymes by coupling the reactions to an optical readout of pH

• Protein engineering efforts will attempt to improve binding and electron transfer between FNR and Fd • High-throughput enzyme evolution • Fusion protein construction

An example of the optical assay. The activity of the enzymes causes a change in bromothymol blue absorbance. The NADPH level is constant because an additional enzyme regenerates it as it is consumed.

A technology to convert glucose to hydrogen rapidly and at high yields could enable a shift from fossil fuels to biomass as a source for the large amount of hydrogen produced each year

1Balat, 2010, Int. J. Hyd. Energy, 2http://en.wikipedia.org/wiki/Hydrogen_economy, 3CO2 calculations with Kunal Mehta, 4www.google.com/publicdata

(Max = 3000)

(Max = 20-200)

H2ase = [FeFe] hydrogenase from Clostridium pasteurianum

• The observed TONs for FNR and the hydrogenase are 3-4 order of magnitude lower than their intrinsic TONs, indicating substantial room for improvement in the hydrogen production rate

• Based off of separate enzyme assays and concentration studies, we believe electron transfer from FNR to Fd is the limiting step in the pathway

• We have designed a synthetic enzyme pathway to convert glucose to hydrogen • Cell-free technology offers a number of advantages over traditional

fermentative approaches to biohydrogen • We have shown proof of principle for the use of this pathway with substantial

improvements in hydrogen production rates • Significant room for improvement still exists in this system, which will be

realized though (1) the development of a kinetic model and (2) protein engineering

• With an order of magnitude improvement in hydrogen productivity this technology would be competitive with US bioethanol (on an energy productivity basis)

• We believe 2 orders of magnitude of productivity improvement is achievable, based on the intrinsic turnover numbers for each enzyme

Acknowledgements: • Alyssa Bingham, Stacey Shiigi, Sylvie Liong • Swartz Lab • GCEP funding!

Visit us at Swartz.openwetware.org