BIOLOGICAL PRODUCTION OF HYDROGEN THROUGH METABOLIC ENGINEERING (Liao)

Production of hydrogen from renewable carbon sources such as glucose and xylose has been demonstrated by many microorganisms. In principle, hydrogen production using biological processes is attractive based on thermodynamic considerations. However, current bioprocesses for hydrogen production are not cost effective for a number of reasons. 1) The organisms used are sensitive to hydrogen itself. Thus the amount of hydrogen produced is very small. 2) The bio-energy required for hydrogen production is too high, which limits the efficiency of the process. 3) The consumption of 5-carbon sugars, such as xylose, is too slow. 4) The biomass pretreatment for releasing glucose and xylose is too costly. Except for the last, these problems can be solved through metabolic engineering to create novel pathways for hydrogen production.  Such hybrid organisms have become possible since the advent of genetic engineering. Recent successes in genome sequencing and high-throughput expression analysis further enhanced the ability in engineering microorganisms for specific metabolic tasks. Our laboratory has demonstrated expertise in both theoretical analysis of metabolic networks as well as genetic construction of metabolically engineered organisms. Therefore, we propose to pursue the following objectives for the design and construction of an efficient microorganism for hydrogen production.

5.1. Develop a metabolic and regulatory model for hydrogen production.

The first task involves building a framework for analysis and optimization of hydrogen producing metabolic networks.  The model will be based largely on the stoichiometry of known organisms. Key regulatory elements will be included to reflect physiological limitations.  We will include the core central metabolism such as glycolysis, TCA cycle, the pentose phosphate pathway, and various fermentative pathways. Hydrogen production through various hydrogenases will be considered. In addition, the hydrogenase activity of nitrogenases will also be analyzed.  Native nitrogenase catalyzes the following reaction: N₂ +16ATP + 8e^- + 10H⁺ → 2NH₄⁺ + 16ADP + 16P_i  + H₂, but a mutant can produce hydrogen without nitrogen fixation. The model developed will be used to design a metabolically engineered organism that produces highest amounts of hydrogen.

5.2. Characterize the gene expression patterns in a hydrogen producing mutant of Rhodopseudomonas palustris.

The second task involves the characterization of a high hydrogen producing R. palustris mutant isolated by F.R. Tabita of Ohio State.  Studies in the Tabita lab have shown that mutant strains of R. palustris, R. sphaeroides, R. capsulatus, and R. rubrum that derepress nitrogenase synthesis in the presence of ammonia can all be selected when the carbon assimilatory (CO₂ fixation) pathway is knocked out. In order to grow in the absence of an exogenous electron acceptor, these bacteria normally use metabolically produced CO₂ as the endogenous electron acceptor for the oxidation of organic carbon under phototrophic growth conditions. To determine the regulatory mechanism used by this mutant, we will perform DNA microarray experiments to obtain the gene expression pattern.  We have designed and constructed the microarray for this organism and are ready to characterize this mutant strain. Information obtained here will be used to develop genetically engineered hydrogen producer.

5.3. Develop methods for transferring the hydrogen producing pathway to E. coli.

Although the R. palustris mutant described above produces a significant amount of hydrogen, it is still too slow to be economical. To harness its high hydrogen producing pathway, we plan to move the key metabolic network from R. palustris to E. coli, which grows much faster under easily commercializable conditions. This task involves significant hurdles, including methods for large DNA transfer, expression of foreign genes, and coupling of native and foreign metabolic networks.  We have already started investigating several aspects related to this task under other supports. The funding of this proposal will leverage other supports. The model developed in task 1 and the regulatory information obtained in task 2 will guide the design and development of the metabolically engineered organism here.