Carbon Utilization

Photoautotrophic carbon fluxomics.

Personnel: Xiao Qian

Our goal is to use flux balance analysis (FBA, left figure) and isotopically nonstationary metabolic flux analysis (INST-MFA, right figure) to quantitatively understand carbon flux distributions and pathway utilizations of the cyanobacterium Synechococcus sp. PCC 7002 during photosynthesis. The outcome also helps discover new roles of existing metabolic pathways.

 

Sustainable BioH2 production in the presence of nitrate

Personnel: Xiao Qian

Our goal is to metabolic engineer Synechococcus sp. PCC 7002 to reroute electrons towards the synthesis of valuable fermentative products, away from nitrate reduction in the presence of nitrate is described. By eliminating the narB-encoded nitrate reductase, we have significantly elevated the fermentative H2 yield in Synechococcus 7002.

 

Carbon utilization towards hydrogen production

Personnel: Kumara Swarmy

The marine cyanobacterium, Synechococcus sp. strain PCC 7002, produces energy rich molecules like ATP and NAD(P)H to power all cell processes. During dark-anoxic conditions these energy resources are produced by metabolic pathways denoted glycolysis and OPP. These pathways share the carbon fluxes of glucose-6-phosphate catabolism and produce ATP and NAD(P)H. The amount of NAD(P)H produced by glycolysis/OPP pathways is balanced mainly by CO2 and organic acids production. When excess NAD(P)H is generated it is used up by a bidirectional NiFe-hydrogenase that produces hydrogen, and thus balancing the intracellular redox poise which is crucial for cell survivability. Although, glucose catabolism through OPP pathway can yield 8 mol of NAD(P)H/glucose, the glycolysis is the preferred fermentative pathway, yielding 4 mol of NAD(P)H /glucose. Through metabolic engineering we are attempting to increase the catabolic flux of these pathways and increase NAD(P)H availability in Synechococcus sp. strain PCC 7002, to further increase the hydrogen production.

 

Contact L. Tiago Guerra 

AMOPs fix atmospheric carbon dioxide, yielding organic molecules through photosynthesis and the Calvin-Benson-Bassham cycle. The initial carbon intermediates are then partitioned into the main carbon based reservoirs of the cell: Carbohydrates (sugars), Proteins and Lipids. Consequently, understanding what controls the partitioning of fixed carbon through the major carbon based molecules is critical for efficient designer biofuel production.

The Dismukes lab is pursuing the following research projects aimed at understanding and manipulating the carbon decision tree by metabolic engineering and environmental manipulation. The ultimate goal is to increase yields of the target chemical fuels without decreasing growth rates.

  1. Metabolic switching in cyanobacteria: regulation of carbohydrate catabolism pathways driven by nutrients and environmental cues. 
  2. Nitrate metabolism: competition for reductant production and utilization 
  3. Triglyceride biosynthesis in algae: de novo vs remodeling of functional lipids. 
  4. Starch metabolism in green algae. 
  5. Choke points in lipid metabolism in diatoms; Regulation of lipid production in diatoms.

Starch metabolism in cyanobacteria

 

In marine cyanobacteria sugars are accumulated mainly in the form of Glycogen (energy storage) and soluble sugars that act as osmoprotectants. As these molecules have the same precursors they are competing pathways. Soluble sugars can be very attractive as a feedstock for other biotechnological processes that use heterotrophic bacteria. In addition, the type of substrates available for fermentation might affect its rates and hydrogen yield. In order to determine if the alteration of the carbohydrate species present would affect metabolism and productivities of osmolytes and hydrogen glycogen mutants were made and analyzed. The main conclusions of these studies were:

 

  1. Prevention of glycogen synthesis leads to excretion of substantial amounts osmolytes to the medium under certain conditions that could be used as feedstock for heterotrophic microorganisms for other biotechnological processes (Xu, 2012) 
  2. Increased levels of glycogen can potentially lead to higher hydrogen yields as it is the main carbon source utilized for autofermentation (Guerra, 2012). 

Choke points in lipid metabolism in diatoms.

Some strains of eukaryotic algae, including Diatoms, accumulate large amounts of lipids and under some stress conditions are capable of reaching even higher levels of lipid accumulation. They are thus regarded with interest as potential biodiesel cell factories.

Nitrogen limitation/depletion severely impairs growth and unbalances the ratios of Proteins, Sugars and Lipids in the cells. We are thus using this perturbation in order to understand what are the metabolic changes that occur and how is the carbon decision tree regulated in response to this stress that leads to higher lipid content by cells. To achieve this goal we are evaluating the transcription levels of target genes, the abundance of several key intracellular metabolites, as well as protein levels in a model strain of a diatom. This study will also allow the identification of potential targets for future metabolic engineering.

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