Exploring the metabolic diversity of cyanobacteria for green energy production

Open Access
Author:
Zhang, Shuyi
Graduate Program:
Biochemistry, Microbiology, and Molecular Biology
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
April 29, 2015
Committee Members:
  • Donald Bryant, Dissertation Advisor
  • Donald Bryant, Committee Chair
  • John H Golbeck, Committee Member
  • Teh Hui Kao, Committee Member
  • Ying Gu, Committee Member
  • Michael Axtell, Committee Member
Keywords:
  • TCA cycle
  • glyoxylate cycle
  • GABA shunt
  • PHB
  • VIPP1
Abstract:
Cyanobacteria absorb and convert sunlight into chemical potential energy by oxygenic photosynthesis. The energy-rich compounds stored during the day provide the fuel to support growth and maintenance energy production at night. In the presence of oxygen, respiration occurs in most organisms, which allows the oxidation of energy-rich substrates for the production of proton-motive force for ATP synthesis and other biochemical work. This dissertation research focused on elucidating metabolic diversities and capacities in cyanobacteria, some of which have potential applications toward green energy and biomaterial production based on these newly defined metabolic pathways. Together with glycolysis and the oxidative pentose phosphate pathway, the tricarboxylic acid (TCA) cycle is one of the three most important pathways of central carbohydrate metabolism. It was long believed that cyanobacteria had an incomplete TCA cycle due to the absence of 2-oxoglutarate dehydrogenase (OGDH). Studies from this dissertation demonstrated that the TCA cycle in most cyanobacteria is completed in manner distinct from the classical TCA cycle through the action of two alternative enzymes, 2-oxoglutarate decarboxylase (2-OGDC) and succinic semialdehyde dehydrogenase (SSADH). Some preliminary studies were also performed in metabolic engineering this newly discovered TCA cycle to produce poly-3-hydroxybutyrate and poly-3-hydroxybutyrate-co-4-hydroxybutyrate from the newly identified intermediate, succinic semialdehyde. Though some previous studies of cyanobacteria reported enzyme activities of the glyoxylate cycle (i.e. isocitrate lyase and malate synthase), recent experimental studies found that these two enzymes are not detectable in Synechocystis sp. PCC 6803. In this study, genes encoding isocitrate lyase and malate synthase from Chlorogloeopsis fritschii PCC 9212 were iiiiv identified and characterized. Furthermore, when the two genes encoding isocitrate lyase and malate synthase were expressed in Synechococcus sp. PCC 7002, the resulting strain was able to assimilate acetate at a higher rate than the wild type strain. Overall, this study demonstrated that the glyoxylate cycle exists in certain cyanobacterial strains, that it plays an essential role in the assimilation of C2 carbon sources (i.e., acetate) for growth, and that it may also be involved in balancing carbon and nitrogen metabolism. The biogenesis of thylakoid membranes in cyanobacteria was also investigated in this study. Previous studies in Synechocystis sp. PCC 6803 reported that vipp1 (sll0617) was essential fro viability. By constructing a fully segregated null mutant in vipp1 (SynPCC7002_A0294) in Synechococcus sp. PCC 7002, we show that Vipp1 is not essential. Thylakoid membranes were still observed in vipp1 mutant cells and resembled those in a psaAB mutant that completely lacks photosystem (PS) I. When the vipp1 mutation was complemented with the orthologous vipp1 gene from Synechocystis sp. PCC 6803 that was expressed from the strong PcpcBA promoter, PS I content and activities were restored to normal levels, and cells again produced thylakoids that were indistinguishable from those of wild type. This study shows that thylakoids are still produced in the absence of Vipp1 and that normal thylakoid biogenesis in Synechococcus sp. PCC 7002 requires expression and biogenesis of PS I, which in turn requires Vipp1.