scholarly journals Increased ethylene production by overexpressing phosphoenolpyruvate carboxylase in the cyanobacterium Synechocystis PCC 6803

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Claudia Durall ◽  
Pia Lindberg ◽  
Jianping Yu ◽  
Peter Lindblad
Keyword(s):  
Heterologous Expression ◽  
Ethylene Production ◽  
Phosphoenolpyruvate Carboxylase ◽  
Carbon Fixation ◽  
Tca Cycle ◽  
Central Carbon Metabolism ◽  
Gene Encoding ◽  
Carbon Supply ◽  
Phosphoenolpyruvate Synthase ◽  
Pcc 6803

Abstract Background Cyanobacteria can be metabolically engineered to convert CO2 to fuels and chemicals such as ethylene. A major challenge in such efforts is to optimize carbon fixation and partition towards target molecules. Results The efe gene encoding an ethylene-forming enzyme was introduced into a strain of the cyanobacterium Synechocystis PCC 6803 with increased phosphoenolpyruvate carboxylase (PEPc) levels. The resulting engineered strain (CD-P) showed significantly increased ethylene production (10.5 ± 3.1 µg mL−1 OD−1 day−1) compared to the control strain (6.4 ± 1.4 µg mL−1 OD−1 day−1). Interestingly, extra copies of the native pepc or the heterologous expression of PEPc from the cyanobacterium Synechococcus PCC 7002 (Synechococcus) in the CD-P, increased ethylene production (19.2 ± 1.3 and 18.3 ± 3.3 µg mL−1 OD−1 day−1, respectively) when the cells were treated with the acetyl-CoA carboxylase inhibitor, cycloxydim. A heterologous expression of phosphoenolpyruvate synthase (PPSA) from Synechococcus in the CD-P also increased ethylene production (16.77 ± 4.48 µg mL−1 OD−1 day−1) showing differences in the regulation of the native and the PPSA from Synechococcus in Synechocystis. Conclusions This work demonstrates that genetic rewiring of cyanobacterial central carbon metabolism can enhance carbon supply to the TCA cycle and thereby further increase ethylene production.

scholarly journals Production of succinate by engineered strains of Synechocystis PCC 6803 overexpressing phosphoenolpyruvate carboxylase and a glyoxylate shunt

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Claudia Durall ◽  
Kateryna Kukil ◽  
Jeffrey A. Hawkes ◽  
Alessia Albergati ◽  
Peter Lindblad ◽  
...  
Keyword(s):  
Phosphoenolpyruvate Carboxylase ◽  
Isocitrate Lyase ◽  
Tricarboxylic Acid Cycle ◽  
Cell Metabolism ◽  
Tca Cycle ◽  
Malate Synthase ◽  
Glyoxylate Shunt ◽  
Control Strain ◽  
Genes Encoding ◽  
Pcc 6803

Abstract Background Cyanobacteria are promising hosts for the production of various industrially important compounds such as succinate. This study focuses on introduction of the glyoxylate shunt, which is naturally present in only a few cyanobacteria, into Synechocystis PCC 6803. In order to test its impact on cell metabolism, engineered strains were evaluated for succinate accumulation under conditions of light, darkness and anoxic darkness. Each condition was complemented by treatments with 2-thenoyltrifluoroacetone, an inhibitor of succinate dehydrogenase enzyme, and acetate, both in nitrogen replete and deplete medium. Results We were able to introduce genes encoding the glyoxylate shunt, aceA and aceB, encoding isocitrate lyase and malate synthase respectively, into a strain of Synechocystis PCC 6803 engineered to overexpress phosphoenolpyruvate carboxylase. Our results show that complete expression of the glyoxylate shunt results in higher extracellular succinate accumulation compared to the wild type control strain after incubation of cells in darkness and anoxic darkness in the presence of nitrate. Addition of the inhibitor 2-thenoyltrifluoroacetone increased succinate titers in all the conditions tested when nitrate was available. Addition of acetate in the presence of the inhibitor further increased the succinate accumulation, resulting in high levels when phosphoenolpyruvate carboxylase was overexpressed, compared to control strain. However, the highest succinate titer was obtained after dark incubation of an engineered strain with a partial glyoxylate shunt overexpressing isocitrate lyase in addition to phosphoenolpyruvate carboxylase, with only 2-thenoyltrifluoroacetone supplementation to the medium. Conclusions Heterologous expression of the glyoxylate shunt with its central link to the tricarboxylic acid cycle (TCA) for acetate assimilation provides insight on the coordination of the carbon metabolism in the cell. Phosphoenolpyruvate carboxylase plays an important role in directing carbon flux towards the TCA cycle.

Predicted CO/CO2 Fixation in Ferroplasma spp. via a Novel Chimaeric Pathway

2009 ◽  
Vol 71-73 ◽  
pp. 219-222 ◽  
Author(s):  
Juan Pablo Cárdenas ◽  
Verónica Martínez ◽  
P. Covarrubias ◽  
David S. Holmes ◽  
Raquel Quatrini
Keyword(s):  
Carbon Dioxide ◽  
Co2 Fixation ◽  
Carbon Fixation ◽  
Mine Drainage ◽  
Tca Cycle ◽  
Central Carbon Metabolism ◽  
Metabolic Reconstruction ◽  
Marker Genes ◽  
Type I ◽  
Genes Encoding

Previous physiological studies of the genus Ferroplasma have indicated that these microorganisms are capable of fixing CO2 in the presence of ferrous iron and low concentrations of yeast extract. Analysis of the gene complement of Ferroplasma acidarmanus fer1 and two partial genomes of Ferroplasma type I and II derived from the Iron Mountain acid mine drainage metagenome revealed the absence of several functional marker genes encoding key enzymes of three know alternative CO2 fixation routes present in archaea, i.e. the 3-hydroxypropionate cycle, the Ljungdahl–Wood pathway and the reverse TCA cycle. It is thus intriguing how these chemoautotrophic archaeal species deal with their requirements for carbon and suggests that they might have a distinct CO2 fixation route, as yet unreported. Using comparative genomics and metabolic reconstruction strategies, a putative pathway was detected for C1 fixation consisting of four main steps: 1) conversion of carbon monoxide to carbon dioxide with gain of energy and/or 2) reduction of carbon dioxide to formate, 3) incorporation of formate to tetrahydrofolate and 4) donation of the carbon moiety of tetrahydrofolate to glycine to produce serine. Steps 1 to 3 involve enzymes that correspond to some of the Ljungdahl–Wood pathway proteins, whereas step 4 resembles the well known “serine cycle”, utilized by methylotrophic microorganisms for formaldehyde fixation. Thus, this chimaeric pathway might represent the missing carbon fixation route in Ferroplasmatales. Herein, we discuss the implications of these findings in the context of central carbon metabolism requirements for biomass production in acidic environments.

scholarly journals Transcriptomic Analysis of the Photosynthetic, Respiration, and Aerenchyma Adaptation Strategies in Bermudagrass (Cynodon dactylon) under Different Submergence Stress

2021 ◽  
Vol 22 (15) ◽  
pp. 7905
Author(s):  
Zhongxun Yuan ◽  
Xilu Ni ◽  
Muhammad Arif ◽  
Zhi Dong ◽  
Limiao Zhang ◽  
...  
Keyword(s):  
Carbon Fixation ◽  
Cynodon Dactylon ◽  
Chlorophyll Biosynthesis ◽  
Tca Cycle ◽  
Ethylene Signaling ◽  
Physiological Mechanisms ◽  
Cell Wall Modification ◽  
Aerenchyma Formation ◽  
Submergence Stress ◽  
Photosynthesis And Respiration

Submergence impedes photosynthesis and respiration but facilitates aerenchyma formation in bermudagrass. Still, the regulatory genes underlying these physiological responses are unclear in the literature. To identify differentially expressed genes (DEGs) related to these physiological mechanisms, we studied the expression of DEGs in aboveground and underground tissues of bermudagrass after a 7 d treatment under control (CK), shallow submergence (SS), and deep submergence (DS). Results show that compared with CK, 12276 and 12559 DEGs were identified under SS and DS, respectively. Among them, the DEGs closely related to the metabolism of chlorophyll biosynthesis, light-harvesting, protein complex, and carbon fixation were down-regulated in SS and DS. Meanwhile, a large number of DEGs involved in starch and sucrose hydrolase activities, glycolysis/gluconeogenesis, tricarboxylic acid (TCA) cycle, and oxidative phosphorylation were down-regulated in aboveground tissues of bermudagrass in SS and DS. Whereas in underground tissues of bermudagrass these DEGs were all up-regulated under SS, only beta-fructofuranosidase and α-amylase related genes were up-regulated under DS. In addition, we found that DEGs associated with ethylene signaling, Ca2+-ROS signaling, and cell wall modification were also up-regulated during aerenchyma formation in underground tissues of bermudagrass under SS and DS. These results provide the basis for further exploration of the regulatory and functional genes related to the adaptability of bermudagrass to submergence.

scholarly journals Biochemical elucidation of citrate accumulation in Synechocystis sp. PCC 6803 via kinetic analysis of aconitase

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Maki Nishii ◽  
Shoki Ito ◽  
Noriaki Katayama ◽  
Takashi Osanai
Keyword(s):  
Kinetic Analysis ◽  
Chemical Equilibrium ◽  
Isocitrate Dehydrogenase ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Unicellular Cyanobacterium ◽  
Citrate Accumulation ◽  
Kinetics Of ◽  
Synechocystis Sp ◽  
Pcc 6803

AbstractA unicellular cyanobacterium Synechocystis sp. PCC 6803 possesses a unique tricarboxylic acid (TCA) cycle, wherein the intracellular citrate levels are approximately 1.5–10 times higher than the levels of other TCA cycle metabolite. Aconitase catalyses the reversible isomerisation of citrate and isocitrate. Herein, we biochemically analysed Synechocystis sp. PCC 6803 aconitase (SyAcnB), using citrate and isocitrate as the substrates. We observed that the activity of SyAcnB for citrate was highest at pH 7.7 and 45 °C and for isocitrate at pH 8.0 and 53 °C. The Km value of SyAcnB for citrate was higher than that for isocitrate under the same conditions. The Km value of SyAcnB for isocitrate was 3.6-fold higher than the reported Km values of isocitrate dehydrogenase for isocitrate. Therefore, we suggest that citrate accumulation depends on the enzyme kinetics of SyAcnB, and 2-oxoglutarate production depends on the chemical equilibrium in this cyanobacterium.

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