What are climate-friendly plants? - PowerPoint Presentation

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2. What are climate-friendly plants? Carbon dioxide concentration in 2021 = 419 ppmDeforestation, industry, increased burning of fossil fuels, agriculture, rapid urbanization…High level of CO2 affect whole biosphereIncrease of temperature, degraded and disappearing ecosystem, extreme weather, acidification of the oceans etc.

3. Why do we need climate-friendly plants? C3 plantsC4 plantsCAM plants

4. Why do we need climate-friendly plants? C3 plantsC4 plantsCAM plantsKhan Academy. Accessed 28.4.2022.  https://www.khanacademy.org.

5. PhotorespirationEnzym rubisco – the most abundant enzyme on the EarthRubisco binds with O2 instead of CO2Only in C3 plants, during hot and dry daysDrains the Calvin cycle =  photosynthetic output, no energy (ATP) is produced, no „food molecules“ (G3P) are producedWasteful process with NO benefits, it even limits the growth rate of plantsCalvin CyclePhotorespirationRubiscoCO2O23PGlycerate (food)Triose PhosphateRibulose-5PRibulose 1,5-BPRibulose 1,5-BP2-PGlycolate3PGlycerateGlycineSerine3PGlycerateCO2 and ammonia -energy

6. Solution: synthetic biology Biological systemsSelf-optimizing Self-repairingSelf-propagatingSustainable Environmental friendly Step 1:Designing theoretical reactions in silicoOutput: Finding/engineering enzymesStep 2:Building and optimizing the pathwayOutput: Synthetic pathwayStep 3:Introducing of the synthetic pathway in living cells

7. 1. Identification of enzymesWhich enzymes are suitable? Measuring of efficiency of enzymes kcat= Turnover number (s-1)Chemical conversion of substrate, molecules per second per active site Km= Michaelis constant(M-1)Substrate concentration at half maximum activityCatalytic efficiencykcat / Km

8. 2. Building and optimizing the pathwayCETCH cycles17 different enzymes from 9 organisms13 reactions that increased the rate of CO2 up to 5-times more efficiently than Calvin cycleLess energy consumptionDRAWBACK: acetyl-CoA must be addedAlternative photorespiration 3Glycolate deghydrogenase (Chlamydomonas reinhardtii) and malate synthase (Cucurbita maxima) Blocking of plastidic glycolate/glycerate transporter 1 (PLGG1) – reducing oxygenase activity of rubiscoCETCH cycle peroxisomePLGG1glycolateglyoxylatemalatepyruvateAcetyl-CoACO2CO2

9. 2. Building and optimizing the pathwayModel 1: Native rubiscoO2CO2RuBPPG+PGAPGAPGARuBP2x 3PGAPLGG1CO2sugarsModel 2: AP3 rubiscoO2CO2RuBP2P-glycolateglyoxalatePGARuBP2x 3PGAPLGG1sugarsglycolatemalatepyruvateAcetyl-CoAModel 3: CETCH + AP3 rubiscoO2CO2RuBP2P-glycolateglyoxalateMCoAC CoAACoAPLGG1sugarsglycolatemalatepyruvateAcetyl-CoAECoACETCHphotorespirationphotorespirationNaseem et al. 2020

10. Optimized modelOnly AP3 model: bad connection and low regeneration rate observedOnly CETCH model: substrate competition, problematic regeneration of NADPH AP3+CETCH + blocking of PLGG1 Repressed enzyme activities of photorespiratory pathwayReduction of oxygenase activity of rubisco Drastically reduced flux of metabolites into the peroxisomesGlycolate is produced, but it is converted into malate in chloroplast which could be used for the another CO2 convertion75% higher enzyme activity

11. Next step: introduction into plants Cloning strategyPreparation of 19 enzymes Gibson assembly Subcloning to the plant vectorTransformation of Agrobacterium tumefaciens Floral dip methodGrowing of the seedlings and selection of vector-bearing plantsAssessment of carbon dioxide uptake

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