Overexpression of sedoheptulose-1,7-bisphosphatase enhances photosynthesis inChlamydomonas reinhardtiiand has no effect on the abundance of other Calvin-Benson cycle enzymes

Abstract

11The productivity of plants and microalgae needs to be increased to feed the growing world population 12 and to promote the development of a low-carbon economy. This goal can be achieved by improving 13 photosynthesis via genetic engineering. In this study, we have employed the Modular Cloning strategy 14to overexpress the Calvin-Benson cycle (CBC) enzyme sedoheptulose-1,7 bisphosphatase (SBP1) up 15 to 3-fold in the unicellular green alga Chlamydomonas reinhardtii. The protein derived from the 16 nuclear transgene represented ~0.3% of total cell protein. Photosynthetic rate and growth were 17 significantly increased in SBP1-overexpressing lines under high-light and high-CO2 conditions. 18Absolute quantification of the abundance of all other CBC enzymes by the QconCAT approach 19 revealed no consistent differences between SBP1-overexpressing lines and the recipient strain. This 20 analysis also revealed that the eleven CBC enzymes represent 11.9% of total cell protein in 21Chlamydomonas. Here the range of concentrations of CBC enzymes turned out to be much larger than 22 estimated earlier, with a 128-fold difference between the most abundant CBC protein (rbcL) and the 23 least abundant (triose phosphate isomerase). Accordingly, the concentrations of the CBC intermediates 24 are often but not always higher than the binding site concentrations of the enzymes for which they act 25 as substrates. The enzymes with highest substrate to binding site ratios might represent good candidates 26 for overexpression in subsequent engineering steps. 27 28 29 30 31 Overexpression of SBP1 in Chlamydomonas 2

Overexpression of Sedoheptulose-1,7-Bisphosphatase Enhances Photosynthesis in Chlamydomonas reinhardtii and Has No Effect on the Abundance of Other Calvin-Benson Cycle Enzymes

“…We are grateful to Karin Gries for technical help. This manuscript has been released as a pre-print at bioRxiv (Hammel et al, 2020).…”

Real-time monitoring of subcellular H2O2distribution inChlamydomonas reinhardtii

“…For high-light exposure, cells were grown to ~1×10 6 cells/ml, transferred to an open 1-L beaker, placed on an orbital shaker, and exposed to 1000 or 1500 μmol photons m −2 s −1 provided by CF Grow® (CXB3590-X4). Transformation was done with the glass beads method (Kindle, 1990) as described previously (Hammel et al, 2020) with constructs linearized with NotI. Transformants were selected on 100 μg ml −1 spectinomycin.…”

“…Protein extractions, SDS–PAGE, semi-dry blotting and immunodetections were carried out as described previously (Hammel et al, 2020). Antibodies used for immunodetection were mouse anti-HA (Sigma H9658, 1:10,000) and rabbit anti-GFP (Roche, Cat.…”

High-Resolution Crystal Structure of Chloroplastic Ribose-5-Phosphate Isomerase from Chlamydomonas reinhardtii—An Enzyme Involved in the Photosynthetic Calvin-Benson Cycle

“…In C. reinhardtii, the estimated CrRPI1 protein quantity is 0.34 amol per cell and 2.5 µmol.L −1 in the chloroplast [12]. Considering the cellular concentration of metabolites [12,13], the approximate molar ratio of substrate or product to CrRPI1 is respectively estimated at 80 and 50.…”

“…In C. reinhardtii, the estimated CrRPI1 protein quantity is 0.34 amol per cell and 2.5 µmol.L −1 in the chloroplast [12]. Considering the cellular concentration of metabolites [12,13], the approximate molar ratio of substrate or product to CrRPI1 is respectively estimated at 80 and 50. In order to adapt to the variations of metabolites concentration in the chloroplast stroma, CBC enzymatic activities are generally regulated according to the environmental light [14,15].…”