Figure 1

Dual HTS assay. (a) NADH-linked enzymatic assay of Rubisco activity. The reaction monitors the rate 3 PGA production from the carboxylation of Ribulose-1,5-bisphosphate (RuBP) by Rubisco. PGK, phosphoglycerokinase; BPGA, 1,3-bisphosphoglycerate; GAPDH, glyceraldehyde-3P dehydrogenase; GAP, glyceraldehyde-3P; TPI, triose-P isomerase; DHAP, dihydroxyacetone-P; GPDH, glycerol-3P dehydrogenase; G3P, glycerol-3P. (b) HPLC-ELSD chromatogram for the detection of 3PGA production/RuBP depletion by Rubisco (+and - represent cationic and anionic compounds in the sample, respectively) and (c), the variation in the concentration of these two compounds during the reaction as measured by HPLC-ELSD. (d) Fitness landscapes for RubRr mutant libraries that are adjusted by varying the MnCl₂ concentrations (in the case of Taq libraries) or by the amount of the DNA template (for mutazyme libraries). The relative RubRr activity of the clones is plotted in descending order and the dashed line shows the activity of the parental type in the assay: red circles, library I (mutazyme, 750 ng DNA template); blue circles, library II (Taq, 0.02 mM MnCl₂); black circles, library III (mutazyme, 100 ng DNA template); white circles, library IV (Taq, 0.03 mM MnCl₂); green circles, library V (Taq, 0.05 mM MnCl₂). (e) Directed evolution landscape for thermostability and activity relative to the parental RubRr enzyme (dashed lines). The mutants selected for further re-screening are contained in the blue rectangle (using thresholds of 0.5- and 1.3-fold over RubRr activity and thermostability, respectively).