Directed Evolution of an Improved Rubisco; In Vitro Analyses to Decipher Fact from Fiction

• Main Authors: Yu Zhou, Spencer Whitney
• Format: Article
• Language: English
• Published: MDPI AG 2019-10-01
• Series: International Journal of Molecular Sciences
• Subjects: photosynthesis; carbon fixation; synthetic biology; metabolic engineering
• Online Access: https://www.mdpi.com/1422-0067/20/20/5019

Inaccuracies in biochemically characterizing the amount and CO₂-fixing properties of the photosynthetic enzyme Ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase continue to hamper an accurate evaluation of Rubisco mutants selected by directed evolution. Here, we outline an analytical pipeline for accurately quantifying Rubisco content and kinetics that averts the misinterpretation of directed evolution outcomes. Our study utilizes a new T7-promoter regulated Rubisco Dependent <i>Escherichia coli</i> (RDE3) screen to successfully select for the first <i>Rhodobacter sphaeroides</i> Rubisco (<i>Rs</i>Rubisco) mutant with improved CO₂-fixing properties. The <i>Rs</i>Rubisco contains four amino acid substitutions in the large subunit (RbcL) and an improved carboxylation rate (<i>k_cat^C</i>, up 27%), carboxylation efficiency (<i>k_cat^C</i>/<i>K_m</i> for CO₂, increased 17%), unchanged CO₂/O₂ specificity and a 40% lower holoenzyme biogenesis capacity. Biochemical analysis of <i>Rs</i>Rubisco chimers coding one to three of the altered amino acids showed Lys-83-Gln and Arg-252-Leu substitutions (plant RbcL numbering) together, but not independently, impaired holoenzyme (L₈S₈) assembly. An N-terminal Val-11-Ile substitution did not affect <i>Rs</i>Rubisco catalysis or assembly, while a Tyr-345-Phe mutation alone conferred the improved kinetics without an effect on <i>Rs</i>Rubisco production. This study confirms the feasibility of improving Rubisco by directed evolution using an analytical pipeline that can identify false positives and reliably discriminate carboxylation enhancing amino acids changes from those influencing Rubisco biogenesis (solubility).