Subategory:

Mutants

Description:

A codon-optimized Arabidopsis rbcL gene was created that encodes a large subunit with 12 Chlamydomonas amino-acid substitutions (S2V, S10G, V11A, E19D, K21R, N442G, E443D, I444V, E447S, T466K, N468E, P470D) and that lacks four C-terminal residues (Asp-476, Gly-477, Gln-478, Glu-479). When the engineered gene was transformed into rbcLΔ/rbcS1-SSSO pf2 mt+ (which lacks Chlamydomonas rbcL and rbcS, but expresses spinach rbcS), photosynthesis-competent transformants were recovered on minimal medium with 5% CO₂ in air. Thus, this mutant expresses a functional Rubisco comprised of spinach small subunits and engineered Arabidopsis large subunits. On minimal medium with 5% CO₂ in air, the mutant grows less than rbcL-LSATNC mt+ and rbcL-LSATNC-T391F mt+, but more than rbcL-LSATNC-T391F/rbcS1-SSAT pf2 mt+. Western analysis indicated that the mutant strain has a decreased level of Rubisco holoenzyme. This strain has been maintained with acetate medium in darkness to prevent selection for secondary mutations that may improve Rubisco function.

Species:

Chlamydomonas

Locus:

rbcL, RBCS1, PF2

Chromosome:

Chloroplast,2,11

Phenotype:

Requires elevated CO₂ for photosynthetic growth