Genkov, Todor , Spreitzer, Robert J. .
Hybrid Rubisco enzymes expressed in Chlamydomonas reinhardtii.
It has been difficult to express and assemble Rubisco large and small subunits from different species in a suitable eukaryotic host. If hybrid holoenzymes could be generated routinely, they may provide new information about the role of the small subunit in Rubisco function and assembly. Attempts to express rbcS cDNA in a Chlamydomonas mutant that lacks the rbcS gene family were unsuccessful because two of the three rbcS introns are required for expression. To circumvent this problem, introns 1 and 2 were engineered into the rbcS region that encodes the transit peptide. When followed by an intronless sequence encoding the mature small subunit, photosynthesis-competent transformants were recovered at frequencies comparable to those obtained with genomic rbcS. However, when cyanobacterial, red algal, or spinach codon-optimized rbcS cDNA was engineered after the intron-containing, transit-peptide region of Chlamydomonas rbcS, no transformants could be obtained. PCR engineering of the amino-terminal region of the mature small subunit of spinach (substituting eight residues with those of Chlamydomonas) produced a spinach rbcS cDNA that could transform the Chlamydomonas rbcS knockout strain to photosynthetic competence. The hybrid Rubisco (with spinach small subunits and Chlamydomonas large subunits) is expressed at a near-normal level. Relative to Chlamydomonas Rubisco, the hybrid holoenzyme has normal CO2/O2 specificity. However, carboxylation Vmax is decreased to a value similar to that of spinach Rubisco, and Km CO2 and O2 are increased to values greater than those of either native enzyme. Work is in progress to substitute more-divergent large and small subunits. Transformants have just recently been obtained with cyanobacterial rbcS. This work is supported by the US DOE.
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University of Nebraska, Department of Biochemistry
1 - University of Nebraska, Department of Biochemistry, Lincoln, NE, 68588, USA
Presentation Type: Plant Biology Abstract
Location: Exhibit Hall (Northeast, Southwest & Southeast)/Hilton
Date: Sunday, July 8th, 2007
Time: 8:00 AM