Functional Hybrid Rubisco Enzymes with Plant Small Subunits and Algal Large Subunits ENGINEERED rbcS cDNA FOR EXPRESSION IN CHLAMYDOMONAS
Functional Hybrid Rubisco Enzymes with Plant Small Subunits and Algal Large Subunits ENGINEERED rbcS cDNA FOR EXPRESSION IN CHLAMYDOMONAS
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DOI：
10.1074/jbc.m110.124230
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发表时间：
2010-06-25
影响因子：
4.8
通讯作者：
Spreitzer, Robert J.
中科院分区：
文献类型：
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作者：
Genkov, Todor;Meyer, Moritz;Spreitzer, Robert J.
人们对叶绿体编码的1，5-二磷酸核酮糖羧化酶/加氧酶（Rubisco）大亚基作为工程增加光合作用中净CO2固定的靶点产生了很大的兴趣。这种酶的改进将导致粮食、纤维和可再生能源产量的增加。虽然大亚基含有活性位点，但rbcS核基因家族编码Rubisco小亚基，这也可以影响该酶的羧化催化效率和CO2/O-2特异性。为了进一步确定小亚基在Rubisco功能中的作用，通过转化缺乏rbcS基因家族的莱茵衣藻突变体，将来自菠菜、拟南芥和向日葵的小亚基与藻类大亚基组装。通过将外源rbcS cDNA与工程化含有rbcS内含子的衣原体rbcS转运肽序列融合，成功地在衣原体中表达。尽管植物Rubisco通常具有比衣原体Rubisco更大的CO2/O-2特异性但更低的羧化Vmax，但杂合酶具有3-11%的CO2/O-2特异性增加，并保持接近正常的Vmax值。因此，小的亚基可以对Rubisco的整体催化性能做出显著贡献。尽管具有正常量的催化活性的Rubisco，杂交突变株显示降低的光合生长水平和缺乏叶绿体蛋白核。看来，小的亚基包含负责靶向Rubisco的藻类蛋白核，这是CO2被集中的最佳光合作用的网站的结构元件。
There has been much interest in the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase ( Rubisco) as a target for engineering an increase in net CO2 fixation in photosynthesis. Improvements in the enzyme would lead to an increase in the production of food, fiber, and renewable energy. Although the large subunit contains the active site, a family of rbcS nuclear genes encodes the Rubisco small subunits, which can also influence the carboxylation catalytic efficiency and CO2/O-2 specificity of the enzyme. To further define the role of the small subunit in Rubisco function, small subunits from spinach, Arabidopsis, and sunflower were assembled with algal large subunits by transformation of a Chlamydomonas reinhardtii mutant that lacks the rbcS gene family. Foreign rbcS cDNAs were successfully expressed in Chlamydomonas by fusing them to a Chlamydomonas rbcS transit peptide sequence engineered to contain rbcS introns. Although plant Rubisco generally has greater CO2/O-2 specificity but a lower carboxylation V-max than Chlamydomonas Rubisco, the hybrid enzymes have 3-11% increases in CO2/O-2 specificity and retain near normal Vmax values. Thus, small subunits may make a significant contribution to the overall catalytic performance of Rubisco. Despite having normal amounts of catalytically proficient Rubisco, the hybrid mutant strains display reduced levels of photosynthetic growth and lack chloroplast pyrenoids. It appears that small subunits contain the structural elements responsible for targeting Rubisco to the algal pyrenoid, which is the site where CO2 is concentrated for optimal photosynthesis.