Liang Feiyan, Lindblad Peter
Microbial Chemistry, Department of Chemistry-Ångström, Uppsala University, Box 523, SE-751 20 Uppsala, Sweden.
Metab Eng Commun. 2017 Feb 20;4:29-36. doi: 10.1016/j.meteno.2017.02.002. eCollection 2017 Jun.
The ribulose-1,5-bisphosphate (RuBP) oxygenation reaction catalyzed by Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is competing with carboxylation, being negative for both energy and carbon balances in photoautotrophic organisms. This makes RuBisCO one of the bottlenecks for oxygenic photosynthesis and carbon fixation. In this study, RuBisCO was overexpressed in the unicellular cyanobacterium PCC 6803. Relative RuBisCO levels in the engineered strains FL50 and FL52 increased 2.1 times and 1.4 times, respectively, and both strains showed increased growth, photosynthesis and RuBisCO activity. The oxygen evolution rate increased by 54% and 42% on per chlorophyll basis, while the RuBisCO activity increased by 52% and 8.6%, respectively. The overexpressed RuBisCO were tagged with a FLAG tag, in strain FL50 on the N terminus of the large subunit while in strain FL52 on the C terminus of the small subunit. The presence of a FLAG tag enhanced transcription of the genes encoding RuBisCO, and, with high possibility, also enhanced the initiation of translation or stability of the enzyme. However, when using a streptavidin-binding tag II (strep-tag II), we did not observe a similar effect. Tagged RuBisCO offers an opportunity for further studying RuBisCO expression and stability. Increased levels of RuBisCO can further improve photosynthesis and growth in the cyanobacterium PCC 6803 under certain growth conditions.
1,5 - 二磷酸核酮糖羧化酶/加氧酶（RuBisCO）催化的1,5 - 二磷酸核酮糖（RuBP）加氧反应与羧化反应相互竞争，这对光合自养生物的能量和碳平衡均产生负面影响。这使得RuBisCO成为产氧光合作用和碳固定的瓶颈之一。在本研究中，RuBisCO在单细胞蓝藻PCC 6803中过表达。工程菌株FL50和FL52中RuBisCO的相对水平分别提高了2.1倍和1.4倍，并且这两个菌株的生长、光合作用和RuBisCO活性均有所增加。以每叶绿素计，放氧速率分别提高了54%和42%，而RuBisCO活性分别提高了52%和8.6%。过表达的RuBisCO用FLAG标签进行标记，在菌株FL50中标记在大亚基的N端，而在菌株FL52中标记在小亚基的C端。FLAG标签的存在增强了编码RuBisCO基因的转录，并且很有可能还增强了该酶的翻译起始或稳定性。然而，当使用链霉亲和素结合标签II（strep - tag II）时，我们未观察到类似的效果。带标签的RuBisCO为进一步研究RuBisCO的表达和稳定性提供了机会。在某些生长条件下，提高RuBisCO的水平可以进一步改善蓝藻PCC 6803的光合作用和生长。
