Satagopan Sriram, Tabita F Robert
Department of Microbiology, The Ohio State University, Columbus, OH, USA.
FEBS J. 2016 Aug;283(15):2869-80. doi: 10.1111/febs.13774. Epub 2016 Jun 27.
Recapturing atmospheric CO2 is key to reducing global warming and increasing biological carbon availability. Ralstonia eutropha is a biotechnologically useful aerobic bacterium that uses the Calvin-Benson-Bassham (CBB) cycle and the enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) for CO2 utilization, suggesting that it may be a useful host to bioselect RubisCO molecules with improved CO2 -capture capabilities. A host strain of R. eutropha was constructed for this purpose after deleting endogenous genes encoding two related RubisCOs. This strain could be complemented for CO2 -dependent growth by introducing native or heterologous RubisCO genes. Mutagenesis and suppressor selection identified amino acid substitutions in a hydrophobic region that specifically influences RubisCO's interaction with its substrates, particularly O2 , which competes with CO2 at the active site. Unlike most RubisCOs, the R. eutropha enzyme has evolved to retain optimal CO2 -fixation rates in a fast-growing host, despite the presence of high levels of competing O2 . Yet its structure-function properties resemble those of several commonly found RubisCOs, including the higher plant enzymes, allowing strategies to engineer analogous enzymes. Because R. eutropha can be cultured rapidly under harsh environmental conditions (e.g., with toxic industrial flue gas), in the presence of near saturation levels of oxygen, artificial selection and directed evolution studies in this organism could potentially impact efforts toward improving RubisCO-dependent biological CO2 utilization in aerobic environments.
d-ribulose 1,5-bisphosphate carboxylase/oxygenase, EC 4.1.1.39; phosphoribulokinase, EC 2.7.1.19.
捕获大气中的二氧化碳是减少全球变暖及增加生物碳可利用性的关键。真养产碱菌是一种具有生物技术用途的需氧细菌，它利用卡尔文-本森-巴斯姆（CBB）循环和1,5-二磷酸核酮糖羧化酶/加氧酶（RubisCO）来利用二氧化碳，这表明它可能是一个用于生物筛选具有改进二氧化碳捕获能力的RubisCO分子的有用宿主。为此，在删除编码两种相关RubisCO的内源基因后构建了真养产碱菌的宿主菌株。通过引入天然或异源RubisCO基因，该菌株可在依赖二氧化碳的情况下生长。诱变和抑制子筛选确定了疏水区域中的氨基酸取代，该区域特异性影响RubisCO与其底物的相互作用，特别是与在活性位点与二氧化碳竞争的氧气的相互作用。与大多数RubisCO不同，尽管存在高水平的竞争性氧气，但真养产碱菌的酶已进化到在快速生长的宿主中保持最佳二氧化碳固定率。然而，其结构-功能特性类似于几种常见的RubisCO，包括高等植物的酶，这使得设计类似酶的策略成为可能。由于真养产碱菌可以在恶劣环境条件下（例如，与有毒工业废气一起）、在接近饱和水平的氧气存在下快速培养，因此在这种生物体中进行人工选择和定向进化研究可能会对改善需氧环境中依赖RubisCO的生物二氧化碳利用的努力产生潜在影响。
1,5-二磷酸核酮糖羧化酶/加氧酶，EC 4.1.1.39；磷酸核酮糖激酶，EC 2.7.1.19。
