COLLABORATIVE PROJECT: MAGIC - A multi-tiered approach to generating increased carbon dioxide in the chloroplast

光合作用几乎是社会各个方面的核心，从食品生产到工业建设。陆地光合作用与我们对其他自然资源的利用密切相关，它对世界上的水、矿物和碳循环施加了重大控制。例如，植物蒸腾作用被认为是最近与全球二氧化碳上升相关的淡水可用性变化的原因之一，它是未来20-30年预计出现的可用性水危机的核心。据估计，在同一时期，全球粮食产量需要增加50%才能跟上人口增长的步伐。直到最近，作物产量才与人口增长相匹配，但绿色革命中培育的谷物品种的收益在10年前就完全实现了。因此，现在寻找进一步提高光合效率的途径是至关重要的。在大多数物种中，二氧化碳在卡尔文-本森循环中被二磷酸核酮糖羧化酶/加氧酶（RuBisCO）固定，生成三碳化合物。RuBisCO的底物选择性非常差，并且会混淆地固定CO2和O2，这一事实使RuBisCO成为光合作用中效率最低的步骤。减少RuBisCO使用O2的一种方法是提高CO2分压（pCO2）。所谓的碳浓缩机制（CCMs）在自然界中已经进化了多次，尽管不是大多数常见作物物种的特征。因此，比较表明，如果在作物中绕过RuBisCO对O2的利用，总产量可能增加大约50%。大量的资源已经投入到RuBisCO的工程中，以提高二氧化碳的选择性，并在水稻中引入单细胞版本的C4光合作用，但这些方法尚未看到光合效率的逐步变化。一套尚待探索的新策略是采用光驱动泵、阴离子交换运输和底物通道向RuBisCO提供二氧化碳。虽然这三个过程都是自然发生的，并在生物学中被综合运用，但迄今为止，还没有发现任何一个过程能促进光合作用。我们的目标是开发一种相当于“两阶段泵”的物质：将(1)运输机制集中在叶绿体中，该机制由来自主要的halobium halobium的光驱动离子泵halorhodopsin （hR）提供动力；(2)叶绿体内的底物通道使用来自Clostridium或blue obacteria的一个或多个分子“构建块”来携带HCO3-或四碳中间体到RuBisCO。这种两阶段策略预计将最大限度地提高由hR吸收的光能独立驱动的CCM增益，并且它具有工程hR开发光合光谱以外未使用的光资产的附加潜力。此外，该方法的一个主要特点是其模块化的性质：可以并行开发两级泵的每个阶段，并在分子、细胞和整体水平上分别评估其功能，然后将组件组合起来进行最终验证。这种模块化方法确保了在三年内实现我们目标的最高效率和速度。

Photosynthesis is at the core of virtually every aspect of society, from food production to industrial construction. Terrestrial photosynthesis is intimately connected with our use of other natural resources, and it exerts major controls on the water, mineral and carbon cycles of the world. For example, plant transpiration is thought to have contributed to recent changes in fresh-water availability associated with the global rise in CO2, and it is at the centre of a crisis in water availability expected over the next 20-30 years. Over this same period it is estimated that a 50% increase in global food production will be required to keep pace with the increase in human population. Crop yields have matched population growth until recently, but the gains from cereal cultivars bred in the Green Revolution were realised in full a decade ago. Thus it is vital that routes to further improvements in photosynthetic efficiency are sought now. In most species, CO2 is fixed by Ribulose Bisphosphate Carboxylase/Oxygenase (RuBisCO) in the Calvin-Benson cycle to generate a three-carbon compound. RuBisCO is remarkably poor in its substrate selectivity and promiscuously fixes both CO2 and O2, a fact that makes RuBisCO arguably the most inefficient step in photosynthesis. One way of reducing O2 use by RuBisCO is to raise the partial pressure of CO2 (pCO2). So-called carbon concentrating mechanisms (CCMs) have evolved multiple times in nature, albeit not as a feature of most common crop species. Thus, comparisons suggest roughly a 50% increase in overall yield might be realised if O2 use by RuBisCO were bypassed in crops. Significant resources have gone into engineering RuBisCO for increased CO2 selectivity and introducing a single-celled version of C4 photosynthesis in rice, but these approaches have yet to see a step change in photosynthetic efficiency. One new set of strategies yet to be explored is to co-opt light-driven pumps, anion exchange transport and substrate channelling to supply CO2 to RuBisCO. To date none of these processes is known to facilitate photosynthesis, although all three occur naturally and have been employed synthetically in biology. It is our goal to develop the equivalent of a 'two-stage pump': placing in series (1) a transport mechanism to concentrate HCO3- in the chloroplast powered by the light-driven ion pump halorhodopsin (hR) from the archeon Halobacterium halobium, and (2) substrate channelling within the chloroplast using one or more molecular 'building blocks' from Clostridium or cyanobacteria to carry HCO3- or a four-carbon intermediate to RuBisCO. This two-stage strategy is expected to maximise CCM gain driven independently with light energy absorbed by hR, and it has the added potential for engineering hR to tap the unused asset of light beyond the photosynthetic spectrum. Furthermore, an overarching feature of this approach is in its modular nature: it will be possible to develop each stage of the two-stage pump in parallel, and to assess its functionality separately at molecular, cellular and whole-organismal levels, combining the components thereafter for final validation. This modular approach ensures the maximum efficiency and speed in realising our goal within the three-year period.