14-PSIL MAGIC: a multi-tiered approach to gaining increased carbon

在植物的卡尔文-本森循环中，酶RuBisCO固定CO2以产生两个分子的3-磷酸甘油酸。大约36亿年前，RuBisCO在高CO2和低O2的大气中进化，几乎不需要区分这两种气体。在今天的大气中，RuBisCO可以修复二氧化碳和氧气。后者产生磷酸乙醇酸，其通过光呼吸回收，但以代表光合效率显著损失的能量为代价。减少RuBisCO固定O2的一种方法是提高CO2的分压。为此目的，碳浓缩机制（CCM）已经发展了多次。例如，C4光合作用使用磷酸烯醇-丙酮酸羧化酶（PEPC），一种不具有加氧酶活性的酶，将HCO 3暂时固定在C4酸中;细胞特化允许释放和浓缩CO 2，以通过RuBisCO重新固定。如果以类似的方式绕过RuBisCO的O2固定，作物产量可能增加50%。大量的资源已经投入到工程RuBisCO中，以提高CO2的选择性，并在水稻中引入C4光合作用的单细胞版本，但光合效率的阶跃变化尚未实现。来自美国大学的调查人员（宾夕法尼亚州立大学的约翰·戈尔贝克（John Golbeck）和密歇根州立大学的谢丽尔·克菲尔德（Cheryl Kerfeld））和英国（迈克布拉特（MB），格拉斯哥;奈杰尔巴勒斯（NB），沃里克;和Julian Hibbern（JH），剑桥）在2010年参加了NSF/BBSRC的想法实验室，在那里他们提出了一个新的策略来解决这个问题，这一提议已经成熟到技术实现的水平。他们现在加入了尼克斯米尔诺夫（NS，埃克塞特）和马尼什库马尔（MK，宾夕法尼亚州立大学），谁带来了额外的和关键的专业知识，该项目。该研究有两个主题：由盐视紫红质和阴离子/HCO 3-交换剂AE 1组成的光驱动离子泵;以及使用人工支架将CO 2引导至RuBisCO。一个平行的目标是重新设计光驱动的离子泵，以直接运输HCO 3-，并吸收光合作用不使用的光能。这些努力是以CO2输送和同化的数学建模为基础的，以围绕以下组件进行直接实验。盐视紫红质（HR）是一种膜蛋白，由7个跨膜α-螺旋和一个结合的视黄醇组成。视网膜经历光驱动的13-顺式和全反式构象之间的键旋转，以驱动离子传输。HR也转运其他卤化物，离子选择性似乎是pHR转运位点的局部特征。pHR是足够混杂的，使工程的光驱动HCO 3- pump成为可能。阴离子/Bicycline交换：红细胞Band 3蛋白（AE 1）促进Cl-/HCO 3-交换通过膜。它产生接近平衡的高通量，并且对pH值很不敏感，使其非常适合设计HCO 3积累机制。AE 1转运蛋白在哺乳动物细胞培养、非洲爪蟾卵母细胞和酵母中具有功能，对体内平衡或生长没有不良影响，这对于合成工程是最有希望的。AE 1的模块化结构提供了一种现实的策略，用于将HCO 3泵送耦合到pHR驱动的Cl- transport.Artificial支架：CO2扩散需要在局部受到足够的时间限制，以使其被RuBisCO固定。基质通道存在于包括植物在内的多种自然系统中。效率的提高来自物理上的接近和“海绵”般的缓冲，使中间体的转移和最大限度地减少流失的基板。

In the Calvin-Benson cycle of plants, the enzyme RuBisCO fixes CO2 to produce two molecules of 3-phosphoglycerate. RuBisCO evolved ~3.6bn years ago in an atmosphere of high CO2 and low O2, with little need to discriminate between the two gases. In today's atmosphere RuBisCO fixes both CO2 and O2. The latter generates phosphoglycolate, which is retrieved by photorespiration but at an energy cost that represents a significant loss in photosynthetic efficiency. One method to reduce O2 fixation by RuBisCO is to raise the partial pressure of CO2. Carbon concentrating mechanisms (CCMs) have evolved multiple times to this end. For example, C4 photosynthesis uses phosphoenol-pyruvate carboxylase (PEPC), an enzyme that does not possess oxygenase activity, to fix HCO3- temporarily in C4 acids; cellular specialization allows release and concentration of CO2 for refixing by RuBisCO. As much as a 50% increase in yield might be realized in crops were O2 fixation by RuBisCO to be bypassed in a similar manner. Significant resources have already gone into engineering RuBisCO for increased CO2 selectivity and into introducing a single-celled version of C4 photosynthesis in rice, but a step change in photosynthetic efficiency has not yet been achieved. Investigators from Universities in the US (John Golbeck (JG), Penn State; and Cheryl Kerfeld (CK), Michigan State) and the UK (Mike Blatt (MB), Glasgow; Nigel Burroughs (NB), Warwick; and Julian Hibberd (JH), Cambridge) participated in an NSF/BBSRC Ideas Laboratory in 2010, at which they proposed a novel strategy to address this problem, a proposal that has since matured to the level of technological implementation. They are now joined by Nick Smirnoff (NS, Exeter) and Manish Kumar (MK, Penn State), who bring additional and key expertise to the project. The research has two themes: a light driven ion pump, composed of halorhodopsin and an anion/HCO3- exchanger, AE1; and the use of artificial scaffolds for channelling CO2 to RuBisCO. A parallel goal is to re-engineer the light-driven ion pump to transport HCO3- directly and to absorb light energy not used by photosynthesis. These efforts are underpinned with mathematical modelling of CO2 delivery and assimilation to direct experimentation based around the following components.Light-Driven Pump. Halorhodopsin (HR) is an integral membrane protein and consists of 7 transmembrane alpha-helices and a bound retinal. The retinal undergoes light-driven bond rotation between 13-cis and all-trans conformations to drive ion transport. HR transports other halides as well, and ion selectivity appears to be a localized feature of the pHR transport site. pHR is sufficiently promiscuous to make engineering a light-driven HCO3- pump a possibility.Anion/Bicarbonate Exchanger: The erythrocyte Band3 protein (AE1) facilitates Cl-/HCO3- exchange across the membrane. It generates a high flux close to equilibrium and is largely insensitive to pH, making it well suited to engineering a HCO3- accumulating mechanism. Most promising for synthetic engineering, the AE1 transporter is functional in mammalian cell cultures, Xenopus oocytes, and yeast without adverse effects on homeostasis or growth. The modular structure of AE1, offers a realistic strategy for coupling HCO3- pumping coupled to pHR-driven Cl- transport.Artificial Scaffolds: CO2 diffusion needs to be constrained locally for sufficient time to allow it to be fixed by RuBisCO. Substrate channelling is found in several natural systems, including plants. Efficiency gains arise from physical proximity and 'sponge'-like buffering that enables transfer of intermediates and minimizes runoff of substrates.