Bilateral NSF/BIO-BBSRC Synthesis of Microcompartments in Plants for Enhanced Carbon Fixation

全球对粮食和燃料的需求正在稳步增长，而近年来，许多主要粮食作物通过传统育种获得的产量增长趋于平稳。作为作物改良源泉的自然变异正逐渐枯竭，因此需要新的努力，包括合成生物学的投入来提高光合作用效率。超过90%的生物量直接来源于光合产物。固定碳酶Rubisco（核酮糖-1:5-二磷酸羧化酶/加氧酶）的性质限制了陆地植物光合作用的效率。Rubisco可以催化RuBP（核酮糖-1,5-二磷酸酯）与CO2的结合，也可以催化RuBP与氧的反应，导致光呼吸，这一过程中先前固定的CO2丢失。蓝藻和一些陆地植物已经进化到通过发展在Rubisco附近集中二氧化碳的机制来处理大气中氧气的增加。然而，许多全球重要作物缺乏这种能力；相反，它们利用Rubisco酶，这种酶具有更高的二氧化碳亲和力，但比具有碳浓缩机制的植物（如玉米）中的Rubisco酶慢。因此，这些植物必须投入相当数量的蛋白质，因此，氮，使Rubisco进行足够数量的碳固定，降低产量和生物量生产。一个突出的千年目标是找到提高光合作用产量的方法，以增强生物质生产。根据已发表的计算模型，用一种速度更快、二氧化碳特异性更低的酶替代内源性Rubisco，以及碳浓缩机制，是显著改善二氧化碳固定的一种方法。我们建议为此目的开展工作。我们将在陆地植物叶绿体中安装一种新的基于蓝藻的碳浓缩机制，并提供必要的分子机制来促进其运作。调控模块将从叶绿体基因组和叶绿体膜靶向碳酸氢盐泵的核基因组中产生，以表达蓝藻羧基体的成分。作为原理证明，这项工作将在烟草中进行，烟草是叶绿体转化最快的物种。我们已经为这一工程壮举奠定了基础，证明了几个组件可以被引入；例如，在烟草叶绿体中产生新的微室。我们希望从该项目中获得的知识将为随后的努力提供信息，以增强其他物种的光合作用，例如，通过将合成微室引入大豆等物种，这些物种的叶绿体和核转化技术已经可用。知识将通过生化和微观研究获得，例如关于蓝藻蛋白质的化学计量对微室大小、形态、碳浓度和光合作用功能的影响。我们将研究合成叶绿体操纵子上基因调控序列的特征，这些操纵子需要以微室组装所需的数量和比例表达蛋白质。我们希望这些发现对未来的项目也有价值，并通过合成人工微室来增加植物的其他能力。

Global demand for food and fuel is steadily increasing, while gains in yield of many major food crops through traditional breeding have leveled off in recent years. The natural variation that has been the source of substantial crop improvement is becoming exhausted, so that new efforts, including input from synthetic biology will be needed to improve photosynthetic efficiency. More than 90% of biomass is derived directly from photosynthetic products. The properties of the carbon-fixing enzyme Rubisco (ribulose-1:5-bisphosphate carboxylase/oxygenase) limit the efficiency of photosynthesis in land plants. Rubisco can catalyze the combination of RuBP (ribulose-1,5-bisphophate) with CO2, but also can catalyze the reaction of RuBP with oxygen, leading to photorespiration, a process in which previously fixed CO2 is lost. Cyanobacteria and some land plants have evolved to deal with an increase of oxygen in the atmosphere by developing mechanisms that concentrate CO2 near Rubisco. However, many of the globally important crop plants lack this ability; instead, they utilise Rubisco enzymes that have higher CO2 affinity but are slower than Rubisco enzymes in plants with carbon-concentrating mechanisms such as maize. Consequently, these plants must devote considerable amounts of protein, and thereby, nitrogen, to allow Rubisco to carry out adequate amounts of carbon fixation, reducing yield and biomass production.One of the outstanding millenial goals is to find ways to improve photosynthetic yields for enhanced biomass production. Replacing endogenous Rubisco with a faster enzyme with less CO2 specificity, along with a carbon concentrating mechanism, is one way to significantly improve CO2 fixation, according to published computational models. We propose to undertake work to this end. We will install a novel cyanobacterial-based carbon-concentrating mechanism in a land plant chloroplast and provide the necessary molecular machinery to facilitate its operation. Regulatory modules will be produced to express components of the cyanobacterial carboxysome from the chloroplast genome and chloroplast membrane-targeted bicarbonate pumps from the nuclear genome. As a proof of principle, this work will be carried out in tobacco, a species in which chloroplast transformants can be most rapidly obtained. We have already established the ground work for this engineering feat, demonstrating that several of the components can be introduced; for example, to generate novel microcompartments within the tobacco chloroplast. We expect the knowledge gained from the project will inform subsequent efforts to enhance photosynthesis in other species, for example by introducing synthetic microcompartments into species such as soybean, in which technology is already available for chloroplast and nuclear transformation. Knowledge will be gained through biochemical and microscopic studies, for example about the effect of stoichiometry of cyanobacterial proteins on microcompartment size, morphology, and function in carbon concentration and photosynthesis. We will examine features of the gene regulatory sequences on the synthetic chloroplast operons needed to express proteins in the amounts and ratios needed for assembly of microcompartments. We expect the findings to be valuable also for future projects and additions of other capabilities to plants by synthesis of artificial microcompartments.