非天然乙醇酸代谢新途径的设计构建与适配机制研究

In C3 crops such as wheat, rice, and soybeans, photorespiration reduces the photosynthetic conversion efficiency of light into biomass by 20 to 50%. The main reason is the low efficiency of glycolate reuse in photorespiration. In the past decade, photosynthesis efficiency can be significantly increased by attenuating the photorespiration pathway of plants themselves, while introducing alternative pathways into the chloroplast to recycle the products of glycolate. The conversion of all or part of glycolate to carbon dioxide can increase the carboxylation activity of RuBisCO and ultimately improve the photosynthesis efficiency. However, these alternative pathways lose the fixed organic carbon and cannot fundamentally solve the problem of low efficiency of glycolate reuse..Here we de novo design and construct a non-natural glycolate metabolic pathway with no carbon loss based on synthetic biology. In 4 steps, glycolate was converted into an important intracellular metabolite, acetyl-CoA. Synthesis of acetyl phosphate from glycolaldehyde is essential for new pathway but is not known to occur naturally. Specifically, we first obtained acetyl phosphate synthase by screening and engineering, which can catalyze the synthesis of acetyl phosphate. Subsequently, we characterized the function of different proteins in the pathway for glycolate metabolism. In addition, the adaptation of different enzymes and the adaptation of reducing power and energy were studied. Finally, NADH and ATP regeneration systems were introduced to achieve efficient conversion of the non-natural glycolate metabolism pathway in vitro. The completion of this project will provide new ideas for solving major scientific problems of low efficiency of glycolic acid reuse.

在小麦，水稻和大豆等C3作物中，光呼吸作用使光合作用的效率降低20%-50%，其主要原因在于，光呼吸途径中乙醇酸再利用效率低。近十几年的研究显示，减弱植物本身的光呼吸途径，在叶绿体中导入新的乙醇酸代谢途径，通过将乙醇酸全部或部分转化为二氧化碳，增加RuBisCO羧化活性，可以提高光合作用效率。然而，这些途径均会损失已固定的有机碳，无法从根本上解决乙醇酸再利用效率低的问题。本研究针对这些问题，基于合成生物学角度设计了一条非天然、且无碳损失的乙醇酸代谢途径，4步将乙醇酸转化成乙酰辅酶A。通过筛选改造并深入研究乙酰磷酸合酶的催化机理，实现自然界不存在的反应——乙醇醛高效合成乙酰磷酸。通过表征途径中不同蛋白元件的功能，研究不同酶的适配以及还原力和能量的适配，导入NADH和ATP再生体系，体外实现乙醇酸代谢新途径的高效转化。本项目的完成将为解决乙醇酸再利用效率低的重大科学问题提供新思路。

在小麦，水稻和大豆等C3作物中，光呼吸作用使光合作用的效率降低20%-50%，其主要原因在于，光呼吸途径中乙醇酸再利用效率低。近十几年的研究显示，减弱植物本身的光呼吸途径，在叶绿体中导入新的乙醇酸代谢途径，通过将乙醇酸全部或部分转化为二氧化碳，增加RuBisCO羧化活性，可以提高光合作用效率。然而，这些途径均会损失已固定的有机碳，无法从根本上解决乙醇酸再利用效率低的问题。本研究针对这些问题，基于合成生物学角度设计了一条非天然、且无碳损失的乙醇酸代谢途径，4步将乙醇酸转化成乙酰辅酶A。通过筛选改造并深入研究乙酰磷酸合酶的催化机理，实现自然界不存在的反应——乙醇醛高效合成乙酰磷酸。通过表征途径中不同蛋白元件的功能，研究不同酶的适配以及还原力和能量的适配，导入NAD(P)H和ATP再生体系，体外实现使乙醇酸到乙酰辅酶A的转化效率达50%。本项目的完成将为解决乙醇酸再利用效率低的重大科学问题提供新思路。