聚苹果酸是出芽短梗霉代谢产生的一种聚酯型聚合物，单体L-苹果酸是应用广泛的有机酸，通过聚苹果酸发酵及酸水解制备L-苹果酸，实现取代化学合成法符合食品安全需求。然而，聚苹果酸合成糖酸转化效率偏低，主要原因在于生物合成模块及适配机制尚不明确。前期研究表明出芽短梗霉具有高效木糖利用能力，乙醛酸支路是聚苹果酸合成代谢流扰动的关键途径；中间代谢物乙醇酸验证发现可明显促进聚苹果酸合成。可见，研究乙醛酸代谢网络与聚苹果酸合成效率的关系，是揭示聚苹果酸合成机制的一条重要线索。本项目拟在底盘细胞优化改造的基础上，设计木糖为底物的乙醛酸外源合成新通路，并解析乙醛酸内外源模块适配差异的分子机制；利用CRISPRai扰动等技术，实现乙醛酸内外源模块与底盘细胞的理性适配。项目的开展有望在聚苹果酸合成途径优化设计及适配分子机制取得突破，为实现代谢工程改造提高菌株的产酸性能提供理论依据。

Polymalic acid （PMA）is a novel polyester polymer produced by the yeast-like fungus Aureobasidium pullulans. Its monomer, L-malic acid, is also an important organic acid that was widely used in the food industry. Compared with the chemical synthesis route, bio-based L-malic acid produced by PMA acid hydrolysis has attracted more attention because of the food safety requirements. However, this process is obviously limited by the low yield of PMA because PMA biosynthesis pathway and fitness was unclear. In our previous study, A. pullulans has the ability to unitize the xylose, and glyoxylate shunt was found and confirmed as the key pathway to regulate the biosynthesis of PMA. Feeding glycollic acid as the intermediate metabolite could increase the PMA titer. Thus, it was an important clue to research the relationship between glyoxylate metabolic network and PMA biosynthesis. In this project, based on the optimized chassis cells, we are going to reconstruct the exogenous module of glyoxylate from xylose as the substrate, and explore the molecular mechanism of fitness difference between endo- and exo-genous modules, following to detect the potential target of module modification. By the aid of the strategy of CRISPRai, etc, we are planned to realize the rational fitness of endo- and exo-genous modules of glyoxylate, synthesis modules and chassis cells. This project is expected to achieve a new breakthrough on the design of PMA biosynthesis pathway, and provide a new theoretical basis for metabolic engineering of A. pullulans for enhancing the PMA production.