聚苹果酸合成乙醛酸代谢网络重构与适配分子机制

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.

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。

项目以木糖等底物利用过程，对出芽短梗霉聚苹果酸合成的乙醛酸关键通路进行新通路设计与适配优化，解析底盘细胞与产物合成适配分子机制。得到如下结论。.1．构建组氨酸营养缺陷型底盘，建立CRISPR/Cas9编辑系统的无痕敲除方法；并对出芽短梗霉基因组进行精简，分别阻断乙醇酸转化、普鲁兰多糖、黑色素、以及Liamocins等关键副产物合成途径，降低底盘细胞杂质副产物，实现出芽短梗霉底盘细胞系统优化改造。.2. 对出芽短梗霉内源木糖代谢利用途径进行精简和验证，敲除木酮糖转化为5-磷酸木酮糖、木糖酸内酯、阿拉伯醇等途径，确定5-磷酸木酮糖途径是内源木糖代谢利用的关键途径；使用人源己酮糖激酶基因(khkC)、醛缩酶基因(aldoB)和细菌源醛脱氢酶基因(aldA)的组合表达，实现木糖为底物驱动的外源乙醛酸合成通路搭建。.3. 转录组学分析等手段考察乙醇为底物的乙醛酸合成通路，可实现产物静息细胞合成，发现乙醇底物条件下乙醇酸氧化酶上调2.28倍，转录因子Cat8可显著上调乙醛酸途径关键基因，造成途径分流；筛选不同表达强度的内外源启动子，利用启动子工程策略对乙醇氧化、乙醛酸循环和糖异生三模块进行精细适配优化，实现聚苹果酸合成最高得率达到0.89 g/g 乙醇。.4. 通过适应性进化、信号调节工程策略调控底盘细胞与合成模块适配性，乙醇耐受适应性进化可提升菌株的乙醇底物4%条件下生长，建立乙醇底物的静息细胞发酵新方法；通过钙信号调控工程策略，发现钙调蛋白cnb基因可有效提升细胞抗逆性能和PMA合成，建立了钙信号调控下的细胞低酸控制及无钙渣排放发酵体系。. 本研究对非模式真菌出芽短梗霉合成聚苹果酸过程及模块功能进行了机制解析，建立了模块适配调控方法，实现了木糖底物的低酸无钙渣碳减排发酵和乙醇底物的静息细胞发酵新方法。项目资助期间，相关研究成果发表在Chem Eng J、Green Chem、Critic Rev Biotechnol、Bioproc Biosyst Eng、微生物学报等期刊（SCI论文9篇，其中IF>10分论文2篇，单篇最高IF为16.744，获授权中国发明专利3项，为工业化高效生产聚苹果酸及单体L-苹果酸提供借鉴。