耐受木质纤维素衍生酚酸抑制物的进化工程菌株构建及耐受机理的研究

The lignocellulose-derived phenolic inhibitors has become the critical bottleneck to the industrial development of biorefinery for their properties of high toxicity, complexity and difficult to remove. For dealing with this issue, an evolutionary engineering strategy was proposed to construct a yeast Saccharomyces cerevisiae with high tolerance to phenolic acids, and then revealed the tolerant mechanism from the level of cell to genome. The real time monitoring adaptive evolution was applied for creating the phenolic acids tolerant mutant pools. Then, the genome shuffling based on the population mating was carried out to accelerate the accumulation of the multiple tolerant mutations from the mutant pools into one genome for constructing an evolutionary strain with high tolerance to phenolic acids. After that, the relationship between the structure and composition of cell membrane and the tolerance to phenolic acids was investigated at cellular level. Finally, the tolerant mechanism was analyzed by whole-genome sequencing followed by transcriptome analysis under the stress of phenolic acids. In addition, the phenolic acids tolerant strain was constructed by over-expressing the key genes corresponding to the phenolic tolerance for further confirming the tolerant mechanism in the real fermentation system with high content of phenolic acids. Based on the above, this evolutionary engineering strategy could shorten the evolutionary cycle and increase the tolerance to phenolic acids. Especially, it will provide the important theoretical basis and technical support for rationally designing and constructing the stress tolerance strain in the biorefinery industry.

木质纤维素衍生的酚类抑制物具有毒性强、种类多和难脱毒等特性，对工业微生物的抑制作用已成为生物炼制产业化发展的主要瓶颈。为此提出一种进化工程策略用于构建耐受酚酸的酿酒酵母，并从细胞到基因水平揭示其酚酸耐受的机理。首先通过实时监测的适应性进化方法构建具有酚酸耐受表型的进化突变株文库，结合基因组改组技术将酚酸耐受表型所对应的多种突变基因重组到一个基因组，实现超强酚酸耐受菌株的构建；其次从细胞水平考察酚酸胁迫下进化菌株的细胞膜微观形态和组成等生理特性与酚酸耐受性的关系；然后通过对酚酸耐受性进化菌株进行全基因组测序和酚酸胁迫下转录调控分析解析酚酸耐受性机制；最后根据酚酸耐受性的关键基因构建工程菌，在富含复杂酚酸抑制物的实际发酵体系中验证该机制。上述研究将缩短构建抗逆菌株的进化周期，增强酚酸耐受能力，为理性设计和构建抗逆菌株用于生物炼制产业提供理论基础和技术支持。

木质纤维素预处理产生的酚酸类抑制物对发酵微生物的抑制作用已经成为限制木质纤维素生物转化的重要问题之一。本研究分别选择不可降解类酚酸（香草酸、p-羟基苯甲酸、丁香酸）和可降解类酚酸（阿魏酸、p-香豆酸、肉桂酸）作为酚酸模式化合物，通过进化工程策略构建耐受复合酚酸的酿酒酵母，并从细胞到基因水平揭示其酚酸耐受机理。（1）酚酸耐受性进化菌株构建方面，可降解类和不可降解类酚酸对细胞生长和乙醇发酵的抑制作用不同。酚酸毒性强弱顺序为：肉桂酸、阿魏酸、p-香豆酸、香草酸、丁香酸和p-羟基苯甲酸。复合酚酸之间存在强烈的协同抑制作用。通过酚酸胁迫下120-160批次的连续转接和培养，获得4种酚酸耐受性状稳定遗传的进化菌株。通过递归式原生质体融合获得耐受混合酚酸的酿酒酵母菌株。（2）细胞生理结构形态方面，混合酚酸作用下适应进化菌株的比生长速率、最大生物量、乙醇得率和乙醇生产速率比原始菌株分别提高18.1%、50.1%、139.8%和71.7%。降解阿魏酸和p-香豆酸的速率提高4-6倍。扫描和投射电镜观察发现酚酸作用下适应进化菌株细胞膜形态、细胞壁厚度、细胞直径明显变化。通过荧光探针、细胞膜电导率和胞内核酸物质外泄检测分析，进化酵母细胞膜渗透性降低，保持良好的完整性。线粒体跨膜电势分析表明线粒体保持正常功能活性。细胞膜脂质成分分析发现磷脂酰乙醇胺与磷脂酰胆碱比值、长链脂肪酸含量和不饱和双键数量提高，这些磷脂重塑结果与细胞膜完整性相关。（3）酚酸耐受性机制探究及应用方面，RNA-seq分析进化菌株酚酸耐受性主要与磷脂合成、脂肪酸链延长和不饱和脂肪酸合成、线粒体呼吸链相关基因的转录调控有关，与酚酸耐受性表型一致。在30%固含量玉米芯残渣（含酚酸抑制物）同步糖化与乙醇发酵体系中进化菌株表现出良好的发酵性能（乙醇浓度62.3g/L，得率55.3%）。