D-乳酸（D-LA）作为L-乳酸的同分异构体，是一种具有高附加值的平台化合物。它可作为手性药物、农药、及其他大宗化学品的中间体，也是可降解塑料聚乳酸（PLA）的合成单体。微生物法生产D-LA可以避免化学法带来的环境污染。酿酒酵母作为一种安全性高的单细胞低等真核生物，具有耐酸性强、易于进行基因编辑等优点，很适合于生产D-LA。但是，外源基因在酿酒酵母中存在表达差异性，重组菌株生长变慢，酸性物质的积累对细胞毒害等问题，严重制约了D-LA在酿酒酵母中的生产。本文用代谢工程的方法从D-乳酸脱氢酶（D-LDH）的表达优化，耐酸性的增强，菌株生长速率的提高等方面构建D-LA高产菌株。首先，以敲除丙酮酸脱羧酶基因的TAMH菌株为宿主菌，组合表达优化筛选D-LA生产菌株。针对4个启动子，5个D-LDH，以及2个终止子的组合表达研究表明，在构建的40个菌株中，含PTEF1-E. coli D-LDH-Tsynth25表达载体的菌株TCSt，D-LA产量最高，达到5.8 g/L，光学纯度达到了99.9%。为了提高D-LA的产量，将该表达载体插入到敲除Pdc1和Pdc6基因的YIP-01菌株的Ty1多拷贝位点，利用双酶偶联方法筛选高产D-LA的整合菌株。基因组重测序结果表明，得到的整合菌株YIP-pTCSt-301含有3个D-LDH拷贝。在分批补料条件下发酵，该菌株产生了35 g/L D-LA，得率为0.45 g/g，产率为0.9 g/L/h。为了进一步提高D-LA产量和得率，敲除了D-LA消耗相关的基因Dld1和Cyb2，转运D-LA到胞内的相关基因Jen1，以及产乙醇的基因Adh1。得到的菌株YIP-JCDA1在分批补料条件下发酵，产生了80 g/L D-LA，得率提高到0.6 g/g，产率提高到1.1 g/L/h。其次，在菌株YIP-JCDA1的基础上引入了Issatchenkia orientalis的IoGas1基因，其编码蛋白具有耐受低pH和高盐特性。得到的菌株YIP-IJCDA1在分批补料条件下发酵， D-LA达到了85.3 g/L，得率提高到0.71 g/g，产率提高到1.20 g/L/h。在此基础上，继续敲除甘油合成途径的Gpd1和Gpd2基因和乙醇合成途径的Adh5基因。得到的YIP-A15G12菌株，其D-LA产量达到92.0 g/L，得率为0.70 g/g，产率为1.21 g/L/h。D-LA光学纯度为99.7%。再次，在YIP-IJCDA1菌株中过表达耐乙酸的Acs2基因和Haa1基因，以及消耗胞质内乙醛的Ald6基因，得到的菌株的D-LA产量却没有显著提高。在含IoGas1基因的YIP-IJCDA1菌株中过表达耐受乙酸的Whi2基因，菌株的葡萄糖发酵能力显著增强。这表明Whi2基因能与IoGas1基因共同作用增强菌株在高浓度乙酸压力下的葡萄糖发酵能力。最后，为了增加胞质乙酰辅酶A的水平，在菌株YIP-A15G12中，引入了大肠杆菌的乙酰化的乙醛脱氢酶（A-ALD）基因。A-ALD基因（mhpF 和eutE）的表达提高了酿酒酵母胞内的乙酰辅酶A水平，提高了菌株的生长速率。含有mhpF基因的YIP-m-ald6菌株，产D-LA的能力显著提高。分批补料发酵55 h，产率从1.21 g/L/h增加到1.68 g/L/h，D-LA产量达到了92.6 g/L，得率为0.70 g/g。D-LA光学纯度达到了99.8%。;D-lactic acid (D-LA), an isomer of L-lactic acid, is one of the most valuable bio-based chemicals, which can be used as an intermediate for producing chiral drugs, pesticides, and other chemicals. Particularly, D-LA is gaining increased attention as it can improve the thermostability of poly lactic acid (PLA). Microbial-production of D-LA is environment friendly and can avoid environmental pollution caused by chemical process. Acid-resistant Saccharomyces cerevisiae, because it is generally recoginzed as safe and easily to be edited, is suitable for industrial-scale production of D-LA. However, S. cerevisiae produces D-LA are limited by the heterologous expression level of the stereospecific D-lactate dehydrogenase (D-LDH), growth retardation of engineered strain, and acidic toxicity. In this work, we optimized the expression level of D-LDH, enhanced the acid- tolerance, and improved the productivity of D-LA by metabolic engineering. Firstly, a synthetic biology approach was used to construct high-producing D-LA strains by optimization of D-LDH expression through combinations of four promoters, two terminators and five D-LDHs. The pyruvate decarboxylase-deficient mutant strain TAMH was used as host strain for optimizing the 40 D-LDH expression cassettes. The TCSt strain harboring PTEF1-E. coli D-LDH-Tsynth25 produced 5.8 g/L D-LA with an optical purity of 99.9%. The D-LA production was further improved by integrating this cassette into the Ty1 transposable element of the YIP-01 strain with the deletion of Pdc1and Pdc6 genes. The integrated strains were screened by a double enzyme-coupled system. Genome sequencing of the resulting strain YIP-pTCSt-301 showed that there were three copies of the D-LDH expression cassette. In fed-batch fermentation, the strain produced 35 g/L D-LA, with a yield of 0.45 g/g and a productivity of 0.9 g/L/h under acid conditions. D-LA production was further improved by deleting the Jen1 (a monocarboxylate transporter), D-Lactate dehydrogenase1 (Dld1), L-lactate cytochrome-c oxidoreductase (Cyb2), and alcohol dehydrogenase 1( Adh1) genes of the YIP-pTCSt-301 strain, and the resulting strain YIP-JCDA1 produced 80.0 g/L D-LA with a yield of 0.6 g/g and a productivity of 1.1 g/L/h in fed-batch fermentation.Then, a heterologous glycosylphosphatidylinositol-anchored protein, IoGas1, required for resistant to low pH and salt stress, was overexpressed in the YIP-JCDA1 strain. D-LA production of the resulting strain YIP-IJCDA1 was improved to 85.3 g/L with a yield of 0.71 g/g and a productivity of 1.20 g/L/h. Next, the D-LA production was further increased by attenuating the ethanol pathway and disrupting glycerol-3-phosphate dehydrogenases (Gpd). The resulting strain YIP-A15G12 with the deletion of Adh1, Adh5, Gpd1and Gpd2 genes, produced 92.0 g/L D-LA with a yield of 0.70 g/g and a productivity of 1.21 g/L/h in fed-batch fermentation under acid conditions. The optical purity of D-LA reached 99.7%.Subsequently, the acetic acid-resistant genes Acs2 and Haa1 were overexpressed in the YIP-IJCDA1 strain, respectively. D-LA production of the resulting strains YIP-J1A and YIP-CH was not improved. Further overexpressing the cytosolic aldehyde dehydrogenase 6 (Ald6) gene in the YIP-J1A strain showed that the D-LA production of the resulting strain was not improved. Overexpression of another acetic acid-resistant gene, Whi2, can significantly improve the glucose fermentation of the resulting strain under acetic acid stress, which suggested that the Whi2 protein combined with IoGas1 protein can significantly improve the glucose fermentation under acetic acid stress. Finally, to increase acetyl-CoA synthesis in S. cerevisiae, the bacterial acetylating acetaldehyde dehydrogenase (A-ALD) genes mhpF and eutE were expressed in the yeast strain which was attenuated acetaldehyde dehydrogenase (Ald)/acetyl-CoA synthetase (Acs). Growth rate of the resulting strains, YIP-m-ald6 and YIP-e-ald6, were significantly increased. Under fed-batch fermentation condition, YIP-m-ald6 produced 92.6 g/L D-LA with a yield of 0.70 g/g and a productivity of 1.68 g/L/h. The productivity of this strain was 1.4 times of YIP-A15G12. The optical purity of D-LA reached 99.8%.