1,3-丙二醇（PDO）是一种重要的化工原料，生物法合成1,3-丙二醇受到研究者的持续关注。本论文围绕K. pneumoniae CGMCC 1.9131有氧发酵甘油生产PDO的代谢途径开展研究，针对旁路径分流碳源、中间产物3-羟基丙醛积累等难题，构建新型工程菌并分析其发酵及代谢特性。分别构建甘油脱水酶（GDHt）和1,3-丙二醇氧化还原酶（PDOR）基因失活菌株，酶学和发酵分析发现K. pneumoniae CGMCC 1.9131胞内存在GDHt和PDOR的同工酶，乙醇和乳酸代谢途径分别是PDO合成前期和后期的主要竞争途径，由此明确代谢调控方向。构建甘油脱氢酶（GDH）基因失活菌株，期望降低氧化支路的通量，但细胞生长受到严重抑制，5 L发酵罐培养16 h 生物量只有0.80 g L-1（野生菌4.60）；还原支路的关键酶GDHt和PDOR活性均降低90 %左右，代谢产物为PDO（2.5 g L-1）和乙酸（0.78 g L-1）。针对发酵前期乙醇的积累，敲除乙醇合成途径的多效基因adhE，摇瓶发酵试验表明乙醇合成降低30 %，PDO合成增加6 %；敲除乙醛脱氢酶的编码基因aldA，乙醇合成降低32 %，PDO合成增加4 %，表明K. pneumoniae CGMCC 1.9131中乙醇合成路径涉及多个酶的调控。针对发酵后期乳酸的积累，构建D-乳酸脱氢酶基因失活的工程菌K.p/L。5 L发酵罐发酵48 h，与野生菌相比：K.p/L乳酸脱氢酶活性降低90 %以上，其他酶活性基本没有受到影响；乳酸合成从40 g L-1降低到3 g L-1以下；PDO合成从92 g L-1提高到100 g L-1；2,3-丁二醇合成从16 g L-1 增加到30 g L-1；胞内氧化还原电势NADH/NAD+ 大幅增加。工程菌K.p/L具有很好的遗传稳定性，能有效利用生物柴油副产物甘油发酵生成PDO，可应用于工业生产和进一步基因改造。PDO代谢路径中间产物3-羟基丙醛的积累易引起甘油代谢停止。1）在K.p/L中分别强化表达非特异性二元醇氧化还原酶和1,3-丙二醇氧化还原酶，3-羟基丙醛的最高积累浓度较野生菌分别降低46 %和50 %，PDO终产量分别为88 g L-1和89 g L-1；2）K.p/L中共表达丙醛脱氢酶和PHA合酶，构建聚3-羟基丙酸的合成路径，3-羟基丙醛的最高积累浓度降低39 %，聚3-羟基丙酸在细胞内积累量为2.92 % （g/g DCW），降低发酵异常终止风险的同时可以有效利用菌体。

1,3-Propanediol (PDO) is an important bulk chemical and its biological production from glycerol has attracted continuous attention. This work investigated the regulation and metabolism of PDO under micro-aerobic condition by K. pneumoniae CGMCC 1.9131, a new screened strain with high productivity. Genetic modifications were applied in order to reduce byproducts formation and 3-hydroxypropionaldehyde (3-HPA) accumulation. Glycerol dehydratase and 1,3-propanediol oxidoreductase were inactivated individually. Enzymatic assay and fermentation results indicated that isoenzymes of glycerol dehydratase and 1,3-propanediol oxidoreductase existed in K. pneumoniae CGMCC 1.9131. Formation of ethanol is the main competitive pathway in the early phase of PDO fermentation, and formation of lactate becomes the main competitive pathway in the late phase. Expressing the isoenzymes and knocking out of ethanol and lactate pathways could be efficient strategy to improve PDO productivity.It is found that glycerol is first catalysed by glycerol dehydrogenase to form dihydroxyactone and further metabolized to such metabolites as ethanol, lactate, acetate, 2,3-butanediol and succinate. Glycerol dehydrogenase was inactivated to eliminate byproducts formation. However, activaties of glycerol dehydratase and 1,3-propanediol oxidoreductase both decreased by more than 90 %, leading to less formation of PDO. Fermentation in 5 L fementor for 16 h showed that biomass decreased by 90 % and concentration of PDO was 2.5 g L-1. Acetate was the only byproduct with concentration of 0.78 g L-1.AdhE has been reported to harbor two enzymatic activites: alcohol dehydrogenase and acetaldehyde dehydragenase. An adhE gene inactiviated strain of K. pneumoniae was constructed. Ethanol formation decreased by 30 %, while PDO synthesis increased by 6 % in the constructed strain compared to the wild strain. Acetaldehyde dehydrogenase encoded gene aldA was aslo inactiviated in K. pneumoniae. Ethanol formation decreased by 32 %, while PDO synthesis increased by 4 %. The results indicated that more than two genes regulate ethanol production in K. pneumoniae CGMCC 1.9131, which is different from E.coli and K. oxytoca. D-lactate dehydrogenase deficient strain K.p/L was constructed by inactivating gene ldhA. In comparison with that of the wild type, activity of lactate dehydrogenase decreased by more than 90 %, while activites of other key enzymes were almost the same. Fermentation results in 5 L fermntor for 48 h showed that lactate formation decreased from more than 40 g L-1 to less than 3 g L-1, PDO formation increased from 92 g L-1 to 100 g L-1, and 2,3-butanediol increased from 16 g L-1 to 30 g L-1, respectiviely. Reducing equivalent NADH increased, leading to the change of metabolism flux. K.p/L had great genetic stability and could effectively utilize crude glycrol from biodiesel production. So it can be applied for the industry production of PDO and further genetic modification. High accumulation of 3-HPA would inhibit cell growth and result in cessation of fermentation process. Enzymatic activity assay indicated that the accumulation of 3-HPA was caused by imbalance of activity between 1,3-propanediol oxidoreductase and glycerol dehydrotase. Gene yqhD encoding the putative alcohol dehydrogenase and gene dhaT encoding 1,3-propanediol oxidoreductase were cloned and overexpressed in K.p/L, respectively. Correspondingly, accumulation of 3-HPA decreased by 46 % and 50 %, respectively. In addition, aldehyde dehydrogenase and PHA synthase were co-expressed in K.p/L to construct the pathway to convert 3-HPA to poly (3-hydroxypropionate). As a result, accumulation of 3-HPA decreased by 39 %, and poly (3-hydroxypropionate) amounted to 2.92 % (g/g DCW).