从自然标本中分离获得一高产甘油的菌株WL2002-5，仅发酵葡萄糖及微发酵蔗糖，能利用葡萄糖、蔗糖、乙醇生长，微利用甘油和柠檬酸，不利用肌醇、硝酸盐、赤藓醇、阿拉伯醇、甘露醇，与DBB显色反应为阴性，可在含500g/L葡萄糖或100mL/L醋酸的培养基中40℃下生长，可在水活度为0.890-0.900的培养基中生长，出芽生殖，易形成“假丝酵母菌型”假菌丝，不进行有性生殖，线粒体DNA的分子量为20kb，是假丝酵母属的一个新种，定名为产甘油假丝酵母（Candida glycerol genesis Zhuge sp. nov.）。

本文对产甘油假丝酵母的甘油代谢关键酶进行了研究，发现产甘油假丝酵母同化甘油能力极弱，少量葡萄糖明显改善其同化甘油的能力；线粒体3-磷酸甘油脱氢酶受3-磷酸甘油的强烈诱导，受葡萄糖代谢的阻遏。在甘油发酵过程中，产甘油假丝酵母胞浆3-磷酸甘油脱氢酶酶活处于较高水平并在36h和60h时出现两次酶活高峰，其中第一次酶活峰值水平决定产甘油假丝酵母的甘油合成和积累水平，成为甘油高速积累期（18～48h）甘油合成的关键 性的限速酶。在甘油发酵18～48h内，3-磷酸甘油酯酶的酶活处于高水平，并在36h时出现酶活峰值；处于缓慢甘油积累阶段的48～72h间，3-磷酸甘油酯酶已处于低水平表达，此时，3-磷酸甘油酯酶则成为甘油合成的限速酶。 产甘油假丝酵母稳定并高表达其胞浆3-磷酸甘油脱氢酶基因并且其所表达的3-磷酸甘油酯酶酶活远高于胞浆3-磷酸甘油脱氢酶这一特征是其高产甘油根本所在。

The glyoxylate bypass genes aceA1 (isocitrate lyase 1, ICL1), aceA2 (isocitrate lyase 2, ICL2) and aceB1 (malate synthase, MS1) of Ralstonia eutropha HF39 were cloned, sequenced and functionally expressed in Escherichia coli. Interposon-mutants of all three genes (vaceA1, vaceA2 and vaceB1) were constructed, and the phenotypes of the respective mutants were investigated. Whereas R. eutropha HF39vaceA1 retained only 19% of ICL activity and failed to grow on acetate, R. eutropha HF39vaceA2 retained 84% of acetate-inducible ICL activity, and growth on acetate was not retarded. These data suggested that ICL1 is in contrast to ICL2 induced by acetate and specific for the glyoxylate cycle. R. eutropha HF39vaceB1 retained on acetate as well as on gluconate about 41-42% of MS activity and exhibited retarded growth on acetate, indicating the presence of a second hitherto not identified MS in R. eutropha HF39. Whereas in R. eutropha HF39vaceA1 and R. eutropha HF39vaceA2 the yields of poly(3-hydroxybutyric acid), using gluconate as carbon source, were significantly reduced, R. eutropha HF39vaceB1 accumulated the same amount of this polyester from gluconate as well as from acetate as R. eutropha HF39.

Candida glycerinogenes, an osmotolerant yeast isolated from a natural sample in an environment of high osmotic pressure, had a modest sugar-tolerance and an extremely high glycerol productivity. The optimum conditions for glycerol formation by C. glycerinogenes were a temperature of 29-33℃ and a pH of 4-6. The optimum medium for glycerol production consisted of 230-250g glucose/l, 2g urea/l and 5ml corn steep liquor/l (55-65mg phosphates/l); the pH was not adjusted. The highest yield of glycerol was 64.5% (w/w) based on consumed glucose from 240g glucose/l, and the highest concentration of glycerol was 137g/l from 260g glucose/l. These results were obtained by using a 30-l agitated fermentor under optimal fermentation conditions. In ten batch-fermentations carried out in a 50,000-l airlift fermentor, an average yield of glycerol of 50.67% (w/w) and an average glycerol concentration of 121.9g/l were obtained from an average 240.6g glucose/l.

利用PCR方法从运动发酵单孢菌染色体DNA扩增出乙醇合成途径的关键酶基因pdc、adhB，分别用tac启动子控制表达，构建了可以在Escherichia coli JMI 09中表达的重组质粒pKK-PA、pEtac-PA。初步的乙醇发酵结果表明，在 E. coli中只引入 adhB基因不能拓宽其中的产乙醇途径，引入pdc基因可以与宿主自身的ADH酶协同作用，使碳流有效导向产乙醇方向。同时引入pdc、adhB基因可以在宿主E. coli中成功建立产乙醇途径。