摘要
A yeast strain R6 was obtained by the method of thiamine（VB1） auxotrophic negative selection from the edible oil-polluted soil in Zibo, China. Physiological and biochemical experiments revealed that strain R6 shared common feature with Rhodotorula mucilaginosa according to the API 20 C AUX yeast identification system which has been tested previously. Furthermore, the 18 S r DNA gene of strain R6 was amplified and sequenced. Phylogenetic analysis based on the 18 S r DNA sequence and the relatives indicated that R6 shared 99% homologies with the members of R. mucilaginosa, suggesting that strain R6 belonged to R. mucilaginosa.Investigation showed that strain R6 possessed the capacity of accumulating exocellular alpha-ketoglutaric acid（alpha-KG）. Finally, the fermentation conditions of R6 to accumulate alpha-KG was optimized by controlling each single fermenting variable and detected through high performance liquid chromatography（HPLC）. Results showed that both VB1 and Ca CO3 in fermentation medium were the key factors influencing the cumulant of alpha-KG. The discovery of natural auxotrophic strain R6 not only broadened the microbial resource which can achieve lots of alpha-KG production through fermentation, but also laid a foundation for further fermentation regulation to achieve excessive alpha-KG accumulation.
该研究通过硫胺素(VB1)营养缺陷型负选择法,从中国淄博食用油污染的土壤中获得一株酵母属菌株R6。该菌株具有累积胞外α-酮戊二酸(α-KG)的能力。生理学和生物化学实验表明该R6菌株与已测试的API 20 C AUX酵母鉴别系统中的胶红酵母具有共同特征。此外,R6菌株的18S r DNA基因已经获得并测序,基于该序列以及同源菌属所得到的系统进化学分析表明菌株R6与胶红酵母属成员具有99%的同源性。因此,菌株R6被鉴别为胶红酵母属的一员。随后,本实验通过控制单一发酵变量以及高效液相色谱法(HPLC)检测优化了R6累积α-KG的发酵条件。当发酵培养基中添加的VB1和Ca CO3的浓度分别为0.03 mg/L和60 g/L时,α-KG达到6.035 g/L的最高产量。天然营养缺陷型的R6菌株的发现不仅丰富了通过发酵生产α-KG的微生物来源,而且为进一步的发酵调控奠定了基础,从而实现过量的α-KG的累积。
基金
Supported by the Natural Science Foundation of Shandong Province,China(ZR2010CQ017)
Program of Young Teachers Development of Shandong University of Technology~~
