Chen Xi, Wang Qin-Qing, Liu Nan-Nan, Liu Guang-Lei, Chi Zhe, Chi Zhen-Ming
College of Marine Life Sciences, Ocean University of China, Yushan Road, No. 5, Qingdao, China.
College of Marine Life Sciences, Ocean University of China, Yushan Road, No. 5, Qingdao, China.
Int J Biol Macromol. 2017 Feb;95:539-549. doi: 10.1016/j.ijbiomac.2016.11.081. Epub 2016 Nov 23.
In this study, one of the glucosyltransferase genes for pullulan production was cloned from Aureobasidum melanogenum P16 and charaterized. It was found that the UGT1 gene had 4774bp with four introns (47, 52, 54 and 46bp). The N-terminal part of the protein displayed a conserved sequence controlling both sugar donor and accepter for substrate specificity whereas its C-terminal part carried a DXD motif that coordinated donor sugar binding. After complete removal of the gene UGT1, the mutant 1152-3 still produced 27.7±3.1g/L of pullulan and 4.6U/g of the specific glucosyltransferase activity while its wild type strain P16 yielded 63.38±2.0g/L of pullulan and 5.7U/g of the specific glucosyltransferase activity. However, after overexpression of the gene UGT1, the transformant G63 could produce 78.0±3.01g/L of pullulan and 19.0U/g of the specific glucosyltransferase activity. It is interesting to note that the molecular weight of the produced pullulan by the wild type strain was 4.6×10 while that of the produced pullulan by the transformant G63 was 6.2×10. During the 10-Litter fermentation, the pullulan titer produced by the transformant G63 reached 80.2±2.0 g/L within 132h.
在本研究中，从黑化短梗霉P16中克隆并鉴定了一个用于支链淀粉生产的葡糖基转移酶基因。发现UGT1基因有4774bp，含四个内含子（47、52、54和46bp）。该蛋白的N端部分显示出一个保守序列，可控制底物特异性的糖供体和受体，而其C端部分带有一个协调供体糖结合的DXD基序。完全去除UGT1基因后，突变体1152 - 3仍能产生27.7±3.1g/L的支链淀粉和4.6U/g的比葡糖基转移酶活性，而其野生型菌株P16产生63.38±2.0g/L的支链淀粉和5.7U/g的比葡糖基转移酶活性。然而，在UGT1基因过表达后，转化体G63可产生78.0±3.01g/L的支链淀粉和19.0U/g的比葡糖基转移酶活性。有趣的是，野生型菌株产生的支链淀粉分子量为4.6×10，而转化体G63产生的支链淀粉分子量为6.2×10。在10升发酵过程中，转化体G63产生的支链淀粉效价在132小时内达到80.2±2.0 g/L。
