Prathumpai Wai, Flitter Simon J, McIntyre Mhairi, Nielsen Jens
BioCentrum-DTU, Center for Microbial Biotechnology, Technical University of Denmark, Building 223, 2800 Lyngby, Denmark.
Appl Microbiol Biotechnol. 2004 Nov;65(6):714-9. doi: 10.1007/s00253-004-1699-y. Epub 2004 Aug 13.
Two recombinant strains of Aspergillus niger (NW 297-14 and NW297-24) producing a heterologous lipase from Thermomyces lanuginosus were constructed. The heterologous lipase was expressed using the TAKA amylase promoter from Aspergillus oryzae. The production kinetics of the two strains on different carbon sources in batch and carbon-limited chemostat cultivations were evaluated. In batch cultivations, the highest total product yield coefficient (Y(xp total)), given as the sum of extracellular and intracellular yields, was obtained during growth on glucose for the transformant strain NW297-24 (5.7+/-0.65 KU/g DW), whereas the highest total product yield coefficient was obtained during growth on maltose for the transformant strain NW297-14 (6.3+/-0.02 KU/g DW). Both transformants were evaluated in glucose-limited chemostat cultures. Strain NW297-14 was found to be the best producer and was thus employed for further analysis of the influence of carbon source in chemostat cultures. Here, the highest total specific lipase productivity (r(p total), the sum of extracellular and intracellular lipase productivity) was found to be 1.60+/-0.81 KU/g DW/h in maltose-limited chemostats at a dilution rate of 0.08 h(-1), compared with a total specific lipase productivity of 1.10+/-0.41 KU/g DW/h in glucose-limited chemostats. At the highest specific productivity obtained in this study, the heterologous enzyme accounted for about 1% of all cellular protein being produced by the cells, which shows that it is possible to obtain high productivities of heterologous fungal enzymes in A. niger. However, SDS-PAGE analysis showed that most of the produced lipase was bound to the cell wall.
构建了两株产嗜热栖热菌异源脂肪酶的黑曲霉重组菌株（NW 297 - 14和NW297 - 24）。该异源脂肪酶利用米曲霉的TAKA淀粉酶启动子进行表达。评估了这两株菌株在分批培养和碳源限制恒化器培养中，于不同碳源上的生产动力学。在分批培养中，转化株NW297 - 24在以葡萄糖为碳源生长期间获得了最高的总产物产率系数（Y(xp total)，以胞外和胞内产率之和表示）（5.7±0.65 KU/g DW），而转化株NW297 - 14在以麦芽糖为碳源生长期间获得了最高的总产物产率系数（6.3±0.02 KU/g DW）。在葡萄糖限制的恒化器培养中对这两种转化株进行了评估。发现菌株NW297 - 14是最佳生产者，因此用于进一步分析恒化器培养中碳源的影响。在此，在稀释率为0.08 h(-1)的麦芽糖限制恒化器中，发现最高的总比脂肪酶生产率（r(p total)，胞外和胞内脂肪酶生产率之和）为1.60±0.81 KU/g DW/h，相比之下，在葡萄糖限制恒化器中的总比脂肪酶生产率为1.10±0.41 KU/g DW/h。在本研究获得的最高比生产率下，异源酶约占细胞产生的所有细胞蛋白的1%，这表明在黑曲霉中有可能获得高产率的异源真菌酶。然而，SDS - PAGE分析表明，产生的大部分脂肪酶与细胞壁结合。
