Area de Microbiología, Departamento de Biología Molecular, Universidad de León, Campus de Vegazana s/n, 24071 León, Spain.
Mol Cell Proteomics. 2010 Jun;9(6):1182-98. doi: 10.1074/mcp.M900327-MCP200. Epub 2010 Feb 12.
Proteomics is a powerful tool to understand the molecular mechanisms causing the production of high penicillin titers by industrial strains of the filamentous fungus Penicillium chrysogenum as the result of strain improvement programs. Penicillin biosynthesis is an excellent model system for many other bioactive microbial metabolites. The recent publication of the P. chrysogenum genome has established the basis to understand the molecular processes underlying penicillin overproduction. We report here the proteome reference map of P. chrysogenum Wisconsin 54-1255 (the genome project reference strain) together with an in-depth study of the changes produced in three different strains of this filamentous fungus during industrial strain improvement. Two-dimensional gel electrophoresis, peptide mass fingerprinting, and tandem mass spectrometry were used for protein identification. Around 1000 spots were visualized by "blue silver" colloidal Coomassie staining in a non-linear pI range from 3 to 10 with high resolution, which allowed the identification of 950 proteins (549 different proteins and isoforms). Comparison among the cytosolic proteomes of the wild-type NRRL 1951, Wisconsin 54-1255 (an improved, moderate penicillin producer), and AS-P-78 (a penicillin high producer) strains indicated that global metabolic reorganizations occurred during the strain improvement program. The main changes observed in the high producer strains were increases of cysteine biosynthesis (a penicillin precursor), enzymes of the pentose phosphate pathway, and stress response proteins together with a reduction in virulence and in the biosynthesis of other secondary metabolites different from penicillin (pigments and isoflavonoids). In the wild-type strain, we identified enzymes to utilize cellulose, sorbitol, and other carbon sources that have been lost in the high penicillin producer strains. Changes in the levels of a few specific proteins correlated well with the improved penicillin biosynthesis in the high producer strains. These results provide useful information to improve the production of many other bioactive secondary metabolites.
蛋白质组学是一种强大的工具，可以了解导致工业丝状真菌产黄青霉菌株产生高青霉素效价的分子机制，这些菌株是通过菌株改良计划得到的。青霉素生物合成是许多其他生物活性微生物代谢物的极好模型系统。最近发表的产黄青霉基因组为理解青霉素高产的分子过程奠定了基础。我们在此报告产黄青霉威斯康星 54-1255（基因组项目参考菌株）的蛋白质组参考图谱，以及对该丝状真菌的三个不同菌株在工业菌株改良过程中产生的变化进行了深入研究。二维凝胶电泳、肽质量指纹图谱和串联质谱用于蛋白质鉴定。在非线性 pI 范围内（3 至 10）使用“蓝色银”胶体考马斯亮蓝染色可观察到约 1000 个斑点，分辨率高，可鉴定 950 种蛋白质（549 种不同的蛋白质和同工型）。野生型 NRRL 1951、威斯康星 54-1255（改良的、中等青霉素生产菌）和 AS-P-78（青霉素高产菌）菌株的细胞质蛋白质组比较表明，在菌株改良计划中发生了全局代谢重排。在高产菌株中观察到的主要变化是半胱氨酸生物合成（青霉素前体）、戊糖磷酸途径的酶以及应激反应蛋白的增加，同时降低了毒性和其他不同于青霉素（色素和异黄酮）的次生代谢物的生物合成。在野生型菌株中，我们鉴定了利用纤维素、山梨醇和其他已在高产青霉素菌株中丢失的碳源的酶。少数特定蛋白质水平的变化与高产菌株中青霉素生物合成的改善密切相关。这些结果为提高许多其他生物活性次生代谢物的产量提供了有用的信息。
