Chemical and Biochemical Process Technology and Control Section (BioTeC), Department of Chemical Engineering, Katholieke Universiteit Leuven, Willem de Croylaan 46, 3001, Leuven, Belgium.
Appl Microbiol Biotechnol. 2012 Oct;96(2):367-72. doi: 10.1007/s00253-012-4182-1. Epub 2012 Jul 14.
Streptomyces lividans has shown potential as an expression system for heterologous proteins. Overexpression of proteic factors important for heterologous protein production is a valuable approach to improve yields of such proteins. Comparative transcriptomic analysis revealed that several genes were differentially expressed in strains involved in heterologous protein production. For instance, the gene-encoding phosphoenolpyruvate carboxykinase (pepck) showed a significant twofold change in recombinant S. lividans producing human tumour necrosis factor-alpha (hTNF-α). The effect of pepck overexpression on S. lividans TK24 and its hTNF-α producing recombinant was thus investigated in bench-top fermenters. Results obtained revealed that pepck overexpression resulted into a twofold increase in specific PEPCK activity during growth. This overexpression is correlated with slower growth rate, reduced excretion of pyruvate and less alkalinisation of the growth medium when compared with the control strain. After 26 h of fermentation, hTNF-α yields were enhanced (up to 1.7-fold) in the pepck-overexpressing S. lividans TK24, demonstrating that this metabolic engineering approach is indeed promising for heterologous protein production.
链霉菌属林奈氏菌已显示出作为异源蛋白表达系统的潜力。过表达对异源蛋白生产重要的蛋白因子是提高此类蛋白产量的一种有价值的方法。比较转录组分析表明，参与异源蛋白生产的菌株中存在几个差异表达的基因。例如，编码磷酸烯醇丙酮酸羧激酶（pepck）的基因在生产人肿瘤坏死因子-α（hTNF-α）的重组链霉菌属林奈氏菌中表达水平显著上调了两倍。因此，在台式发酵罐中研究了 pepck 过表达对链霉菌属林奈氏菌 TK24 及其 hTNF-α 生产重组菌的影响。结果表明，pepck 过表达导致生长过程中特异性 PEPCK 活性增加了两倍。与对照菌株相比，这种过表达与生长速率较慢、丙酮酸排泄减少和生长培养基碱化程度降低相关。发酵 26 小时后，在 pepck 过表达的链霉菌属林奈氏菌 TK24 中 hTNF-α 的产量提高了（高达 1.7 倍），表明这种代谢工程方法确实有望用于异源蛋白生产。
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