Chair of Genetics of Prokaryotes, Bielefeld University, Bielefeld, Germany.
Appl Environ Microbiol. 2013 Apr;79(8):2588-95. doi: 10.1128/AEM.03231-12. Epub 2013 Feb 8.
Corynebacterium glutamicum is particularly known for its industrial application in the production of amino acids. Amino acid overproduction comes along with a high NADPH demand, which is covered mainly by the oxidative part of the pentose phosphate pathway (PPP). In previous studies, the complete redirection of the carbon flux toward the PPP by chromosomal inactivation of the pgi gene, encoding the phosphoglucoisomerase, has been applied for the improvement of C. glutamicum amino acid production strains, but this was accompanied by severe negative effects on the growth characteristics. To investigate these effects in a genetically defined background, we deleted the pgi gene in the type strain C. glutamicum ATCC 13032. The resulting strain, C. glutamicum Δpgi, lacked detectable phosphoglucoisomerase activity and grew poorly with glucose as the sole substrate. Apart from the already reported inhibition of the PPP by NADPH accumulation, we detected a drastic reduction of the phosphotransferase system (PTS)-mediated glucose uptake in C. glutamicum Δpgi. Furthermore, Northern blot analyses revealed that expression of ptsG, which encodes the glucose-specific EII permease of the PTS, was abolished in this mutant. Applying our findings, we optimized l-lysine production in the model strain C. glutamicum DM1729 by deletion of pgi and overexpression of plasmid-encoded ptsG. l-Lysine yields and productivity with C. glutamicum Δpgi(pBB1-ptsG) were significantly higher than those with C. glutamicum Δpgi(pBB1). These results show that ptsG overexpression is required to overcome the repressed activity of PTS-mediated glucose uptake in pgi-deficient C. glutamicum strains, thus enabling efficient as well as fast l-lysine production.
谷氨酸棒杆菌特别以其在生产氨基酸方面的工业应用而闻名。氨基酸的过量生产伴随着对 NADPH 的高需求,这主要由戊糖磷酸途径(PPP)的氧化部分来覆盖。在以前的研究中,通过染色体灭活编码磷酸葡萄糖异构酶的 pgi 基因,将碳通量完全转向 PPP,已应用于改善谷氨酸棒杆菌氨基酸生产菌株,但这伴随着对生长特性的严重负面影响。为了在遗传上定义的背景下研究这些影响,我们在模式菌株 C. glutamicum ATCC 13032 中删除了 pgi 基因。由此产生的菌株 C. glutamicum Δpgi 缺乏可检测的磷酸葡萄糖异构酶活性,并且以葡萄糖为唯一底物时生长不良。除了已经报道的 NADPH 积累对 PPP 的抑制作用外,我们还检测到 C. glutamicum Δpgi 中磷酸转移酶系统(PTS)介导的葡萄糖摄取急剧减少。此外,Northern blot 分析表明,ptsG 的表达,其编码 PTS 的葡萄糖特异性 EII 通透酶,在这个突变体中被废除。应用我们的发现,我们通过删除 pgi 和过表达质粒编码的 ptsG 来优化模型菌株 C. glutamicum DM1729 中的 l-赖氨酸生产。与 C. glutamicum Δpgi(pBB1)相比,C. glutamicum Δpgi(pBB1-ptsG)的 l-赖氨酸产量和生产力明显更高。这些结果表明,ptsG 的过表达是克服 pgi 缺失的谷氨酸棒杆菌菌株中 PTS 介导的葡萄糖摄取活性受到抑制所必需的,从而能够实现高效且快速的 l-赖氨酸生产。