Jayachandran Charumathi, Palanisamy Athiyaman Balakumaran, Sankaranarayanan Meenakshisundaram
Centre for Biotechnology, Anna University, Chennai, India.
PLoS One. 2017 Jul 17;12(7):e0181370. doi: 10.1371/journal.pone.0181370. eCollection 2017.
The cofactor engineering strategy can relieve the metabolic stress induced by expression of recombinant protein in cellular metabolism related to cofactor and energy reactions. To study the effect of cofactor regeneration on recombinant protein expression, NADH oxidase (noxE) was engineered in P. pastoris expressing lipase B (GSCALB). Expression of noxE in P. pastoris (GSCALBNOX) increased NAD+ levels by 85% with a concomitant reduction in NADH/NAD+ ratio of 67% compared to GSCALB. The change in the redox level positively influenced the methanol uptake rate and made 34% augment in CALB activity. The decline in NADH level (44%) by noxE expression had lowered the adenylate energy charge (AEC) and ATP level in GSCALBNOX. In order to regenerate ATP in GSCALBNOX, adenylate kinase (ADK1) gene from S. cerevisiae S288c was co-expressed. Expression of ADK1 showed a remarkable increase in AEC and co-expression of both the genes synergistically improved CALB activity. This study shows the importance of maintenance of cellular redox homeostasis and adenylate energy charge during recombinant CALB expression in P. pastoris.
辅因子工程策略可以缓解细胞代谢中与辅因子和能量反应相关的重组蛋白表达所诱导的代谢应激。为了研究辅因子再生对重组蛋白表达的影响，在表达脂肪酶B（GSCALB）的巴斯德毕赤酵母中对NADH氧化酶（noxE）进行了工程改造。与GSCALB相比，noxE在巴斯德毕赤酵母（GSCALBNOX）中的表达使NAD⁺水平提高了85%，同时NADH/NAD⁺比值降低了67%。氧化还原水平的变化对甲醇摄取率产生了积极影响，并使CALB活性提高了34%。noxE表达导致的NADH水平下降（44%）降低了GSCALBNOX中的腺苷酸能量电荷（AEC）和ATP水平。为了在GSCALBNOX中再生ATP，共表达了来自酿酒酵母S288c的腺苷酸激酶（ADK1）基因。ADK1的表达使AEC显著增加，并使两个基因的共表达协同提高了CALB活性。这项研究表明了在巴斯德毕赤酵母中重组CALB表达过程中维持细胞氧化还原稳态和腺苷酸能量电荷的重要性。
