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Recombinant protein production-associated metabolic burden reflects anabolic constraints and reveals similarities to a carbon overfeeding response.
Biotechnology and Bioengineering ( IF 3.5 ) Pub Date : 2020-09-03 , DOI: 10.1002/bit.27553
Zhaopeng Li 1 , Ursula Rinas 1, 2
Affiliation  

A comparison of the metabolic response of Escherichia coli BL21 (DE3) towards the production of human basic fibroblast growth factor (hFGF‐2) or towards carbon overfeeding revealed similarities which point to constraints in anabolic pathways. Contrary to expectations, neither energy generation (e.g., ATP) nor provision of precursor molecules for nucleotides (e.g., uracil) and amino acids (e.g., pyruvate, glutamate) limit host cell and plasmid‐encoded functions. Growth inhibition is assumed to occur when hampered anabolic capacities do not match with the ongoing and overwhelming carbon catabolism. Excessive carbon uptake leads to by‐product secretion, for example, pyruvate, acetate, glutamate, and energy spillage, for example, accumulation and degradation of adenine nucleotides with concomitant accumulation of extracellular hypoxanthine. The cellular response towards compromised anabolic capacities involves downregulation of cAMP formation, presumably responsible for subsequently better‐controlled glucose uptake and resultant accumulation of glucose in the culture medium. Growth inhibition is neglectable under conditions of reduced carbon availability when hampered anabolic capacities also match with catabolic carbon processing. The growth inhibitory effect with accompanying energy spillage, respectively, hypoxanthine secretion and cessation of cAMP formation is not unique to the production of hFGF‐2 but observed during the production of other proteins and also during overexpression of genes without transcript translation.

中文翻译:

重组蛋白生产相关的代谢负担反映了合成代谢的限制,并揭示了与碳过量喂养反应的相似之处。

大肠杆菌代谢反应的比较BL21 (DE3) 与人类碱性成纤维细胞生长因子 (hFGF-2) 的产生或碳过度喂养揭示了相似之处,这表明合成代谢途径受到限制。与预期相反,能量产生(例如,ATP)和核苷酸(例如,尿嘧啶)和氨基酸(例如,丙酮酸、谷氨酸)的前体分子的提供都不会限制宿主细胞和质粒编码的功能。当合成代谢能力受阻与持续的和压倒性的碳分解代谢不匹配时,假定会发生生长抑制。过多的碳吸收会导致副产物分泌,例如丙酮酸、醋酸盐、谷氨酸盐和能量溢出,例如腺嘌呤核苷酸的积累和降解,伴随着细胞外次黄嘌呤的积累。细胞对合成代谢能力受损的反应涉及 cAMP 形成的下调,这可能是随后更好地控制葡萄糖摄取和由此产生的葡萄糖在培养基中积累的原因。当合成代谢能力受阻也与分解代谢碳处理相匹配时,在碳可用性降低的条件下,生长抑制是可以忽略的。伴随能量溢出、次黄嘌呤分泌和 cAMP 形成停止的生长抑制作用不是 hFGF-2 的生产所独有的,而是在其他蛋白质的生产过程中以及在没有转录翻译的基因过表达过程中观察到的。大概负责随后更好地控制葡萄糖摄取和由此产生的葡萄糖在培养基中的积累。当合成代谢能力受阻也与分解代谢碳处理相匹配时,在碳可用性降低的条件下,生长抑制是可以忽略的。伴随能量溢出、次黄嘌呤分泌和 cAMP 形成停止的生长抑制作用不是 hFGF-2 的生产所独有的,而是在其他蛋白质的生产过程中以及在没有转录翻译的基因过表达过程中观察到的。大概负责随后更好地控制葡萄糖摄取和由此产生的葡萄糖在培养基中的积累。当合成代谢能力受阻也与分解代谢碳处理相匹配时,在碳可用性降低的条件下,生长抑制是可以忽略的。伴随能量溢出、次黄嘌呤分泌和 cAMP 形成停止的生长抑制作用不是 hFGF-2 的生产所独有的,而是在其他蛋白质的生产过程中以及在没有转录翻译的基因过表达过程中观察到的。
更新日期:2020-09-03
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