Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, Mexico.
Microb Cell Fact. 2013 May 2;12:42. doi: 10.1186/1475-2859-12-42.
The bacterium Escherichia coli can be grown employing various carbohydrates as sole carbon and energy source. Among them, glucose affords the highest growth rate. This sugar is nowadays widely employed as raw material in industrial fermentations. When E. coli grows in a medium containing non-limiting concentrations of glucose, a metabolic imbalance occurs whose main consequence is acetate secretion. The production of this toxic organic acid reduces strain productivity and viability. Solutions to this problem include reducing glucose concentration by substrate feeding strategies or the generation of mutant strains with impaired glucose import capacity. In this work, a collection of E. coli strains with inactive genes encoding proteins involved in glucose transport where generated to determine the effects of reduced glucose import capacity on growth rate, biomass yield, acetate and production of an experimental plasmid DNA vaccine (pHN).
A group of 15 isogenic derivatives of E. coli W3110 were generated with single and multiple deletions of genes encoding glucose, mannose, beta-glucoside, maltose and N-acetylglucosamine components of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), as well as the galactose symporter and the Mgl galactose/glucose ABC transporter. These strains were characterized by growing them in mineral salts medium supplemented with 2.5 g/L glucose. Maximum specific rates of glucose consumption (qs) spanning from 1.33 to 0.32 g/g h were displayed by the group of mutants and W3110, which resulted in specific growth rates ranging from 0.65-0.18 h(-1). Acetate accumulation was reduced or abolished in cultures with all mutant strains. W3110 and five selected mutant derivatives were transformed with pHN. A 3.2-fold increase in pHN yield on biomass was observed in cultures of a mutant strain with deletion of genes encoding the glucose and mannose PTS components, as well as Mgl.
The group of E. coli mutants generated in this study displayed a reduction or elimination of overflow metabolism and a linear correlation between qs and the maximum specific growth rate as well as the acetate production rate. By comparing DNA vaccine production parameters among some of these mutants, it was possible to identify a near-optimal glucose import rate value for this particular application. The strains employed in this study should be a useful resource for studying the effects of different predefined qs values on production capacity for various biotechnological products.
大肠杆菌可以利用各种碳水化合物作为唯一的碳源和能源进行生长。其中，葡萄糖可提供最高的生长速率。如今，这种糖被广泛用作工业发酵的原料。当大肠杆菌在含有非限制浓度葡萄糖的培养基中生长时，会发生代谢失衡，其主要后果是分泌乙酸。这种有毒有机酸的产生降低了菌株的生产力和生存能力。解决这个问题的方法包括通过底物进料策略降低葡萄糖浓度，或生成葡萄糖摄取能力受损的突变株。在这项工作中，生成了一组具有失活基因的大肠杆菌菌株，这些基因编码参与葡萄糖运输的蛋白质，以确定降低葡萄糖摄取能力对生长速率、生物量产量、乙酸和实验性 pHN 质粒 DNA 疫苗生产的影响。
生成了一组 15 个同基因大肠杆菌 W3110 的衍生菌株，这些菌株具有单个和多个缺失基因，这些基因编码磷酸烯醇丙酮酸：糖磷酸转移酶系统（PTS）的葡萄糖、甘露糖、β-葡糖苷、麦芽糖和 N-乙酰葡萄糖胺成分、半乳糖协同转运蛋白和 Mgl 半乳糖/葡萄糖 ABC 转运蛋白。这些菌株在补充有 2.5 g/L 葡萄糖的无机盐培养基中进行了表征。突变体组和 W3110 的最大比葡萄糖消耗速率（qs）范围为 1.33-0.32 g/g h，导致比生长速率范围为 0.65-0.18 h（-1）。在所有突变株的培养物中，乙酸积累减少或消除。W3110 和五个选定的突变体衍生株被 pHN 转化。在缺失编码葡萄糖和甘露糖 PTS 成分以及 Mgl 的基因的突变体菌株的培养物中，观察到 pHN 对生物质的产量增加了 3.2 倍。
本研究中生成的大肠杆菌突变体组表现出溢出代谢的减少或消除，以及 qs 与最大比生长速率以及乙酸产生速率之间的线性相关性。通过比较这些突变体中的一些 DNA 疫苗生产参数，确定了这种特定应用的最佳葡萄糖摄取率值。在这项研究中使用的菌株应该是研究不同预定义 qs 值对各种生物技术产品生产能力的影响的有用资源。
