Engineering Escherichia coli to increase plasmid DNA production in high cell-density cultivations in batch mode.
Engineering Escherichia coli to increase plasmid DNA production in high cell-density cultivations in batch mode.
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DOI：
10.1186/1475-2859-11-132
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发表时间：
2012-09-19
影响因子：
6.4
通讯作者：
Lara AR
中科院分区：
文献类型：
--
作者：
Borja GM;Meza Mora E;Barrón B;Gosset G;Ramírez OT;Lara AR
质粒DNA（pDNA）是用于治疗应用的有前途的分子。pDNA由大肠杆菌在高细胞密度培养（HCDC）中使用补料分批模式产生。这种培养的典型局限性，包括由于大规模生物反应器中存在底物梯度而导致的代谢偏差，如需氧乙酸盐生产，仍然是快速和有效pDNA生产的严重挑战。我们以前已经证明，在大肠杆菌中，磷酸转移酶系统被过表达的半乳糖通透酶取代，葡萄糖摄取。大肠杆菌（菌株VH 33）允许有效生长，同时强烈降低乙酸盐生产。在本工作中，对VH 33进行了额外的遗传修饰以进一步改善pDNA生产。从菌株VH 33中删除了几个基因：recA、deoR、nupG和endA基因被单独和组合失活。在摇瓶中评价突变株产生携带抗腮腺炎抗原基因的6.1 kb质粒的性能。在实验室规模的生物反应器中使用100 g/L的葡萄糖培养最佳生产菌株，以分批模式实现HCDC。为了比较，还在相同条件下培养了携带相同质粒的广泛使用的商业菌株DH 5 α。测试的各种突变对比生长速率、葡萄糖摄取速率和pDNA产率（YP/X）具有不同的影响。三重突变体VH 33 Δ（recA deoR nupG）积累了低量的乙酸并产生了最好的YP/X（4.22 mg/g），而菌株VH 33的YP/X仅达到1.16 mg/g。当在高葡萄糖浓度下培养时，三重突变体菌株产生186 mg/L的pDNA、40 g/L的生物量和仅2.2 g/L的乙酸盐。相比之下，DH 5 α仅产生70 mg/L的pDNA，积累9.5 g/L的乙酸盐。此外，由三重突变体产生的pDNA的超螺旋部分在整个培养过程中几乎恒定。据我们所知，用工程菌株VH 33 Δ（recA deoR nupG）获得的pDNA浓度是分批培养中报道的最高浓度，并且其超螺旋分数保持接近80%。菌株VH 33 Δ（recA deoR nupG）及其使用升高的葡萄糖浓度的培养代表了用于快速和有效的pDNA生产的有吸引力的技术，以及商业菌株的补料分批培养的有价值的替代方案。
Plasmid DNA (pDNA) is a promising molecule for therapeutic applications. pDNA is produced by Escherichia coli in high cell-density cultivations (HCDC) using fed-batch mode. The typical limitations of such cultivations, including metabolic deviations like aerobic acetate production due to the existence of substrate gradients in large-scale bioreactors, remain as serious challenges for fast and effective pDNA production. We have previously demonstrated that the substitution of the phosphotransferase system by the over-expressed galactose permease for glucose uptake in E. coli (strain VH33) allows efficient growth, while strongly decreases acetate production. In the present work, additional genetic modifications were made to VH33 to further improve pDNA production. Several genes were deleted from strain VH33: the recA, deoR, nupG and endA genes were inactivated independently and in combination. The performance of the mutant strains was evaluated in shake flasks for the production of a 6.1 kb plasmid bearing an antigen gene against mumps. The best producer strain was cultivated in lab-scale bioreactors using 100 g/L of glucose to achieve HCDC in batch mode. For comparison, the widely used commercial strain DH5α, carrying the same plasmid, was also cultivated under the same conditions. The various mutations tested had different effects on the specific growth rate, glucose uptake rate, and pDNA yields (YP/X). The triple mutant VH33 Δ (recA deoR nupG) accumulated low amounts of acetate and resulted in the best YP/X (4.22 mg/g), whereas YP/X of strain VH33 only reached 1.16 mg/g. When cultivated at high glucose concentrations, the triple mutant strain produced 186 mg/L of pDNA, 40 g/L of biomass and only 2.2 g/L of acetate. In contrast, DH5α produced only 70 mg/L of pDNA and accumulated 9.5 g/L of acetate. Furthermore, the supercoiled fraction of the pDNA produced by the triple mutant was nearly constant throughout the cultivation. The pDNA concentration obtained with the engineered strain VH33 Δ (recA deoR nupG) is, to the best of our knowledge, the highest reported for a batch cultivation, and its supercoiled fraction remained close to 80%. Strain VH33 Δ (recA deoR nupG) and its cultivation using elevated glucose concentrations represent an attractive technology for fast and efficient pDNA production and a valuable alternative to fed-batch cultivations of commercial strains.
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影响因子：
3.8
作者：
Lara, Alvaro R.;Caspeta, Luis;Ramirez, Octavio T.
通讯作者：
Ramirez, Octavio T.
DOI：
10.1007/978-1-61779-433-9_14
发表时间：
2012-01-01
期刊：
RECOMBINANT GENE EXPRESSION: REVIEWS AND PROTOCOLS, THIRD EDITION
影响因子：
--
作者：
Lara, Alvaro R.;Ramirez, Octavio T.
通讯作者：
Ramirez, Octavio T.
DOI：
10.1073/pnas.120163297
发表时间：
2000-06-06
影响因子：
11.1
作者：
Datsenko, KA;Wanner, BL
通讯作者：
Wanner, BL
影响因子：
4.1
作者：
O'Kennedy, RD;Baldwin, C;Keshavarz-Moore, E
通讯作者：
Keshavarz-Moore, E
影响因子：
4.4
作者：
Chavez-Bejar, Maria I.;Lara, Alvaro R.;Gosset, Guillermo
通讯作者：
Gosset, Guillermo