Growth-rate recovery of Escherichia coli cultures carrying a multicopy plasmid, by engineering of the pentose-phosphate pathway
Growth-rate recovery of Escherichia coli cultures carrying a multicopy plasmid, by engineering of the pentose-phosphate pathway
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DOI：
10.1002/bit.20137
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发表时间：
2004-08-20
影响因子：
3.8
通讯作者：
Bolívar, FG
中科院分区：
文献类型：
--
作者：
Flores, S;de Anda-Herrera, R;Bolívar, FG
在细菌中表达质粒编码的基因是生物技术过程中生产特定蛋白质的最常见策略。然而，质粒编码蛋白质的合成和质粒dna的复制通常会给细胞的生化能力带来代谢负荷（代谢负担），这通常会降低生产培养物的生长速度（Glick BR）。生物技术杂志1995；13:247-261)。这种代谢负担可能与细胞提供复制质粒DNA和表达外源多拷贝基因所需的额外构建块和能量的能力有限有关。其中一些必需的阻滞是戊糖磷酸（PP）途径的中间体，例如，5-磷酸核糖，4-磷酸红细胞。由于代谢负担对生物技术过程的重要影响，几个小组已经致力于开发克服这一问题的策略，如减少质粒拷贝数（Seo JH, Bailey JE）。biotechnology Bioeng 1985；27:1668 - 1674;琼斯KL，金S， Keasling JD。Metab engineering 2000；3:328-338)，染色体插入基因的产物，或改变质粒编码的抗生素耐药基因(Hong Y, Pasternak JJ, Glick BR。中华微生物学杂志（英文版）；2004;16(1):1 -6。然而，很少有人试图通过改变中枢代谢途径来克服由于蛋白质过表达而导致的生长速度降低（Chou C-H, Bennett GN, San KY）。生物工程学报（英文版）；1994;44(4):952-960。在诱导型trc启动子（pTRzwf04质粒）的控制下，构建了一个携带葡萄糖-6-磷酸脱氢酶zwf基因的高拷贝数质粒。通过转化野生型菌株并用IPTG诱导，可以将生长速率从0.46 h(-1)（未诱导）恢复到0.64 h(-1)（诱导）。在大肠杆菌zwf（-）中进行同样的转化，可以使生长速率从0.43 h(-1)（非诱导）恢复到0.62 h(-1)（诱导）。我们还将这种效应作为实验室规模生物技术过程的一部分进行了研究：通过将大肠杆菌JM101菌株与pTRzwf07（一种低拷贝数质粒，与pTRzwf04具有相同的诱导结构）和携带TrpLE-proinsulin杂交基因的ptexpi - mmrpi载体共转化，生产重组胰岛素肽。在这个系统中，trple - pro胰岛素的产生强烈地降低了生长速度；然而，TrpLE-proinsulin诱导菌株zwf基因的过表达速度从0.1 h（-1）恢复到0.37 h（-1）。在本文中，我们表明，通过调节zwf基因表达水平来设计戊糖磷酸途径，部分克服了通过PP途径合成的构建块和还原力供应的可能瓶颈，这些构建块和还原力是质粒复制和质粒编码蛋白表达所必需的。(C) 2004 Wiley期刊有限公司
Expression of plasmid-encoded genes in bacteria is the most common strategy for the production of specific proteins in biotechnological processes. However, the synthesis of plasmid-encoded proteins and plasmid-DNA replication often places a metabolic load (metabolic burden) into the cell's biochemical capacities that usually reduces the growth rate of the producing culture (Glick BR. Biotechnol Adv 1995;13:247-261). This metabolic burden may be related to a limited capacity of the cell to supply the extra demand of building blocks and energy required to replicate plasmid DNA and express foreign multicopy genes. Some of these required blocks are intermediaries of the pentose phosphate (PP) pathway, e.g., ribose-5-phosphate, erythrose-4-phosphate. Due to the important impact of metabolic burden on biotechnological processes, several groups have worked on developing strategies to overcome this problem, like reduction of plasmid copy number (Seo JH, Bailey JE. Biotechnol Bioeng 1985; 27:1668-1674; Jones KL, Kim S, Keasling JD. Metab Eng 2000;3:328-338), chromosomal insertion of the gene which product is desired, or changing the plasmid-coded antibiotic resistance gene (Hong Y, Pasternak JJ, Glick BR. Can J Microbiol 1995;41:624-628). However, few efforts have been attempted to overcome the reduction of growth rate due to protein over-expression, by modifying central metabolic pathways (Chou C-H, Bennett GN, San KY. Biotechnol Bioeng 1994;44:952-960). We constructed a high-copy number plasmid carrying the gene for glucose-6-phosphate dehydrogenase, zwf, under the control of an inducible trc promoter (pTRzwf04 plasmid). By transforming a wild-type strain and inducing with IPTG, it was possible to recover growth-rate from 0.46 h(-1) (uninduced) to 0.64 h(-1) (induced). The same transformation in an Escherichia coli zwf(-), allows a growth-rate recovery from 0.43 h(-1) (uninduced) to 0.62 h(-1) (induced). We also studied this effect as part of a laboratory-scale biotechnology process: production of a recombinant insulin peptide by co-transforming E. coli JM101 strain with pTRzwf07, a low-copy-number plasmid that carries the same inducible construction as pTRzwf04, and with the pTEXP-MMRPI vector that carries a TrpLE-proinsulin hybrid gene. In this system, production of TrpLE-proinsulin strongly reduces growth rate; however, overexpression of zwf gene recovers with a growth rate from 0.1 h(-1) in the TrpLE-proinsulin induced strain, to 0.37 h(-1) when both zwf and TrpLE-proinsulin genes were induced. In this paper, we show that the engineering of the pentose phosphate pathway by modulation of the zwf gene expression level partially overcomes the possible bottleneck for the supply of building blocks and reducing power synthesized through the PP pathway, that are required for plasmid replication and plasmid-encoded protein expression. (C) 2004 Wiley Periodicals, Inc.