Low-Copy Plasmids can Perform as Well as or Better Than High-Copy Plasmids for Metabolic Engineering of Bacteria
Low-Copy Plasmids can Perform as Well as or Better Than High-Copy Plasmids for Metabolic Engineering of Bacteria
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DOI：
10.1006/mben.2000.0161
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发表时间：
2000-10-01
影响因子：
8.4
通讯作者：
Keasling, J. D.
中科院分区：
文献类型：
--
作者：
Jones, Kristala L.;Kim, Seon-Won;Keasling, J. D.
在大肠杆菌中表达重组基因时，通常选择多拷贝载体。高拷贝数通常是为了最大限度地表达基因所必需的；然而，在某些代谢工程应用中，通常由多个质粒拷贝引起的代谢负担效应可能被证明不利于最大生产率。在这项研究中，比较了低拷贝mini-F和高拷贝pMB1基质粒在大肠杆菌中产生两种代谢物：聚磷酸盐(PolyP)和由异戊烯基二磷酸(IPP)衍生的番茄红素(番茄红素)。当使用高拷贝或低拷贝质粒时，每个细胞上的息肉的固定相积累增加了约80%，从没有增强的多糖酶(PPK)活性的120mU/g DCW增加到类似于220 mU/g DCW。在稳定期，高拷贝含质粒培养物的细胞密度比低拷贝培养物低24%，比对照培养物低30%。这种细胞密度的差异可能是一种代谢负担效应，并导致高拷贝培养(类似于130mmoL/L培养)的总产物浓度低于低拷贝培养(类似于160mmoL/L培养)。当DXP(1-deoxy-D-xylulose 5-POP)合成酶的基因从tac启动子在多拷贝和低拷贝质粒上表达时，番茄红素的产量比在只表达染色体拷贝的细胞中提高了两到三倍。DXP(1-deoxy-D-xylulose 5-POP)合成酶是IPP甲戊酸非依赖性生物合成途径中的第一种酶。当异丙基-β-D-硫代半乳糖苷酶(IPTG)被添加到含有高拷贝质粒的培养物中时，细胞生长和番茄红素产量显著下降，这表明DXP合成酶的过度表达是一种显著的代谢负担。在含有低拷贝质粒的培养中，没有观察到任何IPTG浓度对细胞生长和番茄红素产量的影响。将DXS置于低拷贝质粒上的阿拉伯糖可诱导启动子(P-BAD)控制下，番茄红素产量与阿拉伯糖浓度成正比，对细胞生长无明显影响。这些结果表明，低拷贝质粒在代谢工程应用中可能是有用的，特别是当重组途径中使用的一种或多种底物是正常细胞代谢所必需的时候。(C)2000年学术出版社
Multicopy plasmids are often chosen for the expression of recombinant genes in Escherichia coli. The high copy number is generally desired for maximum gene expression; however, the metabolic burden effects that usually result from multiple plasmid copies could prove to be detrimental for maximum productivity in certain metabolic engineering applications. In this study, low-copy mini-F plasmids were compared to high-copy pMB1-based plasmids for production of two metabolites in E. coli: polyphosphate (polyP) and lycopene derived from isopentenyl diphosphate (IPP). The stationary-phase accumulation of polyP on a per cell basis was enhanced approximately 80% when either high- or low-copy plasmids were used, from 120 mu mol/g DCW without augmented polyP kinase (PPK) activity to similar to 220 mu mol/g DCW. The cell density of the high-copy plasmid-containing culture at stationary phase was approximately 24% lower than the low-copy culture and 30% lower than the control culture. This difference in cell density is likely a metabolic burden effect and resulted in a lower overall product concentration for the high-copy culture (similar to 130 mu mol/L culture) relative to the low-copy culture (similar to 160 mu mol/L culture). When the gene for DXP (1-deoxy-D-xylulose 5-phosphate) synthase, the first enzyme in the IPP mevalonate-independent biosynthetic pathway, was expressed from the tac promoter on multicopy and low-copy plasmids, lycopene production was enhanced two- to threefold over that found in cells expressing the chromosomal copy only. Cell growth and lycopene production decreased substantially when isopropyl beta-D-thiogalactosidase (IPTG) was added to the high-copy plasmid-containing culture, suggesting that overexpression of DXP synthase was a significant metabolic burden. In the low-copy plasmid-containing culture, no differences in cell growth or lycopene production were observed with any IPTG concentrations. When dxs was placed under the control of the arabinose-inducible promoter (P-BAD) on the low-copy plasmid, the amount of lycopene produced was proportional to the arabinose concentration and no significant changes in cell growth resulted. These results suggest that low-copy plasmids may be useful in metabolic engineering applications, particularly when one or more of the substrates used in the recombinant pathway are required for normal cellular metabolism. (C) 2000 Academic Press