Low-Copy Plasmids can Perform as Well as or Better Than High-Copy Plasmids for Metabolic Engineering of Bacteria

Jones, Kristala L.; Kim, Seon-Won; Keasling, Jay D.

doi:10.1006/mben.2000.0161

Cited by 238 publications

(151 citation statements)

References 25 publications

(18 reference statements)

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“…Indeed, strong enzyme overproduction may considerably exceed the level that is optimal for the metabolic engineering goal. There are several examples of target phenotype optimization achieved by moderate rather than strong multicopy gene expression in S. cerevisiae (147,150,196,345). Moreover, one example even demonstrates that very low enzyme activity can be optimal.…”

Section: Fine-tuning Gene Expressionmentioning

confidence: 99%

Progress in Metabolic Engineering of Saccharomyces cerevisiae

Nevoigt

2008

Microbiol Mol Biol Rev

526

333

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SUMMARY The traditional use of the yeast Saccharomyces cerevisiae in alcoholic fermentation has, over time, resulted in substantial accumulated knowledge concerning genetics, physiology, and biochemistry as well as genetic engineering and fermentation technologies. S. cerevisiae has become a platform organism for developing metabolic engineering strategies, methods, and tools. The current review discusses the relevance of several engineering strategies, such as rational and inverse metabolic engineering, evolutionary engineering, and global transcription machinery engineering, in yeast strain improvement. It also summarizes existing tools for fine-tuning and regulating enzyme activities and thus metabolic pathways. Recent examples of yeast metabolic engineering for food, beverage, and industrial biotechnology (bioethanol and bulk and fine chemicals) follow. S. cerevisiae currently enjoys increasing popularity as a production organism in industrial (“white”) biotechnology due to its inherent tolerance of low pH values and high ethanol and inhibitor concentrations and its ability to grow anaerobically. Attention is paid to utilizing lignocellulosic biomass as a potential substrate.

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Section: Fine-tuning Gene Expressionmentioning

confidence: 99%

Progress in Metabolic Engineering of Saccharomyces cerevisiae

Nevoigt

2008

Microbiol Mol Biol Rev

526

333

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“…Multivariate-modular approach to balance metabolic pathway has been successfully used to systematically search for the optimal conditions for taxadiene production, resulting in 15, 000-fold increase in taxadiene titer over the control (Ajikumar et al 2010). Modulating plasmid copy numbers or engineering different types of promoters (Jones et al 2000) has been used to control and balance the expression levels of multiple genes for more than 10 years. However, these approaches are time consuming and resource demanding and may not cover a sufficiently broad search space to allow the identification of conditions for optimal productivity.…”

Section: Introductionmentioning

confidence: 99%

Experimental design-aided systematic pathway optimization of glucose uptake and deoxyxylulose phosphate pathway for improved amorphadiene production

Zhang

Zou

Chen

et al. 2015

Appl Microbiol Biotechnol

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Artemisinin is a potent antimalarial drug; however, it suffers from unstable and insufficient supply from plant source. Here, we established a novel multivariate-modular approach based on experimental design for systematic pathway optimization that succeeded in improving the production of amorphadiene (AD), the precursor of artemisinin, in Escherichia coli. It was initially found that the AD production was limited by the imbalance of glyceraldehyde 3-phosphate (GAP) and pyruvate (PYR), the two precursors of the 1-deoxy-D-xylulose-5-phosphate (DXP) pathway. Furthermore, it was identified that GAP and PYR could be balanced by replacing the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) with the ATP-dependent galactose permease and glucose kinase system (GGS) and this resulted in fivefold increase in AD titer (11 to 60 mg/L). Subsequently, the experimental design-aided systematic pathway optimization (EDASPO) method was applied to systematically optimize the transcriptional expressions of eight critical genes in the glucose uptake and the DXP and AD synthesis pathways.These genes were classified into four modules and simultaneously controlled by T7 promoter or its variants. A regression model was generated using the four-module experimental data and predicted the optimal expression ratios among these modules, resulting in another threefold increase in AD titer (60 to 201 mg/L). This EDASPO method may be useful for the optimization of other pathways and products beyond the scope of this study.

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“…However, while the ease of manipulating and transferring extrachromosomal DNA makes plasmids an attractive tool for cursory proof-of-concept studies, their use introduces severe inaccuracies when carefully evaluating the performance of engineered biological systems. Indeed, the combined metabolic burden of both plasmid propagation/ maintenance 18,19 and pathway overexpression often results in segregational or genetic population events promoting plasmid loss or allele inactivation 20 . In addition, several studies have shown that cell-to-cell variability in copy number can be significant under different cellular growth states and conditions 21,22 , a phenomenon that can lead to erratic strain performance.…”

mentioning

confidence: 99%

Implementation of stable and complex biological systems through recombinase-assisted genome engineering

Santos

Regitsky²,

Yoshikuni³

2013

Nat Commun

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Evaluating the performance of engineered biological systems with high accuracy and precision is nearly impossible with the use of plasmids due to phenotypic noise generated by genetic instability and natural population dynamics. Minimizing this uncertainty therefore requires a paradigm shift towards engineering at the genomic level. Here, we introduce an advanced design principle for the stable instalment and implementation of complex biological systems through recombinase-assisted genome engineering (RAGE). We apply this concept to the development of a robust strain of Escherichia coli capable of producing ethanol directly from brown macroalgae. RAGE significantly expedites the optimal implementation of a 34 kb heterologous pathway for alginate metabolism based on genetic background, integration locus, copy number and compatibility with two other pathway modules (alginate degradation and ethanol production). The resulting strain achieves a B40% higher titre than its plasmidbased counterpart and enables substantial improvements in titre (B330%) and productivity (B1,200%) after 50 generations.

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Low-Copy Plasmids can Perform as Well as or Better Than High-Copy Plasmids for Metabolic Engineering of Bacteria

Cited by 238 publications

References 25 publications

Progress in Metabolic Engineering of Saccharomyces cerevisiae

Progress in Metabolic Engineering of Saccharomyces cerevisiae

Experimental design-aided systematic pathway optimization of glucose uptake and deoxyxylulose phosphate pathway for improved amorphadiene production

Implementation of stable and complex biological systems through recombinase-assisted genome engineering

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