Accurate DNA Assembly and Genome Engineering with Optimized Uracil Excision Cloning

Cavaleiro, Ana Mafalda; Kim, Se Hyeuk; Seppälä, Susanna; Nielsen, Morten Ørregaard; Nørholm, Morten H. H.

doi:10.1021/acssynbio.5b00113

Cited by 88 publications

(77 citation statements)

References 24 publications

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“…Cells were incubated for 1 hr at 37°C before plating on LB agar with appropriate antibiotics. All plasmids were constructed by USER cloning (Cavaleiro, Kim, Seppälä, Nielsen, & Nørholm, 2015; Nour‐Eldin, Hansen, Nørholm, Jensen, & Halkier, 2006). Briefly, template DNA was amplified using Phusion U polymerase and primers containing a uracil complementary overhang.…”

Section: Methodsmentioning

confidence: 99%

An autoinducible trp‐T7 expression system for production of proteins and biochemicals in Escherichia coli

Landberg

Mundhada

Nielsen

2020

Biotech & Bioengineering

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Inducible expression systems can be applied to control the expression of proteins or biochemical pathways in cell factories. However, several of the established systems require the addition of expensive inducers, making them unfeasible for large‐scale production. Here, we establish a genome integrated trp‐T7 expression system where tryptophan can be used to control the induction of a gene or a metabolic pathway. We show that the initiation of gene expression from low‐ and high‐copy vectors can be tuned by varying the initial concentration of tryptophan or yeast extract, and that expression is tightly regulated and homogenous when compared with the commonly used lac‐T7 system. Finally, we apply the trp‐T7 expression system for the production of l‐serine, where we reach titers of 26 g/L in fed‐batch fermentation.

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Section: Methodsmentioning

confidence: 99%

An autoinducible trp‐T7 expression system for production of proteins and biochemicals in Escherichia coli

Landberg

Mundhada

Nielsen

2020

Biotech & Bioengineering

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“…The third uracil excision protocol adds direct genome integration (clonetegration [19]) to the uracil excision portfolio. The optimal design parameters for multigene assembly and uracil excision combined with clonetegration have recently been explored [18]. Detailed information on clonetegration including vectors and an oligonucleotide list for colony PCR is described in St-Pierre et al [19].…”

Section: Methodsmentioning

confidence: 99%

“…In our experience, purification in some cases enhances the efficiency and fidelity of the assembly reaction, possibly due the removal of interfering oligonucleotides [18], but it also complicates the protocol.…”

Section: Notesmentioning

confidence: 99%

Uracil Excision for Assembly of Complex Pathways

Cavaleiro

Nielsen

Kim

et al. 2015

Springer Protocols Handbooks

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Despite decreasing prices on synthetic DNA constructs, higher-order assembly of PCR-generated DNA continues to be an important exercise in molecular and synthetic biology. Simplicity and robustness are attractive features met by the uracil excision DNA assembly method, which is one of the most inexpensive technologies available. Here, we describe four different protocols for uracil excision-based DNA editing: one for simple manipulations such as site-directed mutagenesis, one for plasmid-based multigene assembly in Escherichia coli, one for one-step assembly and integration of single or multiple genes into the genome, and a standardized assembly pipeline using benchmarked oligonucleotides for pathway assembly and multigene expression optimization.

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“…The three amplified genes were cloned into pUCrop, resulting in plasmids pUCrop_CYP714A2, pUCrop_AtKO, and pUCrop_AtCPR2. Assembly of two or three genes in one vector system, e.g., pUCrop_AtKO_AtCPR2 (Table 1), was carried out via the uracil excision cloning technology (USER) [26].…”

Section: Construction Of Plasmidsmentioning

confidence: 99%

Redesign and reconstruction of a steviol-biosynthetic pathway for enhanced production of steviol in Escherichia coli

Moon

Lee

et al. 2020

Microb Cell Fact

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Background: Steviol glycosides such as stevioside have attracted the attention of the food and beverage industry. Recently, efforts were made to produce these natural sweeteners in microorganisms using metabolic engineering. Nonetheless, the steviol titer is relatively low in metabolically engineered microorganisms, and therefore a steviol-biosynthetic pathway in heterologous microorganisms needs to be metabolically optimized. The purpose of this study was to redesign and reconstruct a steviol-biosynthetic pathway via synthetic-biology approaches in order to overproduce steviol in Escherichia coli. Results: A genome-engineered E. coli strain, which coexpressed 5′ untranslated region (UTR)-engineered geranylgeranyl diphosphate synthase, copalyl diphosphate synthase, and kaurene synthase, produced 623.6 ± 3.0 mg/L ent-kaurene in batch fermentation. Overexpression of 5′-UTR-engineered, N-terminally modified kaurene oxidase of Arabidopsis thaliana yielded 41.4 ± 5 mg/L ent-kaurenoic acid. Enhanced ent-kaurenoic acid production (50.7 ± 9.8 mg/L) was achieved by increasing the cellular NADPH/NADP + ratio. The expression of a fusion protein, UtrCYP714A2-AtCPR2 derived from A. thaliana, where trCYP714A2 was 5′-UTR-engineered and N-terminally modified, gave 38.4 ± 1.7 mg/L steviol in batch fermentation. Conclusions: 5′-UTR engineering, the fusion protein approach, and redox balancing improved the steviol titer in flask fermentation and bioreactor fermentation. The expression engineering of steviol-biosynthetic enzymes and the genome engineering described here can serve as the basis for producing terpenoids-including steviol glycosides and carotenoids-in microorganisms.

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Accurate DNA Assembly and Genome Engineering with Optimized Uracil Excision Cloning

Cited by 88 publications

References 24 publications

An autoinducible trp‐T7 expression system for production of proteins and biochemicals in Escherichia coli

An autoinducible trp‐T7 expression system for production of proteins and biochemicals in Escherichia coli

Uracil Excision for Assembly of Complex Pathways

Redesign and reconstruction of a steviol-biosynthetic pathway for enhanced production of steviol in Escherichia coli

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