Increasing recombinant protein production in Escherichia coli K12 through metabolic engineering

Waegeman, Hendrik; Lausnay, Stijn De; Beauprez, Joeri; Maertens, Jo; Mey, Marjan De; Soetaert, Wim

doi:10.1016/j.nbt.2011.11.008

Cited by 27 publications

(19 citation statements)

References 37 publications

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“…coli in particular) still represent a very common choice for protein production [52]. The improvements in yields and efficiency delivered via metabolic engineering and synthetic biology [53-55] are complemented by more analytical approaches that aim to understand the properties that cause aggregation and its effects on host viability [56-59]. As a result of these efforts, a number of strains, fermentation conditions and co-expression partners (such as chaperones or rare tRNAs) can be used for high-throughput protein expression screening [60].…”

Section: Discussionmentioning

confidence: 99%

A single mutation in the core domain of the lac repressor reduces leakiness

et al. 2013

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BackgroundThe lac operon provides cells with the ability to switch from glucose to lactose metabolism precisely when necessary. This metabolic switch is mediated by the lac repressor (LacI), which in the absence of lactose binds to the operator DNA sequence to inhibit transcription. Allosteric rearrangements triggered by binding of the lactose isomer allolactose to the core domain of the repressor impede DNA binding and lift repression. In Nature, the ability to detect and respond to environmental conditions comes at the cost of the encoded enzymes being constitutively expressed at low levels. The readily-switched regulation provided by LacI has resulted in its widespread use for protein overexpression, and its applications in molecular biology represent early examples of synthetic biology. However, the leakiness of LacI that is essential for the natural function of the lac operon leads to an increased energetic burden, and potentially toxicity, in heterologous protein production.ResultsAnalysis of the features that confer promiscuity to the inducer-binding site of LacI identified tryptophan 220 as a target for saturation mutagenesis. We found that phenylalanine (similarly to tryptophan) affords a functional repressor that is still responsive to IPTG. Characterisation of the W220F mutant, LacIWF, by measuring the time dependence of GFP production at different IPTG concentrations and at various incubation temperatures showed a 10-fold reduction in leakiness and no decrease in GFP production. Cells harbouring a cytotoxic protein under regulatory control of LacIWF showed no decrease in viability in the early phases of cell growth. Changes in responsiveness to IPTG observed in vivo are supported by the thermal shift assay behaviour of purified LacIWF with IPTG and operator DNA.ConclusionsIn LacI, long-range communications are responsible for the transmission of the signal from the inducer binding site to the DNA binding domain and our results are consistent with the involvement of position 220 in modulating these. The mutation of this single tryptophan residue to phenylalanine generated an enhanced repressor with a 10-fold decrease in leakiness. By minimising the energetic burden and cytotoxicity caused by leakiness, LacIWF constitutes a useful switch for protein overproduction and synthetic biology.

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Section: Discussionmentioning

confidence: 99%

A single mutation in the core domain of the lac repressor reduces leakiness

et al. 2013

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“…Although the increased biomass yield and decreased acetate yield in the double knockout beneficially influence recombinant protein production as a higher GFP yield was observed to its wild type E. coli MG1655 (30% increase in the double knockout strain), still a www.intechopen.com striking difference of more than 40% was detected compared to E. coli Bl21. Additionally, we observed that at higher cell densities (>2gL -1 CDW) the GFP concentrations decreases again suggesting proteases activity (Waegeman et al, 2011b).…”

Section: Escherichia Coli Mg1655 ∆Arca ∆Iclr As Potential Candidate Fmentioning

confidence: 72%

Increasing Recombinant Protein Production in E. coli by an Alternative Method to Reduce Acetate

Waegeman¹,

Mey²

2012

Advances in Applied Biotechnology

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“…There are therefore several approaches for the modifications of the E. coli host to achieve the desired goal [23-25]. In our case the design strategy was essentially a high throughput screen which helped us select better producers.…”

Section: Resultsmentioning

confidence: 99%

An inverse metabolic engineering approach for the design of an improved host platform for over-expression of recombinant proteins in Escherichia coli

2012

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BackgroundA useful goal for metabolic engineering would be to generate non-growing but metabolically active quiescent cells which would divert the metabolic fluxes towards product formation rather than growth. However, for products like recombinant proteins, which are intricately coupled to the growth process it is difficult to identify the genes that need to be knocked-out/knocked-in to get this desired phenotype. To circumvent this we adopted an inverse metabolic engineering strategy which would screen for the desired phenotype and thus help in the identification of genetic targets which need to be modified to get overproducers of recombinant protein. Such quiescent cells would obviate the need for high cell density cultures and increase the operational life span of bioprocesses.ResultsA novel strategy for generating a library, consisting of randomly down regulated metabolic pathways in E. coli was designed by cloning small genomic DNA fragments in expression vectors. Some of these DNA fragments got inserted in the reverse orientation thereby generating anti-sense RNA upon induction. These anti-sense fragments would hybridize to the sense mRNA of specific genes leading to gene ‘silencing’. This library was first screened for slow growth phenotype and subsequently for enhanced over-expression ability. Using Green Fluorescent Protein (GFP) as a reporter protein on second plasmid, we were able to identify metabolic blocks which led to significant increase in expression levels. Thus down-regulating the ribB gene (3, 4 dihydroxy-2-butanone-4-phosphate synthase) led to a 7 fold increase in specific product yields while down regulating the gene kdpD (histidine kinase) led to 3.2 fold increase in specific yields.ConclusionWe have designed a high throughput screening approach which is a useful tool in the repertoire of reverse metabolic engineering strategies for the generation of improved hosts for recombinant protein expression.

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Increasing recombinant protein production in Escherichia coli K12 through metabolic engineering

Cited by 27 publications

References 37 publications

A single mutation in the core domain of the lac repressor reduces leakiness

A single mutation in the core domain of the lac repressor reduces leakiness

Increasing Recombinant Protein Production in E. coli by an Alternative Method to Reduce Acetate

An inverse metabolic engineering approach for the design of an improved host platform for over-expression of recombinant proteins in Escherichia coli

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