Efficient strategies to enhance plasmid stability for fermentation of recombinant Escherichia coli harboring tyrosine phenol lyase

Tang, Xiaoling; Hu, Wen-Ye; Wang, Zhichao; Zheng, Ren‐Chao; Zheng, Yu‐Guo

doi:10.1007/s10529-021-03082-5

Cited by 5 publications

(6 citation statements)

References 40 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Original rCTC construct (pTRBAB5-G1S) [18], subcloned to pET30a<>Cer [35] that contains a Cer site to enhance plasmid stability [36], has increased promotor strength from tac (weak promotor) to T7 (strong promotor). The tac promotor regulated by RNA polymerase (RNAP) has to be replaced, before introducing the rCTC-bearing plasmid in the V (V1, V2, and V1 ∆thyA) strains [37,38].…”

Section: Rctc Construct Designmentioning

confidence: 99%

In-Depth Characterization of a Re-Engineered Cholera Toxin Manufacturing Process Using Growth-Decoupled Production in Escherichia coli

Danielewicz

Dai

Rosato

et al. 2022

Toxins

View full text Add to dashboard Cite

Non-toxic derivatives of the cholera toxin are extensively used in neuroscience, as neuronal tracers to reveal the location of cells in the central nervous system. They are, also, being developed as vaccine components and drug-delivery vehicles. Production of cholera-toxin derivatives is often non-reproducible; the quality and quantity require extensive fine-tuning to produce them in lab-scale settings. In our studies, we seek a resolution to this problem, by expanding the molecular toolbox of the Escherichia coli expression system with suitable production, purification, and offline analytics, to critically assess the quality of a probe or drug delivery, based on a non-toxic derivative of the cholera toxin. We present a re-engineered Cholera Toxin Complex (rCTC), wherein its toxic A1 domain was replaced with Maltose Binding Protein (MBP), as a model for an rCTC-based targeted-delivery vehicle. Here, we were able to improve the rCTC production by 11-fold (168 mg/L vs. 15 mg/L), in comparison to a host/vector combination that has been previously used (BL21(DE3) pTRBAB5-G1S). This 11-fold increase in the rCTC production capability was achieved by (1) substantial vector backbone modifications, (2) using Escherichia coli strains capable of growth-decoupling (V strains), (3) implementing a well-tuned fed-batch production protocol at a 1 L scale, and (4) testing the stability of the purified product. By an in-depth characterization of the production process, we revealed that secretion of rCTC across the E. coli Outer Membrane (OM) is processed by the Type II secretion-system general secretory pathway (gsp-operon) and that cholera toxin B-pentamerization is, likely, the rate-limiting step in complex formation. Upon successful manufacturing, we have validated the biological activity of rCTC, by measuring its binding affinity to its carbohydrate receptor GM1 oligosaccharide (Kd = 40 nM), or binding to Jurkat cells (93 pM) and delivering the cargo (MBP) in a retrograde fashion to the cell.

show abstract

Section: Rctc Construct Designmentioning

confidence: 99%

In-Depth Characterization of a Re-Engineered Cholera Toxin Manufacturing Process Using Growth-Decoupled Production in Escherichia coli

Danielewicz

Dai

Rosato

et al. 2022

Toxins

View full text Add to dashboard Cite

show abstract

“…Plasmid loss is a frequent problem in the large-scale cultivation of bacteria carrying episomal plasmids for gene overexpression [15][16][17]. Several strategies have been conducted to improve plasmid stability [18][19][20][21][22].…”

Section: Improving Plasmid Stability By Auxotrophic Complementation A...mentioning

confidence: 99%

Engineering Escherichia coli for l‐homoserine production

et al. 2022

View full text Add to dashboard Cite

l‐homoserine, a nonprotein amino acid, is used to synthesize many active substances in the industry. Here, to develop a robust l‐homoserine‐producing strain, Escherichia coli W3110 was used as a chassis to be engineered. Based on a previous construct with blocked competing routes for l‐homoserine synthesis, five genes were overexpressed by promoter replacement strategy to increase the l‐homoserine production, including enhancement of precursors for l‐homoserine synthesis (ppc, thrA, and asd), reinforcement of the NADPH supply (pntAB) and efflux transporters (rhtA) to improve the l‐homoserine production. However, the plasmid losing was to blame for the wildly fluctuating fermentation performance of engineered strains, ranging between 2.1 and 6.2 g/L. Then, a hok/sok toxin/antitoxin system was introduced into the free plasmid expression cassette to maintain the genetic stability of the episomal plasmid; consequently, the plasmid‐losing rate sharply decreased, resulting in the engineered strain SHL17, which exhibited excellent stability in l‐homoserine production, with 6.3 g/L in shake flasks and 44.4 g/L in a 5‐L fermenter without antibiotic addition. This work verified the effective use of the hok/sok toxin/antitoxin system combined with promoter engineering to improve the genetic stability of E. coli episomal plasmids without antibiotics.

show abstract

“…Several approaches have been conducted to enhance plasmid stability. [9,11,28] Herein, auxotrophic complementation and hok/sok systems were constructed to maintain the stability of pKK-metL plasmid. The auxotroph biosynthetic system, combined with the complementation of essential genes, could enable the stable maintenance of plasmid without the addition of antibiotics, because the essential genes should be expressed by plasmid instead of chromosome to ensure the cell growth.…”

Section: Constructing Auxotrophic Complementation and Hok/sok Systems To Improve The Plasmid Stabilitymentioning

confidence: 99%

“…[1] Additionally, the demand for antibiotics supplementation could increase the costs and result in environmental contamination. [11] Since maintaining the plasmid stability is an important concern for enzyme expression and bioproduction using microbes, constructing an e cient system to maintain plasmid stability is necessary for the competitiveness of the plasmid-containing cells.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Improving the Plasmid Stability by a Hok/Sok System for L-Homoserine Production in Escherichia Coli

Sun

Tao

Sui

et al. 2021

Preprint

View full text Add to dashboard Cite

Background: The production of bioactive compounds using microbial hosts is considered a safe, cost competitive and scalable approach. However, the efficient engineering of cell factories with well stability, such as for the production of L-aspartate family amino acids and derivatives, remains an outstanding challenge.Results: In the work, the toxin/antitoxin system and genome modification strategy were used to construct a stable Escherichia coli strain for L-homoserine production. The metabolic engineering strategies were focused on the enhancement of precursors for L-homoserine synthesis, reinforcement of the NADPH generation and efflux transporters using CRISPR-Cas9 system at the genome level. To improve the plasmid stability, two strategies were explored, including construction of the aspartate-auxotrophic and hok/sok systems. Constructing the auxotrophic complementation system to maintain plasmid stability was failed herein. The plasmid stability was improved by introducing the hok/sok system, resulting in 6.1 g/L (shake flask) and 44.4 g/L (5 L fermenter) L-homoserine production of the final engineered strain SHL19 without antibiotics addition. Moreover, the hok/sok system was also used to improve the plasmid stability for ectoine production, resulting in 36.7% and 46.5% higher titer of ectoine at shake flask and 5L fermenter without antibiotics addition, respectively. Conclusion: This work provides valuable strategies to improve plasmid stability for producing L-aspartate family amino acids and derivatives and eliminate environmental concerns associated with the application of antibiotics.

show abstract

Efficient strategies to enhance plasmid stability for fermentation of recombinant Escherichia coli harboring tyrosine phenol lyase

Cited by 5 publications

References 40 publications

In-Depth Characterization of a Re-Engineered Cholera Toxin Manufacturing Process Using Growth-Decoupled Production in Escherichia coli

In-Depth Characterization of a Re-Engineered Cholera Toxin Manufacturing Process Using Growth-Decoupled Production in Escherichia coli

Engineering Escherichia coli for l‐homoserine production

Improving the Plasmid Stability by a Hok/Sok System for L-Homoserine Production in Escherichia Coli

Contact Info

Product

Resources

About