Characterization of the Lactobacillus plantarum plasmid pCD033 and generation of the plasmid free strain L. plantarum 3NSH

Heiss, Silvia; Grabherr, Reingard; Heinl, Stefan

doi:10.1016/j.plasmid.2015.05.004

Cited by 10 publications

(8 citation statements)

References 44 publications

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“…House-keeping genes involved in the primary metabolism of EFEL2700 were absent in the plasmid, indicating that its removal from the host P-cells would not change the metabolic phenotypes. Some previous studies also reported no phenotypic changes after curing of cryptic plasmids from host cells (Feng et al 2013;Heiss et al 2015).…”

Section: Microbiological Analysismentioning

confidence: 81%

“…Tranformation efficiency (transformants µg DNA -1 ) Therefore, for various applications, plasmid curing has been attempted in bacterial strains, but inconsistent results were obtained depending on the species and cases (Feng et al 2013;Heiss et al 2015). Feng et al (2013) reported that the plasmid-cured strains of Bacillus amyloliquefaciens showed increased levels (6%) of poly-c-glutamic acid production.…”

Section: (A) (B)mentioning

confidence: 99%

“…Feng et al (2013) reported that the plasmid-cured strains of Bacillus amyloliquefaciens showed increased levels (6%) of poly-c-glutamic acid production. Meanwhile, Heiss et al (2015) reported that a plasmid-cured strain of Lactobacillus plantarum showed no significant differences in growth rates or recombinant protein expression. Nevertheless, plasmid-free strains are regarded as preferable expression hosts for microbial cell factories, as naturally existing cryptic plasmids in the host strain may interfere with replication of the engineered plasmid (Heiss et al 2016).…”

Section: (A) (B)mentioning

confidence: 99%

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Plasmid curing resulted in improved heterologous gene expression inLeuconostoc citreumEFEL2700

Jang

Kim

Seo

et al. 2019

Lett Appl Microbiol

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Leuconostoc citreum EFEL2700 isolated from kimchi was used as a host strain for genetic and metabolic engineering in our previous studies, but the cells of EFEL2700 contained a cryptic plasmid (P‐cells). Thus, we created plasmid‐free cells (F‐cells) using the CRISPR/Cas9 system. In this study, we compared the microbial characteristics of P‐ and F‐cells in terms of growth rate, biochemical properties, transformation efficiency, plasmid copy number and protein expression level. When the growth rate was measured in MRS medium at 30°C, no significant difference (P > 0·01) was observed. Biochemical properties, tested using an API 50CHL kit, showed no differences. Transformation efficiency of F‐cells, measured using pCB4270, was higher (1·3 × 104 CFU per μg DNA) than that of P‐cells (5·0 × 103 CFU per μg DNA). Copy number after transformation of pCBBgl was 4‐fold higher for F‐cells than for P‐cells. When β‐glucosidase activity was assayed in the above experiment, F‐cells showed 3·4‐fold higher values than P‐cells. In conclusion, this study demonstrates that plasmid curing in L. citreum EFEL2700 improves its characteristics as a gene expression host. Significance and Impact of the Study Leuconostoc citreum EFEL2700 (P‐cell) isolated from kimchi is a useful food‐grade host for expressing heterologous genes. The presence of a cryptic plasmid is thought to limit efficient gene expression. In this study, we compared the microbial and genetic changes after plasmid curing in this strain. The plasmid‐free strain showed improved levels of transformation efficiency, copy number and heterologous gene expression without alterations in phenotypes such as the growth rates and biochemical properties. The resulting strain of L. citreum EFEL2701 (F‐cell) can be used as an efficient host for genetic engineering.

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Section: Microbiological Analysismentioning

confidence: 81%

Section: (A) (B)mentioning

confidence: 99%

Section: (A) (B)mentioning

confidence: 99%

See 1 more Smart Citation

Plasmid curing resulted in improved heterologous gene expression inLeuconostoc citreumEFEL2700

Jang

Kim

Seo

et al. 2019

Lett Appl Microbiol

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“…However, it was reported that some plasmids without replication protein replicate via an RNA-based replication mechanism, such as pCD033 from L. plantarum 3NSH, and plasmid pColE1 (29)(30)(31). The principle of ddPCR involves subdividing a single PCR reaction mixture into many small partitions and each undergoing the PCR reaction separately (32).…”

Section: Structure Of the Predicted Origin Of Replication In Plasmimentioning

confidence: 99%

Characterization of Four Novel Plasmids from Lactobacillus plantarum BM4

Jie

Zhang

et al. 2017

Jundishapur J Microbiol

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“…Additionally, we tested endogenous lac -operon regulatory sequences from L. plantarum 3NSH. This strain is derived from L. plantarum CD033, which was cured of its native plasmid [ 17 ]. Plasmid free strains are preferable expression hosts, since native plasmids sometimes interfere with expression vector replication.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of novel inducible promoter/repressor systems for recombinant protein expression in Lactobacillus plantarum

et al. 2016

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Background: Engineering lactic acid bacteria (LAB) is of growing importance for food and feed industry as well as for in vivo vaccination or the production of recombinant proteins in food grade organisms. Often, expression of a transgene is only desired at a certain time point or period, e.g. to minimize the metabolic burden for the host cell or to control the expression time span. For this purpose, inducible expression systems are preferred, though cost and availability of the inducing agent must be feasible. We selected the plasmid free strain Lactobacillus plantarum 3NSH for testing and characterization of novel inducible promoters/repressor systems. Their feasibility in recombinant protein production was evaluated. Expression of the reporter protein mCherry was monitored with the BioLector ® micro-fermentation system. Results:Reporter gene mCherry expression was compared under the control of different promoter/repressor systems: P lacA (an endogenous promoter/repressor system derived from L. plantarum 3NSH), P xylA (a promoter/repressor system derived from Bacillus megaterium DSMZ 319) and P lacSynth (synthetic promoter and codon-optimized repressor gene based on the Escherichia coli lac operon). We observed that P lacA was inducible solely by lactose, but not by non-metabolizable allolactose analoga. P xylA was inducible by xylose, yet showed basal expression under non-induced conditions. Growth on galactose (as compared to exponential growth phase on glucose) reduced basal mCherry expression at non-induced conditions. P lacSynth was inducible with TMG (methyl β-D-thiogalactopyranoside) and IPTG (isopropyl β-D-1-thiogalactopyranoside), but also showed basal expression without inducer. The promoter P lacSynth was used for establishment of a dual plasmid expression system, based on T7 RNA polymerase driven expression in L. plantarum. Comparative Western blot supported BioLector ® micro-fermentation measurements. Conclusively, overall expression levels were moderate (compared to a constitutive promoter). Conclusions:We evaluated different inducible promoters, as well as an orthologous expression system, for controlled gene expression in L. plantarum. Furthermore, here we provide proof of concept for a T7 RNA polymerase based expression system for L. plantarum. Thereby we expanded the molecular toolbox for an industrial relevant and generally regarded as safe (GRAS) strain.

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Characterization of the Lactobacillus plantarum plasmid pCD033 and generation of the plasmid free strain L. plantarum 3NSH

Cited by 10 publications

References 44 publications

Plasmid curing resulted in improved heterologous gene expression inLeuconostoc citreumEFEL2700

Plasmid curing resulted in improved heterologous gene expression inLeuconostoc citreumEFEL2700

Characterization of Four Novel Plasmids from Lactobacillus plantarum BM4

Evaluation of novel inducible promoter/repressor systems for recombinant protein expression in Lactobacillus plantarum

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