Lipase and Protease Double-Deletion Mutant of Pseudomonas fluorescens Suitable for Extracellular Protein Production

Son, Myunghan; Moon, Yuseok; Oh, Mi Jin; Han, Seok Joo; Park, Ki Hyun; Kim, Jung-Gon; Ahn, Jung Hoon

doi:10.1128/aem.02476-12

Cited by 27 publications

(31 citation statements)

References 40 publications

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“…Plasmid construction and genetic modifications were performed in E. coli XL1-BLUE. Protein expression and secretion were observed in the P. fluorescens ⌬tliA ⌬prtA strain, which is a double-deletion derivative of P. fluorescens SIK-W1 (37). Microorganisms were cultured in lysogeny broth (LB) with 30 g/ml kanamycin.…”

Section: Bacterial Strains and Growth Mediamentioning

confidence: 99%

A lower isoelectric point increases signal sequence–mediated secretion of recombinant proteins through a bacterial ABC transporter

Byun

Park

Kim

et al. 2017

Journal of Biological Chemistry

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Efficient protein production for industrial and academic purposes often involves engineering microorganisms to produce and secrete target proteins into the culture. has a TliDEF ATP-binding cassette transporter, a type I secretion system, which recognizes C-terminal LARD3 signal sequence of thermostable lipase TliA. Many proteins are secreted by TliDEF when recombined with LARD3, but there are still others that cannot be secreted by TliDEF even when LARD3 is attached. However, the factors that determine whether or not a recombinant protein can be secreted through TliDEF are still unknown. Here, we recombined LARD3 with several proteins and examined their secretion through TliDEF. We found that the proteins secreted via LARD3 are highly negatively charged with highly-acidic isoelectric points (pI) lower than 5.5. Attaching oligo-aspartate to lower the pI of negatively-charged recombinant proteins improved their secretion, and attaching oligo-arginine to negatively-charged proteins blocked their secretion by LARD3. In addition, negatively supercharged green fluorescent protein (GFP) showed improved secretion, whereas positively supercharged GFP did not secrete. These results disclosed that proteins' acidic pI and net negative charge are major factors that determine their secretion through TliDEF. Homology modeling for TliDEF revealed that TliD dimer forms evolutionarily-conserved positively-charged clusters in its pore and substrate entrance site, which also partially explains the pI dependence of the TliDEF-dependent secretions. In conclusion, lowering the isoelectric point improved LARD3-mediated protein secretion, both widening the range of protein targets for efficient production via secretion and signifying an important aspect of ABC transporter-mediated secretions.

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Section: Bacterial Strains and Growth Mediamentioning

confidence: 99%

A lower isoelectric point increases signal sequence–mediated secretion of recombinant proteins through a bacterial ABC transporter

Byun

Park

Kim

et al. 2017

Journal of Biological Chemistry

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“…An nahG deletion mutation strain was constructed according to a previously described homologous recombination gene knockout method using the pK18mobsacB plasmid 52 . The plasmid used for nahG knockout was constructed as follows: 500-bp fragments upstream and downstream of nahG were amplified from strain LZ-4 genomic DNA by PCR.…”

Section: Methodsmentioning

confidence: 99%

The naphthalene catabolic protein NahG plays a key role in hexavalent chromium reduction in Pseudomonas brassicacearum LZ-4

Huang

Tao

Jiang

et al. 2017

Sci Rep

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Soil contamination by PAH and heavy metals is a growing problem. Here, we showed that a new isolate, Pseudomonas brassicacearum strain LZ-4, can simultaneously degrade 98% of 6 mM naphthalene and reduce 92.4% of 500 μM hexavalent chromium [Cr (VI)] within 68 h. A draft genome sequence of strain LZ-4 (6,219,082 bp) revealed all the genes in the naphthalene catabolic pathway and some known Cr (VI) reductases. Interestingly, genes encoding naphthalene pathway components were upregulated in the presence of Cr (VI), and Cr (VI) reduction was elevated in the presence of naphthalene. We cloned and expressed these naphthalene catabolic genes and tested for Cr (VI) reduction, and found that NahG reduced 79% of 100 μM Cr (VI) in 5 minutes. Additionally, an nahG deletion mutant lost 52% of its Cr (VI) reduction ability compared to that of the wild-type strain. As nahG encodes a salicylate hydroxylase with flavin adenine dinucleotide (FAD) as a cofactor for electron transfer, Cr (VI) could obtain electrons from NADH through NahG-associated FAD. To the best of our knowledge, this is the first report of a protein involved in a PAH-degradation pathway that can reduce heavy metals, which provides new insights into heavy metal-PAH contamination remediation.

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“…A previous report indicated a functional role for the secretion pump proteins in the protease activity of Pseudomonas fluorescens (Son et al, 2012). For this reason, we examined the effects of vexAB, vexCD, or acrAB1 deletion on the protease activity of V. vulnificus.…”

Section: Effects Of Vexab Vexcd or Acrab1 Deletion On Total Proteasmentioning

confidence: 99%

Functional analysis of Vibrio vulnificus RND efflux pumps homologous to Vibrio cholerae VexAB and VexCD, and to Escherichia coli AcrAB

et al. 2015

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Resistance-nodulation-division (RND) efflux pumps are associated with multidrug resistance in many gram-negative pathogens. The genome of Vibrio vulnificus encodes 11 putative RND pumps homologous to those of Vibrio cholerae and Escherichia coli. In this study, we analyzed three putative RND efflux pumps, showing homology to V. cholerae VexAB and VexCD and to E. coli AcrAB, for their functional roles in multidrug resistance of V. vulnificus. Deletion of the vexAB homolog resulted in increased susceptibility of V. vulnificus to bile acid, acriflavine, ethidium bromide, and erythromycin, whereas deletion of acrAB homologs rendered V. vulnificus more susceptible to acriflavine only. Deletion of vexCD had no effect on susceptibility of V. vulnificus to these chemicals. Upon exposure to these antibacterial chemicals, expression of tolCV1 and tolCV2, which are putative outer membrane factors of RND efflux pumps, was induced, whereas expression levels of vexAB, vexCD, and acrAB homologs were not significantly changed. Our results show that the V. vulnificus homologs of VexAB largely contributed to in vitro antimicrobial resistance with a broad substrate specificity that was partially redundant with the AcrAB pump homologs.

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Lipase and Protease Double-Deletion Mutant of Pseudomonas fluorescens Suitable for Extracellular Protein Production

Cited by 27 publications

References 40 publications

A lower isoelectric point increases signal sequence–mediated secretion of recombinant proteins through a bacterial ABC transporter

A lower isoelectric point increases signal sequence–mediated secretion of recombinant proteins through a bacterial ABC transporter

The naphthalene catabolic protein NahG plays a key role in hexavalent chromium reduction in Pseudomonas brassicacearum LZ-4

Functional analysis of Vibrio vulnificus RND efflux pumps homologous to Vibrio cholerae VexAB and VexCD, and to Escherichia coli AcrAB

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