Enhanced extracellular production of recombinant proteins in Escherichia coli by co-expression with Bacillus cereus phospholipase C

Su, Lingqia; Jiang, Qi; Yu, Lingang; Wu, Jing

doi:10.1186/s12934-017-0639-3

Cited by 34 publications

(28 citation statements)

References 36 publications

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“…This activity allowed the PLC expressed in E. coli to hydrolyze the phospholipid present in the cell membrane to some extent, increasing membrane permeability and causing the extracellular release of the PLC. These results are similar to those observed previously with Thermobifida fusca cutinase and B. cereus PLC …”

Section: Resultssupporting

confidence: 92%

“…Fusion with super‐folder green fluorescent protein also has been shown to mediate the secretion of recombinant cytoplasmic protein, but the secretion efficiency for different proteins varied significantly . Becaus the extracellular expression of SP Lc presented here involved a nonspecific mechanism of extracellular release, this technique also could result in the extracellular production of other cytoplasmic products, as mentioned in our previous study . The related mechanism and principle will be further explored in the future.…”

Section: Resultsmentioning

confidence: 78%

“…For fermentation in a 3 L fermenter, a seed culture was prepared as described in the previous paragraph, and then diluted into a semisynthetic medium at pH 7.0 and an initial temperature of 37 °C for fed‐batch cultivation. After the initial glycerol was completely consumed, which was signaled by a sudden increase of dissolved oxygen and pH, a feeding solution was continuously fed into the culture . When the dry cell weight (DCW, see next section) of the culture reached a specified level (15 or 25 g L −1 ), lactose was fed at a constant rate (0.1, 0.2 or 0.4 g L −1 h −1 ) to induce protein expression.…”

Section: Methodsmentioning

confidence: 99%

“…No obvious cell lysis was detected, probably because damage to the cell membrane was limited. This strategy also was scaled up in a 3 L fermenter, and the easily controlled fermentation process exhibited great potential for large‐scale extracellular protein production …”

Section: Introductionmentioning

confidence: 99%

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Highly efficient extracellular expression of naturally cytoplasmic Leuconostoc mesenteroides sucrose phosphorylase

Tian

et al. 2018

J of Chemical Tech & Biotech

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BACKGROUND We previously showed that extracellular production of naturally cytoplasmic proteins could be realized through co‐expression with phospholipase C (PLC), which enhances cell membrane permeability through limited hydrolysis of membrane phospholipids. Leuconostoc mesenteroides sucrose phosphorylase (SPLc), a naturally cytoplasmic enzyme, exhibits excellent properties for synthesis of α‐arbutin, but no systemic investigation of its preparation has thus far been reported. RESULTS Three PLCs were selected and expressed, without signal peptide, in Escherichia coli. More than 90% of the PLCs from Listeria monocytogenes and Bacillus cereus were present in the culture medium. When SPLc was co‐expressed with these PLCs, and the location of the PLC and SPLc genes in the co‐expression vector was optimized, the highest production was obtained when SPLc gene was inserted upstream of the B. cereus PLC gene, as in BL21(DE3)/pETDuet‐sp‐bcplc. SPLc production was then scaled up to a 3 L fermenter and fermentation conditions were optimized. When the initial medium contained 0.6 mmol L−1 ZnCl2 and protein expression was induced at a dry cell weight of 15 g L−1 by the addition of lactose at 0.2 g L−1 h−1 at 30 °C, the extracellular SPLc activity reached its highest level of 1445.1 U mL−1 This constituted 81.6% of total activity; the remainder of the enzyme (18.4%) remained inside the cells. CONCLUSION This is the first report describing extracellular expression of sucrose phosphorylase, and the highest yield ever reported was obtained. This provides the basis for industrial‐scale production and application of sucrose phosphorylase. It also provides a potential method for improving the production of other proteins and fine chemicals. © 2018 Society of Chemical Industry

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Section: Resultssupporting

confidence: 92%

Section: Resultsmentioning

confidence: 78%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Highly efficient extracellular expression of naturally cytoplasmic Leuconostoc mesenteroides sucrose phosphorylase

Tian

et al. 2018

J of Chemical Tech & Biotech

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“…It was assumed that proteins are released due to its phospholipid hydrolase activity [211][212][213]. The secretion efficiency of this system depends on the molecular mass of the protein, was up to 4-fold increased for native secretory enzymes and up to nearly 90% of cytosolic enzymes could be secreted without cell lysis [214]. Even cytosolic D-xylose isomerase and trehalose synthase were found to be extracellular with about 65 and 54%, respectively.…”

Section: Induced Permeabilitymentioning

confidence: 99%

Secretion of recombinant proteins from E. coli

Kleiner-Grote

Risse

Friehs

2018

Engineering in Life Sciences

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The microorganism Escherichia coli is commonly used for recombinant protein production. Despite several advantageous characteristics like fast growth and high protein yields, its inability to easily secrete recombinant proteins into the extracellular medium remains a drawback for industrial production processes. To overcome this limitation, a multitude of approaches to enhance the extracellular yield and the secretion efficiency of recombinant proteins have been developed in recent years. Here, a comprehensive overview of secretion mechanisms for recombinant proteins from E. coli is given and divided into three main sections. First, the structure of the E. coli cell envelope and the known natural secretion systems are described. Second, the use and optimization of different one‐ or two‐step secretion systems for recombinant protein production, as well as further permeabilization methods are discussed. Finally, the often‐overlooked role of cell lysis in secretion studies and its analysis are addressed. So far, effective approaches for increasing the extracellular protein concentration to more than 10 g/L and almost 100% secretion efficiency exist, however, the large range of optimization methods and their combinations suggests that the potential for secretory protein production from E. coli has not yet been fully realized.

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Holin‐assisted bacterial recombinant protein export

Guo

Cui

Zhang

et al. 2022

Biotech & Bioengineering

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A simple generic method for enhancing extracellular protein yields in engineered bacteria is still lacking. Here, we demonstrated that phage‐encoded holin can be used to export proteins to the extracellular medium in both Gram‐negative Escherichia coli and ‐positive Lactococcus lactis. When a putative holin gene LLNZ_RS10380 annotated in the genome of L. lactis NZ9000 (hol380) was recombinantly expressed in E. coli BL21(DE3), the Hol380 oligomerized up to hexamer in the cytoplasmic membrane, yielding membrane pore to allow the passage of cytosolic β‐galatosidase (116 kDa), whose extracellular production reached 54.59 U/μl, accounting for 76.37% of the total activity. However, the overexpressed Hol380 could not release cytosolic proteins across the membrane in L. lactis NZ9000, but increased the secretory production of staphylococcal nuclease to 2.55‐fold and fimbrial adhesin FaeG to 2.40‐fold compared with those guided by signal peptide Usp45 alone. By using a combination of proteomics and transcriptional level analysis, we found that overexpression of the Hol380 raised the accumulation of Ffh and YidC involved in the signal recognition particle pathway in L. lactis, suggesting an alternative road participating in protein secretion. This study proposed a new approach by expressing holin in bacterial cell factories to export target proteins of economic or medical interest.

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Enhanced extracellular production of recombinant proteins in Escherichia coli by co-expression with Bacillus cereus phospholipase C

Cited by 34 publications

References 36 publications

Highly efficient extracellular expression of naturally cytoplasmic Leuconostoc mesenteroides sucrose phosphorylase

Highly efficient extracellular expression of naturally cytoplasmic Leuconostoc mesenteroides sucrose phosphorylase

Secretion of recombinant proteins from E. coli

Holin‐assisted bacterial recombinant protein export

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