Construction of a highly active secretory expression system in Bacillus subtilis of a recombinant amidase by promoter and signal peptide engineering

2020 ◽  
Vol 143 ◽  
pp. 833-841 ◽  
Author(s):  
Xue-Mei Kang ◽  
Xue Cai ◽  
Zi-Han Huang ◽  
Zhi-Qiang Liu ◽  
Yu-Guo Zheng
Keyword(s):  
Bacillus Subtilis ◽  
Signal Peptide ◽  
Expression System ◽  
Secretory Expression ◽  
Highly Active ◽  
Peptide Engineering

scholarly journals High efficient extracellular production of recombinant Streptomyces PMF phospholipase D in Escherichia coli

2020 ◽  
Author(s):  
Jing Wang ◽  
Sheng Xu ◽  
Yang Pang ◽  
Xin Wang ◽  
Kequan Chen ◽  
...  
Keyword(s):  
Phospholipase D ◽  
Signal Peptide ◽  
Large Scale ◽  
Expression System ◽  
Cost Effective ◽  
Scale Production ◽  
Secretory Expression ◽  
E Coli ◽  
Cost Effective Method ◽  
High Efficient

Abstract Background Currently, Streptomyces is widely used in the preparation of phospholipase D (PLD) with high transphosphatidylation activity. However, the yield of PLD from Streptomyces was low and the culture period was long. Therefore, an efficient and cost-effective method is needed urgently.Results Firstly, PLDs from Streptomyces PMF and Streptomyces racemochromogenes were separately over-expressed in E. coli to compare their transphosphatidylation activity based on the synthesis of phosphatidylserine (PS), and PLDPMF was determined to have higher activity. To further improve PLDPMF synthesis, a secretory expression system suitable for PLDPMF was constructed and optimized with different signal peptides. The highest secretory efficiency was observed when the PLDPMF gene was expressed together with its native signal peptide (Nat) and the signal peptide PelB from E. coli. For the application of recombinant PLD to PS synthesis, the PLD properties were characterized and 30.2 g/L of PS was produced after 24 h of bioconversion when 50 g/L phosphatidylcholine (PC) was added.Conclusions We succeeded in over-expressing PLD from Streptomyces PMF in E. coli with high transphosphatidylation activity and enhanced the yield by secretory expression. The secreted PLD was successfully used in the production of PS. Our work makes the large-scale production of PLD and PS feasible.

scholarly journals Novel Expression Vector for Secretion of Cecropin AD in Bacillus subtilis with Enhanced Antimicrobial Activity

2009 ◽  
Vol 53 (9) ◽  
pp. 3683-3689 ◽  
Author(s):  
Xiang Chen ◽  
Faming Zhu ◽  
Yunhe Cao ◽  
Shiyan Qiao
Keyword(s):  
Staphylococcus Aureus ◽  
Antimicrobial Activity ◽  
Bacillus Subtilis ◽  
Signal Peptide ◽  
Expression System ◽  
Proteinase K ◽  
Farm Animals ◽  
Commercial Application ◽  
Protease Gene ◽  
Recombinant Product

ABSTRACT Cecropin AD, a chimeric antimicrobial peptide obtained from cecropins, is effective at killing specific microorganisms. However, a highly efficient expression system is still needed to allow for commercial application of cecropin AD. For the exogenous expression of cecropin AD, we fused the cecropin AD gene with a small ubiquitin-like modifier (SUMO) gene and a signal peptide of SacB, while a Bacillus subtilis expression system was constructed based on Bacillus subtilis cells genetically modified by the introduction of an operon including an isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible Spac promoter, a signal peptide of amyQ, and the SUMO protease gene. The recombinant cecropin AD was expressed, and 30.6 mg of pure recombinant protein was purified from 1 liter of culture supernatant. The purified cecropin AD displayed antimicrobial activity against some pathogens, such as Staphylococcus aureus and Escherichia coli, and was especially effective toward Staphylococcus aureus, with MICs of <0.05 μM (0.2 μg/ml). Stability analysis results showed that the activity of cecropin AD was not influenced by temperatures as high as 55°C for 20 min; however, temperatures above 85°C (for 20 min) decreased the antimicrobial activity of cecropin AD. Varying the pH from 4.0 to 9.0 did not appear to affect the activity of cecropin AD, but some loss of potency was observed at pH values lower than pH 4.0. Under the challenge of several proteases (proteinase K, trypsin, and pepsin), cecropin AD maintained functional activity. The results indicated that the recombinant product expressed by the designed Bacillus subtilis expression system was a potent antimicrobial agent and could be applied to control infectious diseases of farm animals or even humans.

scholarly journals Highly Efficient Extracellular Production of Recombinant Streptomyces PMF Phospholipase D in Escherichia coli

Catalysts ◽  
2020 ◽  
Vol 10 (9) ◽  
pp. 1057
Author(s):  
Jing Wang ◽  
Sheng Xu ◽  
Yang Pang ◽  
Xin Wang ◽  
Kequan Chen ◽  
...  
Keyword(s):  
Phospholipase D ◽  
Signal Peptide ◽  
Expression System ◽  
Signal Peptides ◽  
Extracellular Production ◽  
Secretory Expression ◽  
E Coli ◽  
Fusion Signal ◽  
Optimized Conditions ◽  
Native Signal Peptide

To achieve efficient bio-production of phospholipase D (PLD), PLDs from different organisms were expressed in E.coli. An efficient secretory expression system was thereby developed for PLD. First, PLDs from Streptomyces PMF and Streptomyces racemochromogenes were separately over-expressed in E.coli to compare their transphosphatidylation activity based on the synthesis of phosphatidylserine (PS), and PLDPMF was determined to have higher activity. To further improve PLDPMF synthesis, a secretory expression system suitable for PLDPMF was constructed and optimized with different signal peptides. The highest secretory efficiency was observed when the PLD * (PLDPMF with the native signal peptide Nat removed) was expressed fused with the fusion signal peptide PelB-Nat in E. coli. The fermentation conditions were also investigated to increase the production of recombinant PLD and 10.5 U/mL PLD was ultimately obtained under the optimized conditions. For the application of recombinant PLD to PS synthesis, the PLD properties were characterized and 30.2 g/L of PS was produced after 24 h of bioconversion when 50 g/L phosphatidylcholine (PC) was added.

scholarly journals OmpA signal peptide leads to heterogenous secretion of B. subtilis chitosanase enzyme from E. coli expression system

SpringerPlus ◽  
2016 ◽  
Vol 5 (1) ◽  
Author(s):  
Phornsiri Pechsrichuang ◽  
Chomphunuch Songsiriritthigul ◽  
Dietmar Haltrich ◽  
Sittiruk Roytrakul ◽  
Peenida Namvijtr ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Expression System ◽  
E Coli

scholarly journals Antimicrobial genes from Allium sativum and Pinellia ternata revealed by a Bacillus subtilis expression system

2018 ◽  
Vol 8 (1) ◽  
Author(s):  
Xi Kong ◽  
Mei Yang ◽  
Hafiz Muhammad Khalid Abbas ◽  
Jia Wu ◽  
Mengge Li ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Allium Sativum ◽  
Expression System ◽  
Pinellia Ternata ◽  
Bacillus Subtilis Expression
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