Efficient production of extracellular pullulanase in Bacillus subtilis ATCC6051 using the host strain construction and promoter optimization expression system

Abstract The efficient production of antimicrobial peptides (AMPs) for clinical applications has attracted the attention of the scientific community. To develop a novel microbial cell factory for the efficient biosynthesis of a cecropin A-melittin mutant (CAM-W), a recombinant Bacillus subtilis WB700 expression system was genetically modified with a novel vector, including a fusion gene encoding CAM-W, the autoprotease EDDIE and the signal peptide SacB under the control of the maltose-inducible promoter P glv . A total of 159 mg of CAM-W was obtained from 1 L of fermentation supernatant. The purified CAM-W showed a consistent size with the expected molecular weight of 3.2 kDa. Our findings suggest that this novel expression system can be used as a powerful tool for the efficient production of CAM-W.

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Construction of a Super-Folder Fluorescent Protein-Guided Secretory Expression System for the Production of Phospholipase D in Bacillus subtilis

Journal of Agricultural and Food Chemistry ◽

10.1021/acs.jafc.1c02089 ◽

2021 ◽

Author(s):

Haiyang Zhang ◽

Xuehan Li ◽

Qi Liu ◽

Jianan Sun ◽

Francesco Secundo ◽

...

Keyword(s):

Bacillus Subtilis ◽

Phospholipase D ◽

Fluorescent Protein ◽

Expression System ◽

Secretory Expression

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Bombyx mori Pupae Efficiently Produce Recombinant AAV2/HBoV1 Vectors with a Bombyx mori Nuclear Polyhedrosis Virus Expression System

Viruses ◽

10.3390/v13040704 ◽

2021 ◽

Vol 13 (4) ◽

pp. 704

Author(s):

Qian Yu ◽

Pengfei Chang ◽

Xiaoxuan Liu ◽

Peng Lü ◽

Qi Tang ◽

...

Keyword(s):

Gene Therapy ◽

Bombyx Mori ◽

Nuclear Polyhedrosis Virus ◽

Expression System ◽

Efficient Production ◽

Human Bocavirus ◽

Adeno Associated Virus ◽

Silkworm Pupae ◽

Polyhedrosis Virus ◽

Nuclear Polyhedrosis

Recombinant adeno-associated virus (AAV) vectors have broad application prospects in the field of gene therapy. The establishment of low-cost and large-scale manufacturing is now the general agenda for industry. The baculovirus-insect cell/larva expression system has great potential for these applications due to its scalability and predictable biosafety. To establish a more efficient production system, Bombyx mori pupae were used as a new platform and infected with recombinant Bombyx mori nuclear polyhedrosis virus (BmNPV). The production of a chimeric recombinant adeno-associated virus (rAAV) serotype 2/human bocavirus type-1 (HBoV1) vector was used to evaluate the efficiency of this new baculovirus expression vector (BEV)–insect expression system. For this purpose, we constructed two recombinant BmNPVs, which were named rBmNPV/AAV2Rep-HBoV1Cap and rBmNPV/AAV2ITR-eGFP. The yields of rAAV2/HBoV1 derived from the rBmNPV/AAV2Rep-HBoV1Cap and rBmNPV/AAV2ITR-eGFP co-infected BmN cells exceeded 2 × 104 vector genomes (VG) per cell. The rBmNPV/AAV2Rep-HBoV1Cap and rBmNPV/AAV2ITR-eGFP can express stably for at least five passages. Significantly, rAAV2/HBoV1 could be efficiently generated from BmNPV-infected silkworm larvae and pupae at average yields of 2.52 × 1012 VG/larva and 4.6 × 1012 VG/pupa, respectively. However, the vectors produced from the larvae and pupae had a high percentage of empty particles, which suggests that further optimization is required for this platform in the future. Our work shows that silkworm pupae, as an efficient bioreactor, have great potential for application in the production of gene therapy vectors.

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Efficient production of recombinant PP2A at a low temperature using a baculovirus expression system

Biotechnology Reports ◽

10.1016/j.btre.2016.07.004 ◽

2016 ◽

Vol 11 ◽

pp. 86-89 ◽

Cited By ~ 5

Author(s):

Tsuyoshi Ikehara ◽

Shihoko Nakashima ◽

Junichi Nakashima ◽

Tsubasa Kinoshita ◽

Takeshi Yasumoto

Keyword(s):

Low Temperature ◽

Expression System ◽

Baculovirus Expression System ◽

Efficient Production ◽

Baculovirus Expression

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Highly efficient production of Clostridium cellulolyticum H10 d-psicose 3-epimerase in Bacillus subtilis and use of these cells to produce d-psicose

Microbial Cell Factories ◽

10.1186/s12934-018-1037-1 ◽

2018 ◽

Vol 17 (1) ◽

Cited By ~ 4

Author(s):

Lingqia Su ◽

Fan Sun ◽

Zhanzhi Liu ◽

Kang Zhang ◽

Jing Wu

Keyword(s):

Bacillus Subtilis ◽

Efficient Production ◽

Clostridium Cellulolyticum ◽

Highly Efficient

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Construction of a Vitreoscilla Hemoglobin Promoter-Based Tunable Expression System for Corynebacterium glutamicum

Catalysts ◽

10.3390/catal8110561 ◽

2018 ◽

Vol 8 (11) ◽

pp. 561 ◽

Cited By ~ 4

Author(s):

Kei-Anne Baritugo ◽

Hee Taek Kim ◽

Mi Na Rhie ◽

Seo Young Jo ◽

Tae Uk Khang ◽

...

Keyword(s):

Corynebacterium Glutamicum ◽

Fluorescent Protein ◽

Expression System ◽

Vitreoscilla Hemoglobin ◽

Aminobutyric Acid ◽

Vector System ◽

Efficient Production ◽

Gamma Aminobutyric Acid ◽

Recombinant Strains ◽

Gaba Production

Corynebacterium glutamicum is an industrial strain used for the production of valuable chemicals such as L-lysine and L-glutamate. Although C. glutamicum has various industrial applications, a limited number of tunable systems are available to engineer it for efficient production of platform chemicals. Therefore, in this study, we developed a novel tunable promoter system based on repeats of the Vitreoscilla hemoglobin promoter (Pvgb). Tunable expression of green fluorescent protein (GFP) was investigated under one, four, and eight repeats of Pvgb (Pvgb, Pvgb4, and Pvgb8). The intensity of fluorescence in recombinant C. glutamicum strains increased as the number of Pvgb increased from single to eight (Pvgb8) repeats. Furthermore, we demonstrated the application of the new Pvgb promoter-based vector system as a platform for metabolic engineering of C. glutamicum by investigating 5-aminovaleric acid (5-AVA) and gamma-aminobutyric acid (GABA) production in several C. glutamicum strains. The profile of 5-AVA and GABA production by the recombinant strains were evaluated to investigate the tunable expression of key enzymes such as DavBA and GadBmut. We observed that 5-AVA and GABA production by the recombinant strains increased as the number of Pvgb used for the expression of key proteins increased. The recombinant C. glutamicum strain expressing DavBA could produce higher amounts of 5-AVA under the control of Pvgb8 (3.69 ± 0.07 g/L) than the one under the control of Pvgb (3.43 ± 0.10 g/L). The average gamma-aminobutyric acid production also increased in all the tested strains as the number of Pvgb used for GadBmut expression increased from single (4.81–5.31 g/L) to eight repeats (4.94–5.58 g/L).

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A method for the generation of pseudotyped virus particles bearing SARS coronavirus spike protein in high yields

10.1101/2021.07.30.454063 ◽

2021 ◽

Author(s):

Yoichiro Fujioka ◽

Sayaka Kashiwagi ◽

Aiko Yoshida ◽

Aya O. Satoh ◽

Mari Fujioka ◽

...

Keyword(s):

Global Economy ◽

Expression System ◽

Antiviral Agents ◽

Production Method ◽

Spike Protein ◽

High Yield ◽

Pseudotyped Virus ◽

Efficient Production ◽

Live Virus ◽

High Yields

The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has threatened human health and the global economy. Development of additional vaccines and therapeutics is urgently required, but such development with live virus must be conducted with biosafety level 3 confinement. Pseudotyped viruses have been widely adopted for studies of virus entry and pharmaceutical development to overcome this restriction. Here we describe a modified protocol to generate vesicular stomatitis virus (VSV) pseudotyped with SARS-CoV or SARS-CoV-2 Spike protein in high yield. We found that pseudovirions produced with the conventional transient expression system lacked coronavirus Spike protein at their surface as a result of inhibition of parental VSV infection by overexpression of this protein. Establishment of stable cell lines with an optimal expression level of coronavirus Spike protein allowed the efficient production of progeny pseudoviruses decorated with Spike protein. This improved VSV pseudovirus production method should facilitate studies of coronavirus entry and development of antiviral agents.

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Optimised production of disulfide-bonded fungal effectors in E. coli using CyDisCo and FunCyDisCo co-expression approaches

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-08-21-0218-ta ◽

2021 ◽

Author(s):

Daniel Yu ◽

Megan A Outram ◽

Emma Creen ◽

Ashley Smith ◽

Yi-Chang Sung ◽

...

Keyword(s):

Disulfide Bond ◽

Disulfide Bonds ◽

Fungal Pathogens ◽

Sequence Similarity ◽

Expression System ◽

Pathogenic Fungi ◽

Efficient Production ◽

E Coli ◽

Functional Studies ◽

Folded Structure

Effectors are a key part of the arsenal of plant pathogenic fungi and promote pathogen virulence and disease. Effectors typically lack sequence similarity to proteins with known functional domains and motifs, limiting our ability to predict their functions and understand how they are recognised by plant hosts. As a result, cross-disciplinary approaches involving structural biology and protein biochemistry are often required to decipher and better characterise effector function. These approaches are reliant on high yields of relatively pure protein, which often requires protein production using a heterologous expression system. For some effectors, establishing an efficient production system can be difficult, particularly those that require multiple disulfide bonds to achieve their naturally folded structure. Here, we describe the use of a co-expression system within the heterologous host E. coli termed CyDisCo (cytoplasmic disulfide bond formation in E. coli) to produce disulfide bonded fungal effectors. We demonstrate that CyDisCo and a naturalised co-expression approach termed FunCyDisCo (Fungi-CyDisCo) can significantly improve the production yields of numerous disulfide bonded effectors from diverse fungal pathogens. The ability to produce large quantities of functional recombinant protein has facilitated functional studies and crystallisation of several of these reported fungal effectors. We suggest this approach could be broadly useful in the investigation of the function and recognition of a broad range of disulfide-bond containing effectors.

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