Construction and Use of a <i>Bacillus subtilis</i> Mutant Deficient in Multiple Protease Genes for the Expression of Eukaryotic Genes<sup>a</sup>

He, Xiaosong; Shyu, Yuan‐Tay; Nathoo, S.; Wong, Sui‐Lam; Doi, Roy H.

doi:10.1111/j.1749-6632.1991.tb18565.x

Cited by 20 publications

(15 citation statements)

References 23 publications

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“…While it is beyond the scope of this study to identify the molecular basis of these discrepancies, they could stem from the way each strain was created. Notably, unlike the markerless and precise deletions made in the BRB strains, the protease genes in WB800 were disrupted by partial deletions removing the promoter, signal peptide, and part of the pro-region (Wang, Bruckner et al 1989, He, Shyu et al 1991) and by disrupting protease genes with the antibiotic resistance genes blasticidin, bleomycin and hygromycin (Wu, Yeung et al 2002). The additional genetic burden of expressing these antibiotic resistance genes could be responsible for the slightly lower growth and protein production of WB800S.…”

Section: Discussionmentioning

confidence: 99%

Stabilizing displayed proteins on vegetative Bacillus subtilis cells

Huang

Gosschalk

Kim

et al. 2018

Appl Microbiol Biotechnol

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Microbes engineered to display heterologous proteins could be useful biotechnological tools for protein engineering, lignocellulose degradation, biocatalysis, bioremediation and biosensing. Bacillus subtilis is a promising host to display proteins, as this model Gram-positive bacterium is genetically tractable and already used industrially to produce enzymes. To gain insight into the factors that affect displayed protein stability and copy-number, we systematically compared the ability of different protease-deficient B. subtilis strains (WB800, BRB07, BRB08 and BRB14) to display a Cel8A-LysM reporter protein in which the Clostridium thermocellum Cel8A endoglucanase is fused to LysM cell wall binding modules. Whole-cell cellulase measurements and fractionation experiments demonstrate that genetically eliminating extracytoplasmic bacterial proteases improves Cel8A-LysM displays levels. However, upon entering stationary phase, for all protease-deficient strains, the amount of displayed reporter dramatically decreases, presumably as a result of cellular autolysis. This problem can be partially overcome by adding chemical protease inhibitors, which significantly increase protein display levels. We conclude that strain BRB08 is well-suited for stably displaying our reporter protein, as genetic removal of its extracellular and cell wall-associated proteases leads to the highest levels of surface accumulated Cel8A-LysM without causing secretion stress or impairing growth. A two-step procedure is presented that enables the construction of enzyme coated vegetative B. subtilis cells that retain stable cell-associated enzyme activity for nearly 3 days. The results of this work could aid the development of whole cell display systems that have useful biotechnological applications.

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Section: Discussionmentioning

confidence: 99%

Stabilizing displayed proteins on vegetative Bacillus subtilis cells

Huang

Gosschalk

Kim

et al. 2018

Appl Microbiol Biotechnol

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“…B. subtilis DB430, 25 deficient in three extracellular proteases, was used as the engineering host ( kV/cm, capacitance of 25 lF, and resistance of 200 X using a Gene Pulser V R II electroporation apparatus (Bio-Rad, Hercules, CA). Competent transformation of B. subtilis was performed as previously described.…”

Section: Methodsmentioning

confidence: 99%

Chromosomal integration of a synthetic expression control sequence achieves poly‐γ‐glutamate production in a Bacillus subtilis strain

Yeh

Wang

et al. 2010

Biotechnology Progress

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Poly-gamma-glutamate (gamma-PGA) has applications in food, medical, cosmetic, animal feed, and wastewater industries. Bacillus subtilis DB430, which possesses the gamma-PGA synthesis ywsC-ywtAB genes in its chromosome, cannot produce gamma-PGA. An efficient synthetic expression control sequence (SECS) was introduced into the upstream region of the ywtABC genes, and this resulted in gamma-PGA-producing B. subtilis mutant strains. Mutant B. subtilis PGA6-2 stably produces high levels of gamma-PGA in medium A without supplementation of extra glutamic acid or ammonium chloride. The mutant B. subtilis PGA 6-2 is not only a gamma-PGA producer, but it is also a candidate for the genetic and metabolic engineering of gamma-PGA production.

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“…Both classical and molecular genetic approaches have been shown to be very useful in generating protease-reduced or protease-deficient expression strains (Gottesman, 1990;He et al, 1991;Hinnen et al, 1994). In A. niger we have already shown that the classical genetic approach resulted in the isolatioii of at least seven prt (protease mutant) complementation groups (van den Hombergh et al, 1995).…”

Section: Discussionmentioning

confidence: 99%

“…Deletion of the major extracellular aspartic protease from Aspergillus awamori (Berka et a]., 1990) significantly improved heterologous expression of chymosin (Bodie et al, 1994). The use of (multiple) proteasedeficient strains for the expression of recombinant proteins has been described in other expression-secretion systems such as Escherichia coli (Gottesman, 1990), Bacillus (He et al, 1991) and Succharomyces cerevisiae (Hinnen et al, 1994). Studies in these expression hosts have shown that not only the extracellular proteases but also intracellular proteases are involved in the overall degradation of expressed proteins.…”

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confidence: 99%

Disruption of Three Acid Proteases in Aspergillus Niger— Effects on Protease Spectrum, Intracellular Proteolysis, and Degradation of Target Proteins

Hombergh¹,

Gelpke²,

Vondervoort³

et al. 1997

European Journal of Biochemistry

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Three acid protease genes encoding two extracellular proteases (PEPA and PEPB) and one intracellular protease (PEPE) were disrupted in Aspergillus niger. Northern-blot analysis showed the absence of wild-type protease mRNAs in the disruptants while western-blot analysis proved the absence of the encoded proteases. Characterization of the residual proteolytic spectra in the disruptants indicated that the extracellular protease activity was reduced to 16% and 94% for the ApepA and the ApepB disruptants, repectively. In the ApepE disruptant, the total intracellular proteolytic activity was reduced to 32 %. Apart from the reduced intracellular pepstatin-inhibitable aspartyl protease activity, serine protease and serine carboxypeptidase activities were also significantly reduced in the ApepE strain. This may indicate the presence of a cascade activation mechanism for several vacuolar proteases, triggered by the PEPE protein, similar to the situation in Saccharomyces cerevisiae. Disruption of a single protease gene had no effects on the transcription of other non-disrupted protease genes in A. niger. In supernatants of the disruptants, reduced degradation of a proteolytically very susceptible tester protein (PELB) was observed. By recombination, we also constructed ApepAApepB, ApepBApepE and ApepAApepE double disruptants as well as a ApepAApepBApepE triple disruptant, lacking all three acid protease activities. The in vitro residual PELB activity was the highest in the triple disruptant and the ApepAApepB recombinant.Keywords: Aspergillus ; protease ; disruption ; cascade activation ; proteolytic degradation.Aspergillus niger produces a broad spectrum of proteases, both specific and non-specific, which is recognized to be one of the major reasons for the low protein expression yields that are frequently observed, especially when producing heterologous proteins. The genetics of proteolytic degradation in Aspergilli is very complex due the involvement of many regulatory factors in protease expression. Cohen (1972Cohen ( , 1973 showed in a series of pioneering studies that in A. niduluns extracellular proteases were repressed by low-molecular-mass sources of carbon, nitrogen, and sulphur. and van den Hombergh et al. (1994) have recently shown that in A. niger both pathway-specific induction by proteins and wide domain regulatory mechanisms (carbon catabolite repression, nitrogen metabolite repression, and pH regulation) are involved in the overall regulation of extracellular proteases. Several intracellular

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Construction and Use of a Bacillus subtilis Mutant Deficient in Multiple Protease Genes for the Expression of Eukaryotic Genes^a

Cited by 20 publications

References 23 publications

Stabilizing displayed proteins on vegetative Bacillus subtilis cells

Stabilizing displayed proteins on vegetative Bacillus subtilis cells

Chromosomal integration of a synthetic expression control sequence achieves poly‐γ‐glutamate production in a Bacillus subtilis strain

Disruption of Three Acid Proteases in Aspergillus Niger— Effects on Protease Spectrum, Intracellular Proteolysis, and Degradation of Target Proteins

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Construction and Use of a Bacillus subtilis Mutant Deficient in Multiple Protease Genes for the Expression of Eukaryotic Genesa

Cited by 20 publications

References 23 publications

Stabilizing displayed proteins on vegetative Bacillus subtilis cells

Stabilizing displayed proteins on vegetative Bacillus subtilis cells

Chromosomal integration of a synthetic expression control sequence achieves poly‐γ‐glutamate production in a Bacillus subtilis strain

Disruption of Three Acid Proteases in Aspergillus Niger— Effects on Protease Spectrum, Intracellular Proteolysis, and Degradation of Target Proteins

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Construction and Use of a Bacillus subtilis Mutant Deficient in Multiple Protease Genes for the Expression of Eukaryotic Genes^a