Approaches for Improving Protein Production in Multiple Protease-Deficient Bacillus subtilis Host Strains

Kodama, Takeko; Manabe, Kenji; Kageyama, Yasushi; Liu, Shenghao; Ara, Katsutoshi; Ozaki, Katsuya; Sekiguchi, Junichi

doi:10.5772/30092

Cited by 4 publications

(4 citation statements)

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“…BLP did not show self-maturation ability in the culture medium of B. subtilis , and the extracellular proteases of B. subtilis were required for maturation. In many previous studies of protein production in B. subtilis , degradation of products by host extracellular proteases has been regarded as a bottleneck for heterologous protein production [ 23 , 24 ]. In the case of BLP, the focus of the present study, the characteristic result was that the propeptide was cleaved by the proteases from B. subtilis .…”

Section: Discussionmentioning

confidence: 99%

“…In the expression of heterologous proteins in B. subtilis , degradation of the product by endogenous extracellular proteases is often a bottleneck. B. subtilis releases at least nine different proteases into the culture supernatant, and there are many reports that multiple disruptions of the corresponding genes can improve heterologous protein productivity [ 23 , 24 ].…”

Section: Introductionmentioning

confidence: 99%

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Heterologous production of active form of beta-lytic protease by Bacillus subtilis and improvement of staphylolytic activity by protein engineering

et al. 2021

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Background Most of the proteases classified into the M23 family in the MEROPS database exhibit staphylolytic activity and have potential as antibacterial agents. The M23 family is further classified into two subfamilies, M23A and M23B. Proteases of the M23A subfamily are thought to lack the capacity for self-maturation by auto-processing of a propeptide, which has been a challenge in heterologous production and application research. In this study, we investigated the heterologous expression, in Bacillus subtilis, of the Lysobacter enzymogenes beta-lytic protease (BLP), a member of the M23A subfamily. Results We found that B. subtilis can produce BLP in its active form. Two points were shown to be important for the production of BLP in B. subtilis. The first was that the extracellular proteases produced by the B. subtilis host are essential for BLP maturation. When the host strain was deficient in nine extracellular proteases, pro-BLP accumulated in the supernatant. This observation suggested that BLP lacks the capacity for self-maturation and that some protease from B. subtilis contributes to the cleavage of the propeptide of BLP. The second point was that the thiol-disulfide oxidoreductases BdbDC of the B. subtilis host are required for efficient secretory production of BLP. We infer that intramolecular disulfide bonds play an important role in the formation of the correct BLP conformation during secretion. We also achieved efficient protein engineering of BLP by utilizing the secretory expression system in B. subtilis. Saturation mutagenesis of Gln116 resulted in a Q116H mutant with enhanced staphylolytic activity. The minimum bactericidal concentration (MBC) of the wild-type BLP and the Q116H mutant against Staphylococcus aureus NCTC8325 was 0.75 μg/mL and 0.375 μg/mL, respectively, and the MBC against Staphylococcus aureus ATCC43300 was 6 μg/mL and 3 μg/mL, respectively. Conclusions In this study, we succeeded in the secretory production of BLP in B. subtilis. To our knowledge, this work is the first report of the successful heterologous production of BLP in its active form, which opens up the possibility of industrial use of BLP. In addition, this study proposes a new strategy of using the extracellular proteases of B. subtilis for the maturation of heterologous proteins.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Heterologous production of active form of beta-lytic protease by Bacillus subtilis and improvement of staphylolytic activity by protein engineering

et al. 2021

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“…To date two main aspects of protein expression in wild B. subtilis have been investigated. One is the generation of a B. subtilis strain rendered deficient in protease expression via gene knock-out [12,13]. The other is optimization of fermentation processes with respect to high protein expression.…”

Section: Introductionmentioning

confidence: 99%

An effective combination of codon optimization, gene dosage, and process optimization for high-level production of fibrinolytic enzyme in Komagataella phaffii (Pichia pastoris)

et al. 2020

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Background As a main drug for diseased thrombus, some clinically used thrombolytic agents have various disadvantages, safer novel thrombolytic agents are of great demand. This study aimed to achieve high and efficient production of a fibrinolytic enzyme with superior enzymatic properties, by a combination strategy of codon optimization, gene dosage and process optimization in Komagataella phaffii (K. phaffii). Results After codon optimization, the fibase from a marine Bacillus subtilis was expressed and secreted in K. phaffii GS115. Recombinant strains harboring different copies of the fib gene (fib-nc) were successfully obtained via Geneticin (0.25–4 mg/ml) screening on minimal dextrose selection plates and assessment via real-time quantitative PCR. The respective levels of fibase produced by strains expressing fib-5.4c, fib-6c, fib-8c, fib-9c, and fib-12c were 4428, 5781, 7323, 7930, and 2472 U/ml. Levels increased as the copy number increased from 4 to 9, but decreased dramatically at copy number 12. After high cell density fermentation optimization, the highest fibase activity of the strain expressing fib-9c was 7930 U/ml in a shake flask and increased to 12,690 U/ml after 3 days of continuous culture in a 5-L fermenter, which is one of the highest levels of production reported. The recombinant fibase was maximally active at pH 9.0 and 45 °C, and was remarkably stable at pH levels ranging from 5 to 10 and temperatures up to 50 °C. As a metal-dependent serine protease, fibase did not cause hemolysis in vitro and preferentially degraded fibrin directly. Conclusions The combination of codon optimization, gene dosage, and process optimization described herein could be used for the expression of other therapeutic proteins difficult to express. The characteristics of the recombinant fibase suggest that it has potential applications for thrombosis prevention and therapy.

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“…Since the protease production is limited, protease deficient B. subtilis strains have been developed by genome engineering techniques. For example, B. subtilis WB800, deficient in eight extracellular proteases, can serve as an excellent host for the expression of heterologous proteins (Wu et al, 1991;Kodama et al, 2012).…”

Section: Recombinant Technology For Lipolytic-producing Moderately Ha...mentioning

confidence: 99%

Genome, physicochemical properties and esterase gene cloning of novel lentibacillus and halobacillus species

Booncharoen

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A total of 831 moderately halophilic bacteria isolated from various samples in Thailand were screened for their lipolytic activity. Three hundred and forty-nine isolates showed activity on agar plate while 322 isolates exhibited in broth. The grouping and identification of 322 isolates based on DNA-fingerprinting using (GTG)5+ERIC2 primer and 16S rRNA gene sequence, 180 DNA patterns could be generated. The result indicated that the representative isolates belonged to genera Bacillus, Halobacillus, Lentibacillus, Marinobacter, Thallossobacillus, and Virgibacillus. On the basis of polyphasic taxonomic study and genome analysis, a selected moderately halophilic strain SSKP1-9T, Gram-staining-positive, aerobic, endospore-forming, rod-shaped was closely related to Lentibacillus juripiscarius TISTR 1535T and L. halophilus TISTR 1549T, 98.7 and 97.2%, respectively based on 16S rRNA gene sequence similarity. On the basis of these results, the strain represents a novel species of the genus Lentibacillus, for which the name Lentibacillus lipolyticus is proposed (SSKP1-9T=JCM 32625T=TISTR 2597T). A Gram-staining-positive, aerobic, endospore-forming, rod-shaped, strain SKP4-6T was closely related to�Halobacillus salinus JCM 11546T, H. locisalis KCTC 3788T and H. yeomjeoni KCTC 3957T, with 98.6% respectively, based on 16S rRNA gene sequence similarity. On the basis of these results taxonomy and whole-genome analysis, strain SKP4-6T represents a novel species of the genus Halobacillus, for which the name Halobacillus fulvus is proposed (SKP4-6T =JCM 32624T=TISTR 2595T). Based on whole-genome analysis, strain SSKP1-9T and SKP4-6T contained protein-encoding esterase for 8 and 5 genes, respectively. The genes were then cloned into the pSaltExSePR5 plasmid and transferred to Bacillus subtilis WB800. Two recombinant, >APNFEMBD_02578-C112-40 from strain SSKP1-9T and >NKILIEJB_01195-C9-35 from strain SKP4-6T showed the highest esterase activity and were therefore studied. These two genes identified major matches with closely related to a member of family acyl-CoA thioesterase and the alpha/beta hydrolase fold founded in genus Lentibacillus and Halobacillus, respectively. Of the two recombinant enzymes, the first one has a molecular weight of 16.2 kDa, and the other was 27.7 kDa, identical to the predicted protein size from the amino acid sequence. Two recombinant enzymes reached the maximum activity at 15-30% NaCl, and the highest activity was shown at 22 % NaCl. The recombinant >APNFEMBD_02578-C112-40 displayed the activity around 25-60 ?C, and the maximum activity was observed at 40-55 ?C while >NKILIEJB_01195-C9-35 had optimal activity at 55 ?C and lost its activity rapidly above 65 ?C of the remaining activity. They stable in NaCl concentration above 17% (w/v) after incubated at 55 ?C for 1 hr. Two enzymes showed good stability and remained activity about 80% in a temperature range of 5-45 ?C after 1 hr of incubation at 50 ?C with or without NaCl. Theses enzymes reached high activity over a wide range of pH from 6.0 to 9.0, recording maximal activity at pH 7.0-8.0 and displayed excellent stable in with or without NaCl at various pH ranges (pH 6.0 to 9.0). Besides, the two recombinant enzymes were found to have higher esterase activity against p-nitrophenyl butyrate (C4:0) comparing to other esters substrate. Based on finding results, two recombinant enzymes were classified as halophilic and thermophilic esterase�

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Approaches for Improving Protein Production in Multiple Protease-Deficient Bacillus subtilis Host Strains

Cited by 4 publications

References 40 publications

Heterologous production of active form of beta-lytic protease by Bacillus subtilis and improvement of staphylolytic activity by protein engineering

Heterologous production of active form of beta-lytic protease by Bacillus subtilis and improvement of staphylolytic activity by protein engineering

An effective combination of codon optimization, gene dosage, and process optimization for high-level production of fibrinolytic enzyme in Komagataella phaffii (Pichia pastoris)

Genome, physicochemical properties and esterase gene cloning of novel lentibacillus and halobacillus species

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