Expression of transcriptional fusions between the
1. Introduction
2. Inhibition of proteolysis of heterologous and nature proteins after the translocation process by inactivation of multiple proteases
Eight extracellular proteases have been identified in
This complex was also found in the culture supernatant of
2.1. The intracellular protease, AprX is involved in degradation of a heterologous protein
In
2.1.1. Construction of an eight-extracellular-protease-deficient mutant by marker-free deletion in B . subtilis
Antibiotic-resistance marker genes were used to create new bacterial strains. However, the number of markers available for use in
2.1.2. Detection of AprX activity in the culture supernatant with protease zymography
Zymography has been used to detect proteolytic enzymes after electrophoretic separation in gels. Recently, the activities of some proteases, including Vpr have been detected by fibrin zymography of the extracellular proteins of
2.1.3. Intracellular AprX leaked to the culture medium during the late stationary phase
It has been supposed that AprX is a serine protease belonging to the subtilase superfamily, and that it is an intracellular protease, because a canonical signal sequence for secretion has not been found in this protease (Valbuzzi et al.; 1999). However, AprX was detected in the culture medium by gelatin zymography (Fig. 3).
2.1.4. AprX involved in degradation of the α-amylase-A522-PreS2 hybrid protein
AprX in the supernatant was able to degrade gelatin. Therefore, we considered that AprX may affect the production of secreted proteins. pTUBE522-preS2 has already been developed for the extracellular production of small peptides of the human hepatitis B virus preS2 antigen (42 amino acids) fused with
These results indicate that the AprX protease directly degraded the α-amylase-A522-PreS2 protein. One bottleneck of the production of α-amylase-A522-PreS2 was partially solved by the disruption of eight extracellular proteases and AprX, as shown in this chapter. However, the supernatant from the KA8AX culture at 75 h contained not only a small amount of α-amylase-A522-PreS2, but also a large amount of α-amylase protein (determined by Western blotting with the anti-α-amylase antibody; data not shown). On other hand, no PreS2 peptide was detected by Western blotting with the anti-PreS2 antibody (Fig. 5A). These results indicate that the degradation of α-amylase-A522-PreS2 was not inhibited completely in the KA8AX strain, and that there were as yet unidentified protease(s) involved in the proteolysis of the PreS2 region. Therefore, there is still room for improving the inhibition of hybrid protein degradation. It has been reported that IspA (Isp) was identified as a major intracellular serine protease (Koide et al., 1986). We evaluated the inhibition of the degradation of α-amylase-A522-PreS2 by the inactivation of IspA in the KA8AX strain. However, the productivity of α-amylase-A522-PreS2 in the ten-protease deficient mutant was almost same as that in the KA8AX strain. A search in the GenoList database for
2.2. The effect of HtrA and HtrB on the degradation of secreted proteins
In this section we describe the effects of membrane-bound proteases and a two-component system on degradation of secreted proteins, and transcriptional regulation of the membrane-bound protease genes.
2.2.1. Cell envelope-associated quality control proteases
In
2.2.2. High-level lipase A (LipA) production in eleven proteases mutant
We examined the production of lipase A (LipA) of
We constructed Dpr9∆htrA, Dpr9∆htrB, and Dpr9∆htrA/B (with eleven inactivated proteases), and evaluated each strain for the production of LipA. No effect on LipA production was observed in Dpr9∆htrA and Dpr9∆htrB. However, the production of LipA by the Dpr9∆htrA/B strain was at 1100 mg/L, which is 1.2-times higher than that of the Dpr9 strain (Fig. 8). These results suggest that inactivation of both
2.2.3. Transcriptional regulation of htrB and htrA by reciprocal cross regulation
We predicted that there was no difference between the productivities of LipA in the Dpr9∆htrA and Dpr9∆htrB strains, because the inactivation of either
Expressed gene | Strain | Plasmid | Expression a |
htrB-lacZ | Dpr9 | pHY300PLK | 0.22±0.01 |
Dpr9∆htrA | pHY300PLK | 1.46± 0.11 | |
Dpr9 | pHYLApm | 0.51± 0.11 | |
Dpr9∆htrA | pHYLApm | 2.30± 0.02 | |
htrA-lacZ | Dpr9 | pHY300PLK | 0.38± 0.03 |
Dpr9∆htrB | pHY300PLK | 1.29± 0.01 | |
Dpr9 | pHYLApm | 0.41± 0.03 | |
Dpr9∆htrB | pHYLApm | 4.26± 0.02 | |
a One activity unit is defined as 1 nmol of o-nitrophenyl-ß-d-galactopyranoside hydrolysed per min per µg of OD600. The results presented are the average of three individual experiments. Plus/minus values represent standard deviations. |
3. Conclusion
This chapter focused on biotechnological approaches to optimization of heterologous protein and enzyme production by multiple protease-deficient mutations in
Acknowledgments
We would like to thank Mr. Keiji Endo, Mr. Kazuhisa Sawada, Dr. Koji Nakamura, Dr. Yasutaro Fujita, Dr. Fujio Kawamura, and Dr. Naotake Ogasawara for useful advice and discussions, and Dr. Hiroshi Kakeshita and Dr. Kunio Yamane for their generous gift of plasmid of pTUBE522-PreS2, and for useful advice and discussions. This work was supported by the New Energy and Industrial Technology Development Organization (NEDO).
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