Heterologous production of Clostridium cellulovorans engB, using protease-deficient Bacillus subtilis, and preparation of active recombinant cellulosomes

Abstract

In cellulosomes produced by Clostridium spp., the high-affinity interaction between the dockerin domain and the cohesin domain is responsible for the assembly of enzymatic subunits into the complex. Thus, heterologous expression of full-length enzymatic subunits containing the dockerin domains and of the scaffolding unit is essential for the in vitro assembly of a "designer" cellulosome, or a recombinant cellulosome with a specific function. We report the preparation of Clostridium cellulovorans recombinant cellulosomes containing the enzymatic subunit EngB and the scaffolding unit, mini-CbpA, containing a cellulose binding domain, a putative cell wall binding domain, and two cohesin units. The full-length EngB containing the dockerin domain was expressed by Bacillus subtilis WB800, which is deficient in eight extracellular proteases, to prevent the proteolytic cleavage of the enzymatic subunit between the catalytic and dockerin domains that was observed in previous attempts to express EngB with Escherichia coli. The assembly of recombinant EngB with the mini-CbpA was confirmed by immunostaining, a cellulose binding experiment, and native polyacrylamide gel electrophoresis analysis.

FIG. 1.

Schematic diagram of mini-CbpA. The signal sequence, a cellulose binding domain, hydrophilic domains, and cohesin domains of CbpA (14) are shown. Numbers are amino acid residues counted from the translation start of CbpA. The amplified mini-CbpA fragment is also indicated.

FIG. 2.

Construction of pWB980-EngB. Plasmid pWB980 carries both the constitutively expressed P43 promoter (20) and the engineered levansucrase signal sequence (22). The engB gene was inserted into pWB980 to generate pWB980-EngB by a two-step cloning strategy as described in Materials and Methods. The sequences coding for the kanamycin resistance marker and levansucrase signal sequence are represented by kan and sacB SP, respectively. The arrows show the transcription directions for the genes.

FIG. 3.

Silver-stained SDS-polyacrylamide gel showing the purified mini-CbpA (lane 1) and the purified rEngB-B (lane 2).

FIG. 4.

SDS-PAGE analysis of the culture broth of recombinant B. subtilis WB800 harboring pWB980-EngB (A) and the periplasmic fraction of recombinant E. coli BL21(DE3) harboring pET-B2 (B). The gels were immunostained with anti-EngB. Culture periods in panel A: lane 1, 12 h; lane 2, 18 h; lane 3, 24 h; lane 4, 30 h; lane 5, 36 h; lane 6, 42 h; lane 7, 48 h.

FIG. 5.

Interaction between mini-CbpA and rEngB expressed by B. subtilis WB800 (A) and E. coli (B). The mini-CbpA (2 μg) was electroblotted onto a polyvinylidene difluoride membrane. The membrane was incubated with crude EngB expressed by B. subtilis (A) or with EngB found in the periplasmic fraction of E. coli (B). Bound proteins were immunostained with anti-EngB.

FIG. 6.

Cellulose binding of rEngB-B mixed with mini-CbpA. rEngB-B (2 pmol) was mixed with various amounts of mini-CbpA, and then the mixtures of rEngB-B and mini-CbpA were bound to 500 μg of Avicel. The amounts of rEngB-B bound to Avicel were determined as described in Materials and Methods.

FIG. 7.

Native PAGE analysis of the rEngB-B-mini-CbpA complex. Lane 1, rEngB-B; lane 2, mixture of rEngB-B and mini-CbpA (molar ratio, 1:1); lane 3, mini-CbpA.