Heterologous expression of endo-beta-1,4-D-glucanase from Clostridium cellulovorans in Clostridium acetobutylicum ATCC 824 following transformation of the engB gene

Abstract

Heterologous expression of the Clostridium celhulovorans engB gene by Clostridium acetobutylicum BKW-1 was detected as zones of hydrolysis on carboxymethyl cellulose (CMC) Trypticase glucose yeast plates stained with Congo red. The extracellular cellulase preparation from C. acetobutylicum BKW-1 has a specific activity towards CMC which is more than fourfold that present in C. acetobutylicum ATCC 824. Western blot (immunoblot) analysis using the C. cellulovorans anti-EngB primary antibody demonstrated that an additional 44-kDa protein band was present in the supernatant derived from C. acetobutylicum BKW-1 but was not present in ATCC 824 or ATCC 824(pMTLS00E).

Genetic systems development in the clostridia

“…Furthermore, this extracellular cellulase preparation from C. beijerinckii BKW-1 was active against acid-swollen cellulose and Sigmacell, but not filter paper, whereas the extracellular cellulase preparation from C. beijerinckii NCIMB 8052 did not show activity against these substrates. Additional verification of heterologous expression of C. cellulovorans engB gene in C. beijerinckii was carried out using Westem blot analysis [25]. Results of this work confirms heterologous expression of the engB gene in C. beijerinckii BKW-1.…”

“…However, the strain specific nature of transformation and the instability of various shuttle vectors necessitated the development of a more versatile genetic system for Clostridium [3,4,23]. Progress has been made in the development of simple electroporation protocols for the clostridia using a 10% PEG solution [9,24,25]. We suggested earlier [21] that the cell wall structure represents an additional barrier to DNA uptake in C. perfringens.…”

“…Using the above developed protocols, we were able to bring about the introduction into C. beijerinckii NCIMB 8052 of plasmid DNA which contains E. coli derived DNA [25]. C. beijerinckii 8052 lacks an active restriction system that is found in C. acetobutylicum ATCC 824 [27] and may account for the former being more amenable to DNA uptake.…”

“…in C. beijerinckii following transformation of the engB gene [25]. Initially, a phage h genomic DNA library from C. cellulovorans ATCC 35296 was generated in A ZAP II and screened for cellulase components.…”

Heterologous expression of aClostridiumminicellulosome inSaccharomyces cerevisiae

“…The concept of expressing designer cellulosomes, containing a mix-andmatch configuration of parts from different cellulosomes, in a suitable industrial host cell system has also drawn considerable attention as an attractive strategy to produce large quantities of highly active cellulases or cellulosomes with obvious benefits for processes such as simultaneous saccharification and fermentation and consolidated bioprocessing (Lynd et al, 2005;Bayer et al, 2007). Host microorganisms to date engineered with designer cellulosomes include Escherichia coli (Fierobe et al, 2001(Fierobe et al, , 2002, Bacillus subtilis (Cho et al, 2004), Clostridium acetobutylicum (Kim et al, 1994;Sabathe & Soucaille, 2003;Perret et al, 2004a, b;Mingardon et al, 2005) and Aspergillus niger (Levasseur et al, 2004). The major drive for applying a cellulosomebased strategy for heterologous expression of carbohydrateactive enzymes lies in the fact that the success of engineering a microorganism for efficient cellulose degradation is defined not by the improvement of individual enzymes, but by how the different cellulases work in synergy to overcome the recalcitrant nature of cellulose.…”