Intergeneric Protoplast Fusion between Ruminococcus albus and an Anaerobic Recombinant, FE7

摘要

Intergeneric protoplast fusion between Ruminococcus albus, a cellulolytic, gram-positive, anaerobic bacterium (Pcs Smr Kms), and an anaerobic recombinant, FE7 (Pcr Sms Kmr), having lignin-related compound-degrading activities, was performed under strictly anaerobic conditions to introduce cellulase genes into strain FE7. The fusion frequency varied with different selected markers from 3.0 × 10?6 to 3.3 × 10?7. Two fusants, obtained from a synthetic medium with selective pressures of penicillin and streptomycin and with cellooli-gomer as the sole carbon source, were gram-negative rods. One of them, named FE7R2, showed 45 to 47% of the β-glucosidase and cellobiosidase activities of its parent R. albus and still maintained a level of degradation activity against dehydrodivanillin, a lignin-related compound, of up to 87% of that of the parent strain FE7. To verify that the cellulolytic activities expressed in the fusant FE7R2 originated from R. albus cellulase genes, the β-glucosidase gene of R. albus was cloned into Escherichia coli HB101 with plasmid pBR322. Cells bearing a recombinant plasmid, pRAII, produced high enzyme activities against both p-nitrophenyl-β-d-glucoside and p-nitrophenyl-β-d-cellobioside and could degrade cellobiose to glucose. Southern blot results showed that the cloned DNA fragment could hybridize with chromosomal DNAs of both R. albus and FE7R2, but did not with the chromosomal DNA of FE7, indicating that the β-glucosidase gene fragment was introduced into the chromosome of FE7R2 from R. albus via the protoplast fusion. The fusant FE7R2 could utilize simultaneously both cellobiose and dehydrodivanillin. These results gave evidence that the fusion product FE7R2 is a recombinant strain between its parents R. albus and FE7. This recombinant has stably kept the above properties for about 2 years.