Saccharification of Cellulose by Recombinant Rhodococcus opacus PD630 Strains

Abstract

ABSTRACTThe noncellulolytic actinomyceteRhodococcus opacusstrain PD630 is the model oleaginous prokaryote with regard to the accumulation and biosynthesis of lipids, which serve as carbon and energy storage compounds and can account for as much as 87% of the dry mass of the cell in this strain. In order to establish cellulose degradation inR. opacusPD630, we engineered strains that episomally expressed six different cellulase genes fromCellulomonas fimiATCC 484 (cenABC,cex,cbhA) andThermobifida fuscaDSM43792 (cel6A), thereby enablingR. opacusPD630 to degrade cellulosic substrates to cellobiose. Of all the enzymes tested, five exhibited a cellulase activity toward carboxymethyl cellulose (CMC) and/or microcrystalline cellulose (MCC) as high as 0.313 ± 0.01 U · ml−1, but recombinant strains also hydrolyzed cotton, birch cellulose, copy paper, and wheat straw. Cocultivations of recombinant strains expressing different cellulase genes with MCC as the substrate were carried out to identify an appropriate set of cellulases for efficient hydrolysis of cellulose byR. opacus. Based on these experiments, the multicellulase gene expression plasmid pCellulose was constructed, which enabledR. opacusPD630 to hydrolyze as much as 9.3% ± 0.6% (wt/vol) of the cellulose provided. For the direct production of lipids from birch cellulose, a two-step cocultivation experiment was carried out. In the first step, 20% (wt/vol) of the substrate was hydrolyzed by recombinant strains expressing the whole set of cellulase genes. The second step was performed by a recombinant cellobiose-utilizing strain ofR. opacusPD630, which accumulated 15.1% (wt/wt) fatty acids from the cellobiose formed in the first step.