Cellulosic biomass, such as wood waste and rice straw, remains unexploited despite the high content of cellulose. Streptomycetes are producers of industrially important antibiotics and most of the antibioticproducing strains are unable to utilize cellulose. We have, therefore, aimed to breed streptomycetes that produce antibiotics from cellulosic biomass. Two cellulolytic streptomycetes, i.e. Streptomyces thermocarboxydus C42 and Streptomyces argenteolus M178, were selected on the basis of carboxymethyl cellulose (CMC)-degrading activity from approximately 1,000 soil-isolated streptomycetes. Shotgun cloning experiments of cellulase genes from these genomic DNAs were performed in Streptomyces lividans as a host to find putative four cellulase genes, i.e. cel9A C and cel5A C from the C42 strain and cel5A M and cel12B M from the M178 strain. The transformants of S. lividans harboring the four genes subcloned in an expression plasmid showed secreted CMC-degrading cellulase (CMCase) activities, thus demonstrating successful cloning and heterologous expression of the cellulase genes from the cellulolytic streptomycetes. 2.2 CMC assay and Measurement of CMC-degrading cellulase (CMCase) activity Streptomyces isolates were cultivated in 2 ml of GPY and CPY liquid media at 30˚C in a test tube containing a glass bead on a reciprocal shaker at 130 strokes/min for 3 to 4 days. A portion (50 µl) of the supernatant of culture broth was placed in wells (6 mm diameter and 5 mm depth) on 1.5% (w/v) agar plates containing 0.5% (w/v) CMC sodium salt and the plates were incubated at 37˚C for 12 hours for CMC degradation [14]. Staining of the plates with 0.1% (w/v) Congo red solution and the following destaining with 1 M NaCl visualized CMC-digested clear zones formed around the wells by the diffusion of CMCase. On the other hand, the CMCase activity was measured using CMC as the substrate as described by Okada et al. [15] with some modifications. The supernatant (20 µL) of the Streptomyces culture was mixed with 80 µL of 1.25% CMC solution in 50 mM acetate buffer (pH 6.0) for incubation at 37˚C for 60 min, and the reducing sugar released was determined by the 3,5-dinitrosalicylic acid method [16]. The experiment was conducted in triplicate. One unit of CMCase activity was defined as the amount of enzyme releasing 1 µmol of glucose equivalent per minute.2.3 DNA manipulation DNA manipulations using E. coli as a host were carried out as described by Sambrook et al. [18]. DNA manipulations in Streptomyces cells were conducted as described in a manual by Kieser et al. [16]. Enzymes and kits for DNA recombination techniques were purchased from Takara Bio Inc. and Toyobo Co. Ltd., and used according to the manufacturers' instructions. Polymerase chain reaction (PCR) was conducted using an iCycler thermal cycler (Bio-Rad Laboratories Inc.) with Takara LA Taq with GC buffer (Takara Bio Inc.) and KOD-Plus-(Toyobo Co. Ltd.). All PCR primers used for this study were synthesized commercially (Hokkaido System Science Co. Ltd.) as listed in Table...

Cellulosic biomass such as wood waste and rice straw remains unexploited despite its high cellulose content. Streptomyces thermocarboxydus C42, which grows on cellulosic compounds including microcrystalline cellulose, was isolated from soil for genetic breeding of streptomycetes that produce antibiotics from cellulosic biomass. Draft genome sequencing revealed putative genes encoding nine cellulases and one xyloglucanase dispersed on the chromosome. All these genes were isolated and rearranged on a chromosome-integration vector for streptomycetes pTYM19 to construct cellulase-expression plasmids pBOM51 and pBOM66 for streptomycete host strains. The cellulase gene cluster on pBOM66 was further introduced into pTYM18, another Streptomyces integration vector, to yield pBOM67. To investigate cellulase secretion and antibiotic production, the resulting plasmids were introduced into Streptomyces avermitilis K139, the producer of avermectin and oligomycin. Remarkable cellulose-degrading activity for fi lter paper was observed by pBOM66-carrying transformants. Oligomycin alone was produced by the transformed strain with pBOM67 only when cultivated in a medium containing glucose: not in a medium containing microcrystalline cellulose as a carbon source. Using antibiotic-producing streptomycetes for antibiotic production from cellulosic biomass therefore appears to be infeasible, even when transformed with cellulase genes of Streptomyces origin.