Cellulolytic Enzyme Production and Enzymatic Hydrolysis for Second-Generation Bioethanol Production - Biotechnology in China: Biofuels and Bioenergy - page 15

with enhanced observed cellulase and hemicellulase production in glucose-
containing media. Genomic analysis of this strain revealed a single nucleotide
deletion at the +1205 position in the creA gene, which encodes a carbon catabolite
repressor protein. This frameshift mutation changes the amino acid sequence
downstream from the site of the mutation (unpublished data). Numerous other
mutations have also been identified from this mutant strain through genomic
analysis, in addition to the changes in the creA gene.
Another focus for Chinese scientists is identifying inducers for cellulolytic
enzyme production, which could potentially benefit the cellulase industry. Wang
et al. [ 100 ] observed that the concentrations of ATP and cyclic AMP (cAMP)
influence cellulase production. Cellulase synthesis is repressed by high concen-
trations of intracellular ATP, whereas exogenous cAMP increases cellulase
synthesis. The effects of wheat bran on the hydrolysis of extracellular biomass
were investigated in P. decumbens by Sun et al. [ 96 ]. The soluble cello-
oligosaccharide composition of wheat bran was shown to be one of the most
significant factors in cellulase production. This significant discovery may be
critical in the cellulase industry because wheat bran, as an inducer in cellulase and
xylanase production, is inexpensive.

3.3 Optimization of Cellulase-Producing Strains, Cellulases,
and Their Production

Traditional random mutagenesis techniques, such as ultraviolet (UV) irradiation,
N-methyl-N 0 -nitro-N-nitrosoguanidine treatment, low-energy ion beam implanta-
tion, and atmospheric pressure nonequilibrium discharge plasma, are useful and
effective approaches for the development of fungal strains with increased cellulase
production. Through these approaches, mutants of T. reesei and P. decumbens with
highly increased cellulolytic capabilities have been obtained (Fig. 5 )[ 16 , 24 , 101 ].
Genome shuffling is another effective approach for the rapid engineering of
microbial strains with desirable industrial phenotypes, and it is thus used for the
development of cellulase-producing strains. Cheng et al. [ 91 ] used this technique
to enhance cellulase production by repeated protoplast fusions, and the GS2-15,
GS2-21, and GS2-22 fusants obtained showed 100, 109, and 94% increased filter
paperase activity compared with their parent strain. Xu et al. [ 68 ] used genome
shuffling to improve the cellulase production of the wild-type strain T. viride
TL-124. The initial mutants were generated through random mutagenesis and were
then subjected to recursive protoplast fusion. The cellulase activity of the resulting
strains was assayed after solid-state fermentation using wheat straw as the sub-
strate. The shuffled strain T. viride F161, which was selected from among
approximately 2,000 strains after two rounds of genome shuffling, exhibited a total
cellulase activity of 4.17 U g -1 dry weight, which was 1.97-fold higher than that of
wild-type strain T. viride TL-124 (2.12 U g -1 dry weight).