11.5.3 Improvement of Fungal Strains Adapted
to SSF or SmF Processes
Shankaranand et al. (1992) wrote about ‘‘the need for an extensive screening
program for the selection of a potent culture most suited for SSF system.’’ Antier
et al. (1993) obtained DG-resistant mutants of A. niger in two culture media one
with low water activity (a w = 0.96) and other with high water activity
(a w = 0.99). The former were better adapted to produce pectinase in coffee pulp
than the latter which were better for pectinase production in shake flasks suggesting that different genes were coding for the production of enzymes adapted to
SmF or to SSF. Ishida et al. (1998) showed that the gluco-amylase gene, glaB, is
specifically induced in SSF and not in SmF. In a similar way, Biesebeke et al.
(2002) found that expression of gene brlA, necessary for sporulation, is specifically
expressed by SSF of A. oryzae. Furthermore, Machida et al. (2008) indicated that
the genome of A. oryzae used in koji processes ‘‘contains 12,074 genes’’ and Wang
et al. (2010) published the transcriptome of A. oryzae, showing that ‘‘4,628 genes
were differentially expressed between LC (liquid culture) and SC (solid culture),
including 2,355 and 2,273 genes up- and downregulated on SC, respectively.’’
Altogether, such information is conclusive that nearly 40 % of the genome of
A. oryzae is specialized for SSF and, consequently, the techniques for strain
improvement of enzyme producers should be different whether their industrial use is
for SSF or for SmF technologies. For example, the gene glaB is selectively activated
when A. oryzae is grown on agar plates provided with a Millipore membrane
opposing hyphal extension, whereas gene glaB is not (Ishida et al. 1998). These
authors simulated the actual SSF environment on agar plates provided with starch,
low a w , high temperature, and Membrane Millipore, and obtained 6,300 IU/mg
protein that considered equivalent to koji SSF. Biesebeke et al. (2005) also found
differences on the expressions of glucoamylases and proteases between SSF and
SmF and atributed to differences in carbohydrate metabolism.
Furthermore, Tsuchiya et al. (1994) made a fusion between pro-chymosin and
glaA genes in A. oryzae and found that chymosin was expressed in SmF with
levels on wheat bran 500-fold higher in SSF as compared to SmF, later, Ishida
et al. (2006) suggested that the glaB promoter also can be used for recombinant
protein production by SSF. Thus, a combination of empirical, physiological, and
molecular knowledge seems to be quite relevant for new areas of SSF in the
production of conventional enzymes and recombinant proteins.
11.5.4 Combinatorial Analysis of Regulatory Mutants
in Complex Genetic Networks
Recent work indicates that improvement of fungal strains requires multiple mutations in domains such as, gene dosage, protein folding, glycosylation, protein
11 New Horizons for the Production of Industrial Enzymes
329
to SSF or SmF Processes
Shankaranand et al. (1992) wrote about ‘‘the need for an extensive screening
program for the selection of a potent culture most suited for SSF system.’’ Antier
et al. (1993) obtained DG-resistant mutants of A. niger in two culture media one
with low water activity (a w = 0.96) and other with high water activity
(a w = 0.99). The former were better adapted to produce pectinase in coffee pulp
than the latter which were better for pectinase production in shake flasks suggesting that different genes were coding for the production of enzymes adapted to
SmF or to SSF. Ishida et al. (1998) showed that the gluco-amylase gene, glaB, is
specifically induced in SSF and not in SmF. In a similar way, Biesebeke et al.
(2002) found that expression of gene brlA, necessary for sporulation, is specifically
expressed by SSF of A. oryzae. Furthermore, Machida et al. (2008) indicated that
the genome of A. oryzae used in koji processes ‘‘contains 12,074 genes’’ and Wang
et al. (2010) published the transcriptome of A. oryzae, showing that ‘‘4,628 genes
were differentially expressed between LC (liquid culture) and SC (solid culture),
including 2,355 and 2,273 genes up- and downregulated on SC, respectively.’’
Altogether, such information is conclusive that nearly 40 % of the genome of
A. oryzae is specialized for SSF and, consequently, the techniques for strain
improvement of enzyme producers should be different whether their industrial use is
for SSF or for SmF technologies. For example, the gene glaB is selectively activated
when A. oryzae is grown on agar plates provided with a Millipore membrane
opposing hyphal extension, whereas gene glaB is not (Ishida et al. 1998). These
authors simulated the actual SSF environment on agar plates provided with starch,
low a w , high temperature, and Membrane Millipore, and obtained 6,300 IU/mg
protein that considered equivalent to koji SSF. Biesebeke et al. (2005) also found
differences on the expressions of glucoamylases and proteases between SSF and
SmF and atributed to differences in carbohydrate metabolism.
Furthermore, Tsuchiya et al. (1994) made a fusion between pro-chymosin and
glaA genes in A. oryzae and found that chymosin was expressed in SmF with
levels on wheat bran 500-fold higher in SSF as compared to SmF, later, Ishida
et al. (2006) suggested that the glaB promoter also can be used for recombinant
protein production by SSF. Thus, a combination of empirical, physiological, and
molecular knowledge seems to be quite relevant for new areas of SSF in the
production of conventional enzymes and recombinant proteins.
11.5.4 Combinatorial Analysis of Regulatory Mutants
in Complex Genetic Networks
Recent work indicates that improvement of fungal strains requires multiple mutations in domains such as, gene dosage, protein folding, glycosylation, protein
11 New Horizons for the Production of Industrial Enzymes
329