years in order to isolate a superior microbial strain (Demain and Davis 1998). In
Western countries with much higher labor costs, screening robots were developed
called High Throughput Screens (HTS). They are effective and require high capital
investments in the range of millions of dollars, since robot systems are not mass
produced but tailor-made for that purpose (Parekh et al. 2000).
An interesting alternative is a combination of genetic engineering with random
mutation. It usually takes two steps (a) A chosen gene, coding for an enzyme with
interesting commercial applications is cloned from an organism with undesirable
production features to a microbial strain with desired fermentation ability, and (b)
the transformed strain with low potency is random mutated and screened by an
HTS until a desired industrial strain is obtained.
A landmark development was the cloning of calf chymosin traditionally used
for curd making but available in very small quantities. The gene was transferred
and expressed by an improved strain of A. oryzae. Dunncoleman et al. (1991)
reported the achievement of industrial fermentations with titers higher than
E L = 1 g L
-1 and claimed to have used ‘‘a mutagenesis protocol with a novel
robotic screening program.’’ Such development was the basis of a series of patents
assigned by the US Patent Office in 1992 to International Genencor Inc. Such
breakthroughs paved the way to mass production of chymosin without the problem
of a limited supply of slaughtered calves. It is worth recalling that glucoamylase,
one of the most important bulk enzymes, is produced from an amplified copy of
the corresponding gene of A. niger (van Brunt 1986). Thus, enzyme production by
SmF processes has reached the level of commodity production.
An interesting possibility is to link a biochemical screen, such as resistance
toward a toxic chemical, or a pleiotropic (multiple action) mutation that enhances
the performance of a complex regulatory network. One interesting case is the
discovery that microbial mutants resistant to 2-deoxyglucose (2-DG) very often
become enzyme overproducers. This is related to the discovery that catabolite
repression in yeast is associated to hexokinase PII (Entian 1980) and resistance to
2-deoxyglucose (2-DG) of many microbial organisms yield complex phenotypes,
including de-repression of many hydrolases and phosphorylases and also,
according to Carlson M (1999) dgr mutants are related to Snf1 protein kinase that
is a ‘‘central component of the signaling pathway for glucose repression in yeast.’’
Apparently this happens because dgr mutants, often have a pletiotropic effect on
phosphorylases genes that reduce the toxicity of phosporylated DG and, at the
same time, they de-repress regulatory networks (Ashokkumar et al. 2004). This
technique has been applied to the selection of improved A. niger strains producing
higher titers of pectinases as compared to the wild type (Antier et al. 1993 Similar
results have been applied to A. niger invertase production (Montiel-González et al.
2002; Ashokkumar et al. 2004; Rajoka and Yasmeen 2005), xylanases (Bokhari
et al. 2010), xylosidase (Rajoka and Khan 2005), beta-glucosidase (Bokhari et al.
2008), and cellulases (Rajoka 2005). But to the best of our knowledge none of
those reported dgr strains have overcome the benchmark of E S [ 10
4 IU g
-1 .
Perhaps, a mixed approach of 2-DG resistance and THS is required to obtain
strains comparable to such benchmark.
328
G. Viniegra-González
Western countries with much higher labor costs, screening robots were developed
called High Throughput Screens (HTS). They are effective and require high capital
investments in the range of millions of dollars, since robot systems are not mass
produced but tailor-made for that purpose (Parekh et al. 2000).
An interesting alternative is a combination of genetic engineering with random
mutation. It usually takes two steps (a) A chosen gene, coding for an enzyme with
interesting commercial applications is cloned from an organism with undesirable
production features to a microbial strain with desired fermentation ability, and (b)
the transformed strain with low potency is random mutated and screened by an
HTS until a desired industrial strain is obtained.
A landmark development was the cloning of calf chymosin traditionally used
for curd making but available in very small quantities. The gene was transferred
and expressed by an improved strain of A. oryzae. Dunncoleman et al. (1991)
reported the achievement of industrial fermentations with titers higher than
E L = 1 g L
-1 and claimed to have used ‘‘a mutagenesis protocol with a novel
robotic screening program.’’ Such development was the basis of a series of patents
assigned by the US Patent Office in 1992 to International Genencor Inc. Such
breakthroughs paved the way to mass production of chymosin without the problem
of a limited supply of slaughtered calves. It is worth recalling that glucoamylase,
one of the most important bulk enzymes, is produced from an amplified copy of
the corresponding gene of A. niger (van Brunt 1986). Thus, enzyme production by
SmF processes has reached the level of commodity production.
An interesting possibility is to link a biochemical screen, such as resistance
toward a toxic chemical, or a pleiotropic (multiple action) mutation that enhances
the performance of a complex regulatory network. One interesting case is the
discovery that microbial mutants resistant to 2-deoxyglucose (2-DG) very often
become enzyme overproducers. This is related to the discovery that catabolite
repression in yeast is associated to hexokinase PII (Entian 1980) and resistance to
2-deoxyglucose (2-DG) of many microbial organisms yield complex phenotypes,
including de-repression of many hydrolases and phosphorylases and also,
according to Carlson M (1999) dgr mutants are related to Snf1 protein kinase that
is a ‘‘central component of the signaling pathway for glucose repression in yeast.’’
Apparently this happens because dgr mutants, often have a pletiotropic effect on
phosphorylases genes that reduce the toxicity of phosporylated DG and, at the
same time, they de-repress regulatory networks (Ashokkumar et al. 2004). This
technique has been applied to the selection of improved A. niger strains producing
higher titers of pectinases as compared to the wild type (Antier et al. 1993 Similar
results have been applied to A. niger invertase production (Montiel-González et al.
2002; Ashokkumar et al. 2004; Rajoka and Yasmeen 2005), xylanases (Bokhari
et al. 2010), xylosidase (Rajoka and Khan 2005), beta-glucosidase (Bokhari et al.
2008), and cellulases (Rajoka 2005). But to the best of our knowledge none of
those reported dgr strains have overcome the benchmark of E S [ 10
4 IU g
-1 .
Perhaps, a mixed approach of 2-DG resistance and THS is required to obtain
strains comparable to such benchmark.
328
G. Viniegra-González