6.14.3 Microbial Cells as Production Factories
The production level of cold-active (heat-labile) proteins by wild-type strains is
usually too low for the production on an industrial scale. To facilitate biotechnological applications of psychrophiles and of their products, a recombinant protein
secretion system is a way to produce large-scale production. Therefore, genes
encoding for cold-active (heat-labile) proteins have been cloned and expressed in
host bacteria, such as Escherichia coli, for which efficient expression systems have
been designed to obtain high enzyme yields.
Tutino et al. (2001a) described the first recombinant production of a cold-active
enzyme (α-amylase from Antarctic Pseudoalteromonas haloplanktis) in an Antarctic
host bacterium of the same species. The cold gene-expression system was further
developed and optimized for the recombinant extracellular secretion of heterologous
proteins in P. Haloplanktis, with enzymes originating from various Antarctic
P. haloplanktis strains and a mesophilic yeast (Cusano et al. 2006; Papa et al.
2007). The simultaneous secretion of proteolytic enzymes that degraded the recombinant products could be considerably reduced by inactivating the secretion system
with the use of a gene insertion strategy; the mutant strain still secreted the coldactive enzyme (α-amylase) as efficiently as the wildtype and in a stable form (Parrilli
et al. 2008). Another recombinant protein expression system working at low temperature was developed by using an Antarctic Shewanella sp. strain and was based
on the selection of a suitable promoter and a broad host-range plasmid. High yields
of ß-lactamase were produced in the Shewanella sp. strain at 4
C; the enzyme yield
produced at 4
C was 64% of that obtained at 18
C. The efficiency of the system was
demonstrated by the production of foreign proteins (putative peptidases and a
glucosidase) from the psychrophile Desulfotalea psychrophila (Miyake et al. 2007).
Cold-active chaperones have also found very useful application in the production
of recombinant proteins. High-level expression of heterologous proteins in E. coli
can result in the production of large amounts of incorrectly folded proteins,
generating aggregates of inactive protein generally in the form of inclusion bodies.
To circumvent this insolubility problem, low temperature cultivation of E. coli
represents a classical strategy and co-expression of chaperones also frequently
improves the recovery of soluble proteins. Chaperones are a ubiquitous class of
proteins that assist the folding of nascent polypeptides, preventing misfolding or
even repairing misfolding. In this context, the chaperonins Cpn10 and Cpn60
(homologous to GroES and GroEL in E. coli) from the Antarctic bacterium Oleispira
antarctica were shown to improve the growth of E. coli at low temperatures and to
remain optimally active as folding catalysts at these low temperatures (Ferrer et al.
2003). Taking advantage of these properties, the Arctic Express E. coli cells from
Stratagene (USA) have been engineered to co-express the cold-active chaperonins
with the recombinant protein of interest, therefore improving protein processing at
low temperatures and increasing the yield of active, soluble recombinant protein.
214
P. K. Mishra et al.
The production level of cold-active (heat-labile) proteins by wild-type strains is
usually too low for the production on an industrial scale. To facilitate biotechnological applications of psychrophiles and of their products, a recombinant protein
secretion system is a way to produce large-scale production. Therefore, genes
encoding for cold-active (heat-labile) proteins have been cloned and expressed in
host bacteria, such as Escherichia coli, for which efficient expression systems have
been designed to obtain high enzyme yields.
Tutino et al. (2001a) described the first recombinant production of a cold-active
enzyme (α-amylase from Antarctic Pseudoalteromonas haloplanktis) in an Antarctic
host bacterium of the same species. The cold gene-expression system was further
developed and optimized for the recombinant extracellular secretion of heterologous
proteins in P. Haloplanktis, with enzymes originating from various Antarctic
P. haloplanktis strains and a mesophilic yeast (Cusano et al. 2006; Papa et al.
2007). The simultaneous secretion of proteolytic enzymes that degraded the recombinant products could be considerably reduced by inactivating the secretion system
with the use of a gene insertion strategy; the mutant strain still secreted the coldactive enzyme (α-amylase) as efficiently as the wildtype and in a stable form (Parrilli
et al. 2008). Another recombinant protein expression system working at low temperature was developed by using an Antarctic Shewanella sp. strain and was based
on the selection of a suitable promoter and a broad host-range plasmid. High yields
of ß-lactamase were produced in the Shewanella sp. strain at 4
C; the enzyme yield
produced at 4
C was 64% of that obtained at 18
C. The efficiency of the system was
demonstrated by the production of foreign proteins (putative peptidases and a
glucosidase) from the psychrophile Desulfotalea psychrophila (Miyake et al. 2007).
Cold-active chaperones have also found very useful application in the production
of recombinant proteins. High-level expression of heterologous proteins in E. coli
can result in the production of large amounts of incorrectly folded proteins,
generating aggregates of inactive protein generally in the form of inclusion bodies.
To circumvent this insolubility problem, low temperature cultivation of E. coli
represents a classical strategy and co-expression of chaperones also frequently
improves the recovery of soluble proteins. Chaperones are a ubiquitous class of
proteins that assist the folding of nascent polypeptides, preventing misfolding or
even repairing misfolding. In this context, the chaperonins Cpn10 and Cpn60
(homologous to GroES and GroEL in E. coli) from the Antarctic bacterium Oleispira
antarctica were shown to improve the growth of E. coli at low temperatures and to
remain optimally active as folding catalysts at these low temperatures (Ferrer et al.
2003). Taking advantage of these properties, the Arctic Express E. coli cells from
Stratagene (USA) have been engineered to co-express the cold-active chaperonins
with the recombinant protein of interest, therefore improving protein processing at
low temperatures and increasing the yield of active, soluble recombinant protein.
214
P. K. Mishra et al.