Page 28 - Advances in Textile Biotechnology

Design and engineering of novel enzymes for textile applications 7

1.2 Production of enzymes: searching for effi cient
production systems

Various expression hosts (Escherichia coli, Bacillus sp., Saccharomyces cer-
evisiae, Pichia pastoris, fi lamentous fungi, insect and mammalian cell lines)
have been developed to express heterologous proteins (Chelikani et al.,
2006; Huynh and Zieler, 1999; Li et al., 2007; Makrides, 1996; Ogay et al.,
2006; Silbersack et al., 2006). Among the many systems available for heter-
ologous protein production, the enteric Gram-negative bacterium E. coli
remains one of the most attractive. Compared with other established and
emerging expression systems, E. coli offers several advantages including its
capacity to grow rapidly and at high density on economical carbon sources,
simple scale-up process, its well-characterized genetics and the availability
of an increasingly large number of commercial cloning vectors and opti-
mized host strains (Baneyx, 1999). However, depending on protein to be
expressed, the use of E. coli is not always suitable so there is no guarantee
that a recombinant gene product will accumulate in E. coli at high levels in
a full-length and biologically active form (Makrides, 1996). To circumvent
such constraints and as an alternative, the genes have to be cloned back
into species similar to those from which they were derived, although with
an expression cassette driven towards an increasing of production. In these
instances, bacteria from the unrelated genera Bacillus (Biedendieck et al.,
2007; Silbersack et al., 2006), Clostridium (Girbal et al., 2005) and Staphy-
lococcus, and the lactic acid bacteria Streptococcus (Arnau et al., 2006),
Lactococcus (Miyoshi et al., 2002) and Lactobacillus (Miyoshi et al., 2004),
can be an alternative.
If heterologous produced proteins require complex post-translational
modifications and are not expressed in the soluble form using prokaryotic

expression systems, yeasts can be an efficient choice because they present
several advantages over bacteria for the production of eukaryotic proteins.
Among yeast species, the methylotrophic yeast Pichia pastoris is a particu-
larly well suited host for this purpose, offering a number of important benefi ts:

(a) high levels of recombinant protein expression are reached under the
alcohol oxidase 1 gene (aox 1) promoter;
(b) high-density cell cultures can be obtained;
(c) scaled-up fermentation methods without loss of yield have been
developed;
(d) efficient secretion of the recombinant product together with a very

low level of endogenous protein secretion represents a very simple
and convenient pre-purifi cation step;
(e) accurate post-translational modifications are possible (such as proteo-

lytic processing and glycosylation).

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