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Metabolic Engineering 393

FIGURE 2 The cycle of metabolic engineering.

recent years. Thus, genetic engineering techniques have formation with a high transformation efficiency. For a few
been facilitated and the sequencing of several complete microorganisms, e.g., S. cerevisiae and E. coli, there are
genomes and developments in bioinformatics has speeded efficient transformation vectors available that enable ei-
up the process of gene cloning and transformation. To- ther rapid chromosal integration of DNA or expression of
day many different microorganisms have been completely genes through the use of high copy number plasmids. Fur-
sequenced—including several industrially important mi- thermore, for these organisms there are many auxotrophic
croorganisms like Bacillus subtilis, E. coli, and S. cere- strains available, which facilitates the genetic engineering
visiae, and the list is rapidly growing (for a up to date significantly. For S. cerevisiae there is a very high de-
list, see www.genome.ad.jp). Table I compiles some of gree of homologous recombination, and this enables high
the tools that are often recruited in metabolic engineering, frequency of directed DNA integration into the chromo-
and in the following some of these tools are discussed in some. For E. coli many special tools have also been de-
further details. veloped, e.g., segregationally stable plasmids present in
low copy numbers that enables rapid cloning of different
genes and methods for stabilization of mRNA of heterol-
III. MOLECULAR BIOLOGY TOOLS
ogous genes through introduction of specific hairpins. For
other microorganisms the necessary tools may be avail-
For introduction of genetic modifications it is pivotal to
able, but the transformation efficiency is often low, and
have suitable strains and vectors that enable rapid trans-
the genetic engineering is much more cumbersome. For
many industrially important organisms, e.g., P. chryso-
TABLE I Tools of Metabolic Engineering genum, Aspergillus species, and Corynebacterium glu-
tamicum, there are also suitable transformation vectors,
Molecular biological tools Analytical tools
and even though there are less auxotrophic strains avail-
able (at least in industrial strain backgrounds) there are
Methods for rapid gene cloning Gene expression analysis (DNA
arrays, Northern’s) typically several dominant markers available.
Stable plasmids Protein level analysis (2D gels, Anotherimportanttoolrequiredformetabolicengineer-
Western’s) ing is access to promoters with varying strength. Often
Different promoters (inducible, Metabolite profiling it is of interest to increase expression of a certain gene,
strong, etc.)
and availability of strong promoters is therefore desir-
Site-directed chromosomal Metabolic network analysis
able. Glycolytic promoters are generally a good choice
integration of genes
for strong, constitutive promoters, e.g., the promoter of
Error prone PCR Metabolic control analysis
thegeneencodingglyceraldehyde-3-Pdehydrogenasethat
Gene shuffling
has been cloned in many different microorganisms. For