Biology Reference
In-Depth Information
erythrose-4-phosphate (E4P) to 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP),
catalyzed by DAHP synthase. To avoid competition for PEP by the
E. coli
phosphotransferase system (PTS) and therefore improve flux to DAHP during growth on
glucose, the authors sought to replace PEP with pyruvate in a novel enzyme-catalyzed
condensation with E4P. 2-Keto-3-deoxy-phosphogalactonate (KDPGal) aldolase catalyzes
the reversible condensation of pyruvate and glyceraldehyde-3-phosphate (G3P) to KDPGal,
and the authors reasoned this enzyme could be engineered to use E4P instead of G3P, thus
producing DAHP. Directed evolution of KDPGal aldolase activity toward E4P was
accomplished by screening mutants based on their ability to improve growth of an
E. coli
strain engineered to be deficient in DAHP synthase. Starting from three different parent gene
sequences encoding KDPGal aldolase (from
E. coli
,
K. pneumoniae
, and
S. typhimurium
),
a combination of random mutagenesis and DNA shuffling, and later site-directed
mutagenesis based on homology models, were used to create mutant libraries and evolve
activity toward E4P utilization and DAHP production. Ultimately, a variant with
60-fold
improved catalytic activity on E4P compared to wild-type
E. coli
KDPGal aldolase was
created, resulting in an effective bypass route to DAHP and its downstream products.
.
CONCLUSIONS
This chapter summarizes key methods in protein engineering, and describes noteworthy
and thought-provoking examples in which they were effectively applied to achieve improved
or novel protein properties, leading to improved or novel cellular properties. Molecular
modeling and other computational tools are improving, advancing rational and semirational
protein design efforts. Owing to their relative ease of genetic manipulation and growth,
microbial systems have served as the workhorses for protein engineers. More recently,
advances in the in vitro production and screening of proteins have provided a new
experimental framework. Continuous development of genetic and protein expression systems
in more complex cell lines offer protein engineers opportunities to design more complex
protein functions within the context of eukaryotic hosts. As increasingly complex synthetic
biological systems are sought, the diversity and complexity of required protein properties also
increase. Protein engineering is thus taking on a central role in synthetic biology.
39
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