Evolution and Genetic Engineering - Bioprocess Engineering: Kinetics, Biosystems, Sustainability and Reactor Design - page 737

the polymer, polytrimethylene terepthalate, is poised for commercialization. A process to
make 2-keto- L -gluconic acid as a precursor for vitamin C production is in the pilot stage
(30,000 L), using metabolically engineered bacteria with glucose as a substrate. Other prod-
ucts from metabolically engineered cells may be new polyketides, modified polyhydroxyal-
kanoates (as biodegradable polymers), indigo, xylitol, and hybrid antibiotics.
14.10. PROTEIN ENGINEERING
Not only can cells be engineered to make high levels of naturally occurring proteins or to
introduce new pathways, but we can also make novel proteins. It is possible to make
synthetic genes encoding for totally new proteins. We are beginning to understand the rules
by which a protein's primary structure is converted into its three-dimensional form. We are
just learning how to relate a protein's shape to its functional properties, stability, and catalytic
activity. It may become possible to customize protein design to a particular well-defined
purpose.
Protein engineering at present mainly involves the modification of existing proteins to
improve their stability, substrate and inhibitor affinity and specificity, and catalytic rate.
Generally, the protein structure must be known from X ray crystallography. Key amino acids
in the structure are selected for alteration based on computer modeling, on interactions of the
protein with substrates, or by analogy to proteins of related structure. The technique used to
generate genes encoding the desired changes in protein structure is called site-directed muta-
genesis . Using this approach, any desired amino acid can be inserted precisely into the desired
position.
Site-directed mutagenesis is preferred to simple mutation-selection procedures. One
reason is that mutation followed by selection for particular properties may be difficult
when the alterations in protein properties are subtle and confer no advantages or disadvan-
tages on the mutant cell. A second reason is that site-directed mutagenesis can be used to
generate the insertion of an amino acid in a particular location, while a random mutation
giving the same result would occur so infrequently as to be unobtainable. To make this
point more evident, consider the degenerate nature of the genetic code. Each codon consists
of three letters. The odds for mutation in one of these three letters are about 10 8 per gener-
ation. The odds that two letters would simultaneously be altered are much lower (order of
10 16 ). The codon UAC (for tyrosine) could be altered by single letter substitutions to give
AAC (asparagine), GAC (aspartate), CAC (histidine), UCC (serine), UUC (phenylalanine),
UGC (cysteine), UAA (stop signal), UAG (stop signal), and UAU (tyrosine). Random
mutants in this case are very unlikely to carry substitutions for 13 of the 20 amino acids.
Thus, most of the potential insertions can be generated reliably only by using site-directed
mutagenesis.
The above approaches are directed toward the rational design of proteins. An alternative,
and often complementary, approach is that of directed evolution . This process is based on
random mutagenesis of a gene and the subsequent selection of proteins with desired prop-
erties. Large libraries of mutant genes must be made so that the rare beneficial forms are
present. A rapid screen or selection must be available to select those mutants with the desired
function or characteristics. One technique to generate mutants is the use of “error-prone”