Protein Engineering as an Enabling Tool for Synthetic Biology - Synthetic Biology - page 35

2-ketoisocaproate by the LeuACDB pathway ( Fig. 2.3 ). The authors started from an E. coli
strain previously designed to overproduce the precursor threonine. KIVD and ADH6 are
naturally promiscuous, so to reduce byproduct formation and drive flux to the C6 alcohol,
KIVD was rationally engineered to have higher selectivity toward 2-keto-4-methylhexanoate.
Based on homology models of the KIVD substrate-binding region built from structures
of homologues, the authors identified residue positions that could influence selectivity.
After testing a handful of site-directed point mutations, a variant with two amino acid
substitutions that produced the highest titer of 3-methyl-1-pentanol was selected, and this
variant was shown to have greatly enhanced specificity towards 2-keto-4-methylhexanoate
versus 2-ketoisovalerate.

Also using homology modeling and site-directed mutagenesis, the enzyme LeuA, which
naturally catalyzes the condensation of acetyl-CoA with 2-ketoisovalerate, was engineered to
have a larger binding pocket, thus better accommodating the nonnative substrate 2-keto-
3-methylvalerate and further increasing production of the C6 alcohol. It is also worth noting
that the LeuA enzyme used in this study additionally carried a single amino acid
substitution previously identified to reduce feedback inhibition by leucine. 87

A result of increasing tolerance to larger substrates by LeuA is the ability of these variants
to carry out additional condensations of acetyl-CoA with the carboxylate product of the
LeuCDB pathway, effectively adding an additional carbon per cycle. Thus, in a follow-up
study, a combination of quantum mechanical modeling, protein substrate complex

modeling, and structure-based protein engineering were used to further expand the substrate
range of LeuA from branched-chain ketoacids to linear-chain and even aromatic-chain
2-ketoacids, allowing for the production of their respective decarboxylated alcohols. 88
A variant with the largest active site volume has six amino acid substitutions and produced
80 mg L 2 1 1-heptanol and 2 mg L 2 1 1-octanol.

38

NONNATURAL AMINO ACID: L-HOMOALANINE
L-homoalanine is a nonnatural amino acid that can serve as a chiral precursor to a variety
of important pharmaceutical products. Glutamate dehydrogenase (GDH) catalyzes
the reductive amination of 2-ketoglutarate with ammonia, producing glutamate. Zhang
and coworkers sought a GDH variant capable of catalyzing reductive amination of
2-ketobutyrate for the production of L-homoalanine. 89 While L-valine is synthesized in
E. coli via transamination between glutamate and 2-ketoisovalerate catalyzed by AvtA and
IlvE (see Fig. 2.3 ), an alternate route could involve reductive amination of 2-ketoisovalerate.
Since 2-ketobutyrate is similar in structure to 2-ketoisovalerate, the authors reasoned that a
GDH variant capable of acting on 2-ketoisovalerate to make L-valine would also synthesize
L-homoalanine from 2-ketobutyrate. Auxotrophic complementation was therefore used to
isolate GDH variants with altered substrate specificity. Based on analysis of the GDH crystal
structure from Clostridium symbiosum and sequence comparison with the enzyme from E. coli ,
four E. coli GDH substrate binding pocket resides were targeted for simultaneous saturation
mutagenesis. A valine auxotroph E. coli strain was constructed (by deleting genes avtA and
ilvE ) and used to isolate GDH variants conferring valine synthesis and growth on selective
media. A resulting GDH variant was characterized and shown to have greatly enhanced
activity on 2-ketobutyrate, allowing for 5.4 g L 2 1 of L-homoalanine to be produced from
E. coli growing on glucose.

ENGINEERING PRODUCTS OF THE AROMATIC AMINO ACID PATHWAY
In a final example of using protein engineering to create synthetic pathways, Frost and
coworkers sought increased production of shikimic acid by E. coli , which naturally produces
shikimic acid as an intermediate toward aromatic amino acids and vitamins. 90 Shikimic
acid can be used to synthesize Tamiflu s , an orally effective antiinfluenza agent. 91,92 The
shikimate pathway starts with the condensation of phosphoenolpyruvate (PEP) and