Biology Reference
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little of the expressed HUM (
15%) resided in the soluble fraction of the cell, suggesting
that much of the enzyme was misfolding or was insufficiently solublized. An adaptive
evolution approach that involved replacing amino acid residues in HUM with glycine and
proline residues improved the solubility of the enzyme (
B
60% in the soluble fraction),
resulting in a 220-fold increased production of sesquiterpines. A biochemical analysis of
wild-type and mutant HUM enzymes revealed that the catalytic properties ( k cat or K m )of
the mutant enzyme remained the same, implying that the production improvement was
achieved by increasing concentration of folded, active enzyme. Other methods adapted from
the protein overexpression field may help to solubilize an unfolded enzyme, 16,17 and thus
help improve final product titer by increasing the flux it catalyzes.
B
Many enzymes require post-translation modifications for activity,
e.g. phosphopantethenylation. 18 20 Often the proteins responsible for catalyzing these
modifications need to be transplanted from their host organism, along with the pathway
enzyme, if the modification activity is absent from the host or incompatible with the
pathway enzyme. Other accessory proteins whose activities are absent in the host cell may
also need to be present, and their activity levels optimized, in order to demonstrate activity.
For instance, a key intermediate step in the microbial production of 1,3-propanediol using
E. coli is the dehydration of glycerol to 3-hydroxypropionaldehyde by a glycerol dehydratase
gene dhaB1-3 . 21 The enzyme can also be deactivated by glycerol, requiring reactivation by a
glycerol dehydratase reactivase enzyme. 22 An engineered strain capable of converting glucose
to 1,3-propanediol on an industrial scale uses glycerol dehydratase and its reactivating
factors from Klebsiella pneumonia. Other examples of accessory proteins are the cytochrome
P450 reductases (CPR) that are required to reduce the heme group in cytochrome P450s. 23
Because commonly used platform organisms for biosynthesis lack CPR enzymes, CPR
activity often needs to be imported with the P450 genes, and itself optimized to
improve titer. 14
211
Flux through a pathway may also be modulated by feedback regulation of the enzymes by
metabolites or other small molecules. Feedback regulation can strongly affect enzymatic
activity through product or allosteric inhibition. This mode of regulation reacts to
metabolite concentration far quicker than either translational or transcriptional control, 24,25
and maintains the concentrations of intermediates within a narrow range to prevent
osmotic stress or other toxicity resulting from over-accumulation of small molecules. 26
Negative feedback regulation on an enzyme decreases its activity, effectively lowering the
concentration of the enzyme. When production pathways contain allosterically regulated
enzymes, these enzymes can become the bottleneck if the regulating metabolite reaches
inhibitory levels. Thus, engineering feedback-resistant enzymes often enables higher
production. Because our knowledge of allosteric inhibition and similar modes of regulation
by metabolites has not kept pace with our vast catalogue of genes, 27 it seems likely that
careful biochemical measurements of pathway enzymes would reveal such feedback
mechanisms.
Relieving feedback inhibition in the biosynthetic pathway of 2-keto acids, 28 a production
intermediate in the biosynthesis of amino acids, enabled workers in the Liao group to
greatly increase their titers of alcohols. Work by Shen and Liao 29 found that production of
threonine, a precursor to 2-ketobutyrate, was a bottleneck in their alcohol production
pathway. The first two steps in threonine production from aspartate are catalyzed by
the enzyme aspartate kinase/homoserine dehydrogenase (ThrA), which is inhibited by
the downstream product threonine, an effect first discovered in 1976. 30 A feedback-
resistant ThrA enzyme was cloned from a threonine-overproducing E. coli strain and
expressed under control of an inducible promoter. Expressing the feedback-resistant
ThrA resulted in a three- to four-fold increase in the titers of both 1-propanol and
1-butanol. 29
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