Biology Reference
In-Depth Information
HMG-CoA reductase. Clones with increased LacZ activity (expressed from PBAD) also
produced up to four-fold increased titers of mevalonate in liquid culture. 81
A third example from this group is depicted in Figure 2.2 . 82 Here, an AraC library was
screened (using FACS) to isolate a variant responding to 4-hydroxy-6-methyl-2-pyrone
(triacetic acid lactone; TAL), which is a precursor to industrially useful chemicals and
bioactive compounds. 83 85 The TAL reporter was then used for screening in the directed
evolution of TAL production ( Fig. 2.2B ). Random mutation libraries of the TAL-synthesizing
enzyme 2-pyrone synthase (2-PS) from Gerbera hybrida were expressed in E. coli containing
the reporter system. As in the case for mevalonate, clones with increased reporter activity
also produced increased titers of TAL.
New Biosynthetic Pathways
Synthetic biology now plays an important role in the metabolic engineering of
microorganisms for novel or enhanced production of biochemicals. Here we provide
examples in which protein engineering was critical to the success of designing new
biosynthetic pathways.
LONG-CHAIN ALIPHATIC ALCOHOLS
Liao and coworkers sought a nonnatural metabolic pathway in E. coli to produce aliphatic
alcohols with carbon chain lengths greater than 5 (attractive biofuel targets) during
growth on glucose. As depicted in Figure 2.3 , they developed a strategy to produce 7- (C7)
to 9-carbon (C9) 2-keto-acids, which could then be decarboxylated (by 2-keto acid
decarboxylase KIVD from Lactococcus lactis ) and reduced to the alcohol by ADH6 from
Saccharomyces cerevisiae . 86 E. coli naturally produces 2-keto-3-methylvalerate from
L-threonine, as a precursor to L-isoleucine. The L-valine analogue of 2-keto-3-methylvalerate
is 2-ketoisovalerate (containing one less methyl group), which is converted to
37
FIGURE 2.3
Synthetic metabolic pathway engineered for the production of the C6 alcohol 3-methyl-1-pentanol by E. coli. The native
L-leucine biosynthetic pathway from pyruvate uses the enzymes LeuA, LeuC, LeuD, and LeuB. Heterologous expression of
the nonnative decarboxylase KIVD and dehydrogenase ADH6 results in production of 3-methyl-1-butanol (C5). As described
in the text, C6 production from 2-keto-3-methylvalerate was achieved by enhancing the selectivity of KIVD and enlarging
the substrate binding pocket of LeuA using protein engineering.
Search WWH ::




Custom Search