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glutamate. The pathways were ranked based on maximum theoretical yield, pathway length,
number of nonnative steps, number of novel steps, and thermodynamic feasibility. The
highest-ranking pathways were found to proceed through the 4-hydroxybutyrate
intermediate. The optimal pathway determined is shown in
Figure 3.2B
. In initial results,
the expression of the six genes under a single strong promoter in a multicopy plasmid
resulted in a substantial metabolic burden on the cell. The subsequent experiments
55
FIGURE 3.2
(A) Novel heterologous pathway for the production of glucaric acid. (B) BDO biosynthetic pathways introduced into E. coli. Enzymes for each numbered step
are: (1) succinyl-CoA synthetase; (2) CoA-dependent succinate semialdehyde dehydrogenase; (3) 2-oxoglutarate decarboxylase; (4) 4-hydroxybutyrate
dehydrogenase; (5) 4-hydroxybutyryl-CoA transferase; (6) 4-hydroxybutyryl-CoA reductase; (7) alcohol dehydrogenase. Steps 1 and 7 occur naturally in
E. coli, but the rest were heterologously expressed. (C) The engineered metabolic pathway from D-glucose to 1,3-propanediol
