parameters – ambient temperature, (near) neutral pH, aqueous solvent – and are
nicely compatible with each other.
6 After all, 6144 proteins work nicely side-byside to manage the whole metabolism of a yeast cell [405]. These properties make
enzymes ideal for the construction of biocatalytic cascades [406]. Two different
philosophies are currently persued:
Metabolic engineering employs a top-down approach, whereby the metabolism
of a living cell is redirected by minimizing (but maintaining) its vital pathways
while directing the bulk of its metabolic flux from a cheap carbon source (usually a
carbohydrate) into a desired metabolite. This powerful technology enables the
large-scale production of fermentation products, not only simple carboxylic acids
(citric, succinic, fumaric, itaconic, lactic acid) and alcohols (ethanol, 1,2- and
1,3-propanediol, 1-butanol, 1,4-butanediol), but also complex natural products
(vitamins, antibiotics, terpenoids, steroids, etc.). Overall, this technology relies on
a (minimized) living system – a ‘cell factory’ – and is only applicable to natural
compounds, which are non-toxic and hence do not disrupt vital pathways. Problems
to overcome are material erosion by competing metabolic pathways, kinetic restrictions by physical (membrane) barriers and the re-wiring of regulating circuits.
Cascade biocatalysis, also denoted as systems biocatalysis [407] or artificial
metabolism [408], is a bottom-up approach, where individual enzymes are combined to an artificial biocatalytic network in analogy to the assembly of Legoblocks by imitating the metabolism occurring in living cells. Since this is a ‘dead’
system, toxicity is not a major problem and the adjustment of the overall kinetics is
comparatively simple. The most striking difference to metabolic engineering is the
possibility to convert non-natural ‘foreign’ compounds.
During the past years, the impressive potential of combining enzymes into
biocatalytic cascades has been recognized and several systems have been proposed
(Fig. 3.4) [409].
• Linear cascades aim at the straightforward synthesis of a target compound
through sequential combination of n steps.
• Parallel cascades are the most common type and are applied in cofactorrecycling using the ‘coupled-substrate’ or the ‘coupled enzyme’ approach
(Sect. 2.2.1); thus, product formation by reduction (or oxidation) is coupled
with a second parallel reaction proceeding in the opposite oxidation
(or reduction) direction to provide redox equivalents from a sacrificial
co-substrate. Alternatively, two reactions are joined in a concomitant fashion
to yield two products, e.g. in transamination using a sacrificial amine donor
(Sect. 2.6.2).
• Closely related to parallel cascades are orthogonal cascades, where a sacrificial
cosubstrate yields a co-product, which is further utilized in the cascade or
decomposes into a final waste product, for instance, by decarboxylation. This
circumvents co-product inhibition or enables to shift equilibria.
6 With the rare exception of proteases.
3.2 Cascade-Reactions
359
nicely compatible with each other.
6 After all, 6144 proteins work nicely side-byside to manage the whole metabolism of a yeast cell [405]. These properties make
enzymes ideal for the construction of biocatalytic cascades [406]. Two different
philosophies are currently persued:
Metabolic engineering employs a top-down approach, whereby the metabolism
of a living cell is redirected by minimizing (but maintaining) its vital pathways
while directing the bulk of its metabolic flux from a cheap carbon source (usually a
carbohydrate) into a desired metabolite. This powerful technology enables the
large-scale production of fermentation products, not only simple carboxylic acids
(citric, succinic, fumaric, itaconic, lactic acid) and alcohols (ethanol, 1,2- and
1,3-propanediol, 1-butanol, 1,4-butanediol), but also complex natural products
(vitamins, antibiotics, terpenoids, steroids, etc.). Overall, this technology relies on
a (minimized) living system – a ‘cell factory’ – and is only applicable to natural
compounds, which are non-toxic and hence do not disrupt vital pathways. Problems
to overcome are material erosion by competing metabolic pathways, kinetic restrictions by physical (membrane) barriers and the re-wiring of regulating circuits.
Cascade biocatalysis, also denoted as systems biocatalysis [407] or artificial
metabolism [408], is a bottom-up approach, where individual enzymes are combined to an artificial biocatalytic network in analogy to the assembly of Legoblocks by imitating the metabolism occurring in living cells. Since this is a ‘dead’
system, toxicity is not a major problem and the adjustment of the overall kinetics is
comparatively simple. The most striking difference to metabolic engineering is the
possibility to convert non-natural ‘foreign’ compounds.
During the past years, the impressive potential of combining enzymes into
biocatalytic cascades has been recognized and several systems have been proposed
(Fig. 3.4) [409].
• Linear cascades aim at the straightforward synthesis of a target compound
through sequential combination of n steps.
• Parallel cascades are the most common type and are applied in cofactorrecycling using the ‘coupled-substrate’ or the ‘coupled enzyme’ approach
(Sect. 2.2.1); thus, product formation by reduction (or oxidation) is coupled
with a second parallel reaction proceeding in the opposite oxidation
(or reduction) direction to provide redox equivalents from a sacrificial
co-substrate. Alternatively, two reactions are joined in a concomitant fashion
to yield two products, e.g. in transamination using a sacrificial amine donor
(Sect. 2.6.2).
• Closely related to parallel cascades are orthogonal cascades, where a sacrificial
cosubstrate yields a co-product, which is further utilized in the cascade or
decomposes into a final waste product, for instance, by decarboxylation. This
circumvents co-product inhibition or enables to shift equilibria.
6 With the rare exception of proteases.
3.2 Cascade-Reactions
359