239Synthetic Biology: Parts, Devices and Applications, First Edition. Edited by Christina Smolke.
© 2018 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2018 by Wiley-VCH Verlag GmbH & Co. KGaA.
12
12.1 Introduction
Increasing numbers of chemicals are produced by various genetically engineered
organisms. Those organisms possess biosynthetic pathways composed of
enzymes that act successively on the emerging substrate, in order to produce the
final product molecule. The efficiency of biosynthetic pathways is crucial for
industrial processes, and various strategies for the optimization of production
strains have been undertaken thus far. The most common strategies include
(i) increasing the pool of available substrates and/or overexpression of the
enzymes of the limiting biosynthetic steps [1–3], (ii) introducing heterologous
enzymes with preferred kinetic characteristics [4], and (iii) inhibition of the non‐
desired branching of biosynthetic pathways [5, 6].
Although diverse, none of aforementioned approaches guarantee the optimal
arrangement of the enzymes of biosynthetic pathways inside the producing
strain. Even if overexpressed, the enzymes still float randomly in the cytoplasm,
which results in nonoptimal metabolite flow. In living cells, biosynthetic pathway
enzymes or other functional polypeptides are often brought together into
multienzyme complexes through specific interactions, membrane anchoring, or
organelle targeting mechanisms. This type of organization increases the local
concentration of enzymes and their substrates and products and minimizes the
concentration of intermediates that may be toxic or unstable or may represent
substrates for branching reactions. We can view such multienzyme complexes as
autonomous units, where the evolving substrate travels from one enzyme to
another without dissociating into the bulk solution. Therefore, reaction interme
diates cannot be used by other competing biosynthetic pathways that synthesize
non‐desired side products. Due to the smaller characteristic distances between
the consecutive enzymes in the pathway, reactions can run more efficiently.
DNA scaffolding is an artificial approach to the design formation of multien
zyme complexes and will be described here. Similarly, the RNA molecule has
been used as a scaffold for biosynthetic pathway enzymes [7]. Protein scaffolding
12 Metabolic Channeling Using DNA as a Scaffold
Mojca Benčina1,2, Jerneja Mori1,2, Rok Gaber1,2, and Roman Jerala1,2
(^1) National Institute of Chemistry, Department of Biotechnology, Hajdrihova 19, SI1000 Ljubljana, Slovenia
(^2) Centre of Excellence EN‐FIST, Trg Osvobodilne fronte 13, SI1000 Ljubljana, Slovenia