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Directed evolution of artificial metalloenzymes for in vivo metathesis
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Journal Article
Directed evolution of artificial metalloenzymes for in vivo metathesis
2016
Overview
An artificial metalloenzyme is compartmentalized and evolved
in vivo
for olefin metathesis—an archetypal organometallic reaction without equivalent in nature; the evolved metathase reveals broad substrate scope and compares favourably with commercial catalysts.
Directed evolution of an artificial metalloenzyme
Artificial metalloenzymes—made by incorporating an abiotic cofactor within a protein scaffold—have the potential to engineer non-natural
in vivo
reactions. To be of practical use, such catalysts must maintain their activity in a cellular environment, which means overcoming the tendency of introduced metal cofactors to be inhibited by cellular components. This paper demonstrates that directed evolution can overcome this difficulty. Markus Jeschek
et al
. report on the
in vivo
evolution of a ruthenium–protein complex that can catalyse olefin metathesis—an archetypal organometallic reaction with no equivalent in nature—in the periplasm of
Escherichia coli
. The evolved metathase compares favorably with commercial catalysts, shows activity for different metathesis substrates and can be further evolved in different directions by adjusting the protocol.
The field of biocatalysis has advanced from harnessing natural enzymes to using directed evolution to obtain new biocatalysts with tailor-made functions
1
. Several tools have recently been developed to expand the natural enzymatic repertoire with abiotic reactions
2
,
3
. For example, artificial metalloenzymes, which combine the versatile reaction scope of transition metals with the beneficial catalytic features of enzymes, offer an attractive means to engineer new reactions. Three complementary strategies exist
3
: repurposing natural metalloenzymes for abiotic transformations
2
,
4
;
in silico
metalloenzyme (re-)design
5
,
6
,
7
; and incorporation of abiotic cofactors into proteins
8
,
9
,
10
,
11
. The third strategy offers the opportunity to design a wide variety of artificial metalloenzymes for non-natural reactions. However, many metal cofactors are inhibited by cellular components and therefore require purification of the scaffold protein
12
,
13
,
14
,
15
. This limits the throughput of genetic optimization schemes applied to artificial metalloenzymes and their applicability
in vivo
to expand natural metabolism. Here we report the compartmentalization and
in vivo
evolution of an artificial metalloenzyme for olefin metathesis, which represents an archetypal organometallic reaction
16
,
17
,
18
,
19
,
20
,
21
,
22
without equivalent in nature. Building on previous work
6
on an artificial metallohydrolase, we exploit the periplasm of
Escherichia coli
as a reaction compartment for the ‘metathase’ because it offers an auspicious environment for artificial metalloenzymes, mainly owing to low concentrations of inhibitors such as glutathione, which has recently been identified as a major inhibitor
15
. This strategy facilitated the assembly of a functional metathase
in vivo
and its directed evolution with substantially increased throughput compared to conventional approaches that rely on purified protein variants. The evolved metathase compares favourably with commercial catalysts, shows activity for different metathesis substrates and can be further evolved in different directions by adjusting the workflow. Our results represent the systematic implementation and evolution of an artificial metalloenzyme that catalyses an abiotic reaction
in vivo
, with potential applications in, for example, non-natural metabolism.
Publisher
Nature Publishing Group UK,Nature Publishing Group
