title: Developing in vitro tools for engineering allosteric transcription factors, enzymes and metabolic systems
title: Entwicklung von in vitro Methoden für das Engineering von allosterischen Transkriptionsfaktoren, Enzymen und Stoffwechselsystemen
titleType: AlternativeTitle
Creators: name: Barthel, Sebastian
Publisher: name: Philipps-Universität Marburg
Year: 2024
Subjects: Life sciences
Biowissenschaften, Biologie
in vitro evolution
biosensing
cell-free systems
biocatalysis
method development

description: Anthropogenic climate change, caused by the emission of more greenhouse gases
such as carbon dioxide than the global carbon cycle can fix, is drastically affecting our
planet and life on it. While humanity can decarbonize its energy needs, we will continue
to rely on carbon-based molecules for food, medicine, and materials of all kinds. 34%
of anthropogenic greenhouse gas emissions come from industrial processes such as
the production of chemicals, plastics and fertilizers, and are primarily caused by the
need for high heat and pressure in industrial processes. Biocatalysis allows the
production of chemicals at ambient pressure and lower temperatures, as well as the
use of substrates derived from waste streams, biomass or atmospheric carbon
dioxide. In recent years, many methods have been developed to tailor enzymes to the
needs of industrial biocatalysis, but low throughput in testing enzyme variants limits
progress.
The aim of this work was to develop tools to increase the throughput of enzyme
engineering and the testing of metabolic systems such as enzyme cascades. Allosteric
transcription factor (aTF)-based biosensors allow the rapid and label-free detection of
metabolites using common laboratory equipment, making them attractive for highthroughput enzyme assays. aTFs transduce the formation of the aTF ligand by the
enzyme variant tested (the input) into the generation of a reporter signal such as
fluorescence or DNA replication (the output).
To demonstrate the rapid and inexpensive testing of a complex enzyme cascade and
its individual enzymes, we developed an in vitro transcription (IVT)-based biosensor
to read out the product formation of the CETCH cycle, a complex synthetic CO2 fixation
cycle that produces glycolate from CO2. Notably, several cofactors of the CETCH cycle
inhibited IVT, but we still achieved high correlations of r = 0.94-0.98 between IVT
output and LC-MS quantification.
However, suitable aTFs that recognize metabolites of interest in the required
concentration range are rare, limiting the widespread application of biosensing. To
address this deficiency, I have developed a new concept for rapidly engineering aTFs
for new substrate specificities and other optimized properties such as sensitivity. The
concept is based on one major principle. Despite the many advantages of living cells,2
their active metabolism, metabolome and membrane pose challenges to the
engineering of aTFs, such as product degradation, interference with the native
metabolome leading to low sensitivity and crosstalk with structurally similar
compounds, and exclusion of membrane-impermeable ligands. To overcome these
limitations, the PURE system, an in vitro transcription-translation system reconstituted
from purified components, allows the testing of protein variants without competing
metabolism, metabolome and membrane, allowing the use of metabolites that cannot
be tested in living cells. Recently, a powerful in vitro compartmentalization method for
engineering transcription-translation-related enzymes, called compartmentalized
partnered replication (CPR), has been demonstrated. In CPR, the activity of an aTF is
coupled to the production of a thermostable DNA polymerase in Escherichia coli, the
cells are encapsulated in water-in-oil emulsions, and functional aTF genes are
replicated by emulsion PCR. The better the encoded aTF, the more DNA polymerase
is produced and the more the aTF gene is enriched in the gene population. However,
the previously published CPR approach is not compatible with the PURE system.
We have laid the foundation for CPR in the PURE system by establishing the
genetically encoded control of DNA replication in the PURE system, called
transcription-translation coupled DNA replication (TTcDR). We present data on
several TTcDR systems, general design rules for genetic circuits in the PURE system,
and improved DNA polymerase mutants that yield a genetic circuit based on the model
aTF TetR with &gt;1000-fold DNA replication under non-repressing conditions, ~150-fold
repression by TetR, and ~4-fold derepression by the ligand anhydrotetracycline.
We will use the genetic circuit-controlled TTcDR system to establish selection for the
evolution of allosteric transcription factors in the first place, and enzymes and
metabolic systems in the future.