1
Metabolic engineering
Engineering and optimizing genetic, epigenetic and
biochemical processes to increase the cells' production
Dr. Martin Marek
Loschmidt Laboratories
Faculty of Science, MUNI
Kamenice 5, bld. A13, room 332
martin.marek@recetox.muni.cz
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What will we talk about
• Metabolic engineering
• definition, goals and applications
• Metabolomics and systems biology
• concepts, methodology, limitations,
• Synthetic epigenetics, epigenetic therapy
• definition, goals and applications
• Synthetic biology in drug discovery
• Emerging technologies, unmet challenges
3
What is metabolic engineering?
https://doi.org/10.1016/j.tibs.2018.05.004
• Metabolic engineering is basically meant for the production of chemicals, fuels, pharmaceuticals, and
medicine by altering the metabolic pathways.
• Metabolic engineering is motivated by commercial applications by which we can improve the
developing strains for production of useful metabolites. This method requires overexpression or
downregulation of certain proteins in a metabolic pathway, such that the cell produces a new product.
4
What is metabolic engineering?
• Metabolic engineering is basically meant for the production of chemicals, fuels, pharmaceuticals, and
medicine by altering the metabolic pathways.
• Metabolic engineering is motivated by commercial applications by which we can improve the
developing strains for production of useful metabolites. This method requires overexpression or
downregulation of certain proteins in a metabolic pathway, such that the cell produces a new product.
• First step for successful engineering requires the complete understanding of host cell for genetic
modifications. The effect of genetic manipulation on growth should also be examined. The genetic
manipulation may negatively effect on metabolic burden.
• To achieve the product several methods, such as elimination of competitive pathway and toxic
byproduct and expression of a heterologous enzyme to improve the synthesis of products, are used for
the modification of the host cell.
• In classical metabolic engineering approach, genetic manipulation is based on the prior knowledge of
enzyme network pathway and its kinetics; on the other hand, in inverse metabolic engineering the
environmental or genetic conditions are considered for the desired phenotype for genetic manipulation.
• A number of approaches are used for secondary metabolites improvement using bacteria in metabolic
engineering, such as (1) heterologous expression of gene clusters, (2) regulatory networks pathway
engineering, (3) gene insertion or deletion, and (4) stimulation using certain substrates.
• Therefore, metabolic engineering is currently used as cell factories for production of amino acids,
biofuels, pharmaceuticals, bioplastics, platform chemicals, silk proteins, etc.
https://doi.org/10.1016/j.tibs.2018.05.004
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Goals of metabolic engineering
https://www.mdpi.com/2227-9717/7/4/213
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Metabolic engineering workflow
7
Why do we need to build a synthetic
metabolism of the future?
8
The power of synthetic biology
Synthetic chemistry 20th century
Biological systems are:
self-optimizing
self-repairing
self-propagating
Biological systems operate:
in a sustainable fashion
environmentally friendly
Synthetic biology 21st century
9
Five different levels of metabolic engineering
• The enzyme solution space
describes the number of possible
enzymes reactions available for a
given strategy while the pathway
solution space corresponds to the
number of possible pathways that
can be constructed.
• While level 1, 2 and 3 metabolic
engineering efforts do not differ in
enzyme solution space, because
they all rely on known enzymes,
level 4 and 5 metabolic engineering
efforts provide new enzymes
created through enzyme
engineering or de novo protein
design.
https://www.sciencedirect.com/science/article/pii/S1367593116302071
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Central questions of metabolic engineering
How to design synthetic metabolic networks?
How to realize synthetic metabolic networks?
How to optimize synthetic metabolic networks?
How to transplant synthetic metabolic networks?
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Measuring the efficiency of enzymes
kcat = Turnover number (s-1)
Chemical conversions of substrate
molecules per second per active site
Km = Michaelis constant (M-1)
Substrate concentration at
half maximum activity
kcat / Km (M-1 s-1)
Catalytic efficiency
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Examples of metabolic engineering
Example 1: a fixation of carbon dioxide
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A fixation of carbon dioxide in vitro: an example of ME
Carbon dioxide (CO2) is an important carbon feedstock for a future green economy. This
requires the development of efficient strategies for its conversion into multicarbon compounds.
We describe a synthetic cycle for the continuous fixation of CO2 in vitro. The crotonylcoenzyme
A (CoA)/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle is a reaction network
of 17 enzymes that converts CO2 into organic molecules at a rate of 5 nanomoles of CO2 per
minute per milligram of protein. The CETCH cycle was drafted by metabolic retrosynthesis,
established with enzymes originating from nine different organisms of all three domains of
life, and optimized in several rounds by enzyme engineering and metabolic proofreading.
The CETCH cycle adds a seventh, synthetic alternative to the six naturally evolved CO2
fixation pathways, thereby opening the way for in vitro and in vivo applications.
Enoyl-CoA Carboxylases/reductases
are up to 4-fold efficient than RuBisco
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Expanding the biosynthetic space of ECR reactions
Enzyme engineering Genome mining
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Designing and realizing a new synthetic metabolic pathway
https://science.sciencemag.org/content/354/6314/900
Evaluation criteria:
• Kinetically favoured
Fast and efficient enzyme reactions
• Thermodynamically favoured
Energy per CO2 molecule fixed
• Thermodynamically feasible
All equilibrium constants ≥1
The CETCH cycle for CO2 fixation. Important enzymes that were engineered to
establish the cycle are highlighted in purple.
Design principles:
• Cyclic topology
• Carbon-splitting reactions
• Biochemically possible reactions
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Designing and realizing a new synthetic metabolic pathway
https://science.sciencemag.org/content/354/6314/900
Finding the parts:
• Searching enzyme databases
• Testing enzyme homologs
• (Re)-engineering enzymes
The CETCH cycle for CO2 fixation. Important enzymes that were engineered to
establish the cycle are highlighted in purple.
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Key role of protein design in metabolic engineering
A) Active site comparison of the human
short-chain acyl-CoA dehydrogenase
(PDB: 2VIG, green backbone) with a
model of Mcd from Rhodobacter
sphaeroides (blue backbone, modeled
by the SWISS-MODEL server with 2VIG
as template) and the short-chain acylCoA
oxidase 4 from Arabidopsis thaliana
(PDB: 2IX5, orange backbone). The
three residues that were targeted to
introduce oxidase activity into Mcd are
highlighted.
B) HPLC-MS based analysis of wild-type
and different single active-site mutants
for oxidation of methylsuccinyl-CoA into
mesaconyl-CoA with molecular oxygen
as electron acceptor. The screen
identified three substitutions (W315F,
T317G and E377N) that were combined
into double and triple mutants.
C) Michaelis-Menten graphs of single,
double and triple mutants characterized
in more detail. The triple mutant showed
best kinetic parameters.https://science.sciencemag.org/content/354/6314/900
Structure guided engineering of Mcd into a Mco
(Methysuccinyl-CoA Dehydrogenase into Methylsuccinyl-CoA oxidase)
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The CETCH cycle
The crotonyl-coenzyme A (CoA)/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle
(A) Topology of the CETCH cycle (version 5.4), including
proofreading and cofactor regenerating enzymes.
https://science.sciencemag.org/content/354/6314/900
• 17 different enzymes
• 9 different organisms
Methylobacterium
Rhodobacter
Clostridium
E. coli
Nitrosopumilus
Human liver
etc.
• 3 engineered enzymes
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Dynamics of key intermediates of CETCH over 90 minutes
B) Shown are the levels of six different intermediates, as well as their fractional labeling patterns from the
incorporation of 13CO2 for each turn of the cycle.
C) Left x-axis: CO2-fixation efficiency of the CETCH cycle over the course of its optimization. CO2-fixation
efficiency is defined as the CO2-equivalents fixed per acceptor molecule in the cycle (i.e., starting amount of
propionyl-CoA). Right x-axis: Absolute malate concentration formed over the course of 90 minutes in CETCH
5.4. The final assay (0.52 ml) contained 2.3 mg ml-1 protein of cycle core enzymes plus 0.8 mg ml-1 auxillary
enzymes and produced 540 μM malate over 90 min, which corresponds to 1080 μM fixed CO2.
https://science.sciencemag.org/content/354/6314/900
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How to optimize metabolic pathways?
https://doi.org/10.1016/j.tibs.2018.05.004
(A) Topology of the CETCH cycle version 3.0. The newly added enzymes Fdh (NADPH
regeneration ) and Mas (read-out module) converting the primary CO2-fixation product
glyoxylate into malate are boxed in red.
CETCH 3.0
(addition of
NADPH
regeneration &
read-out module)
Production of
dead-end
metabolites
from unwanted
side reaction
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Use of synthetic regulators to modulate metabolic
pathways that have a toxic intermediate
https://www.ncbi.nlm.nih.gov/books/NBK84444/
Regulatory proteins or RNAs bind the toxic metabolite and down-regulate the biosynthetic
pathway and up-regulate the consumption pathway.
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Strategy for the design and realization of the CETCH cycle,
a synthetic pathway for CO2-fixation
https://www.beilstein-journals.org/bjoc/articles/15/49
CETCH cycle:
4 ATP molecules per CO2 (pyruvate)
Calvin cycle:
7 ATP molecules per CO2 (pyruvate)
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Next steps: Transplanting the CETCH cycle
https://www.beilstein-journals.org/bjoc/articles/15/49
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Artemisinin: a metabolic engineering success story
Malaria is caused by parasites of the Plasmodium species, primarily Plasmodium falciparum and
Plasmodium vivax. Almost one million deaths from malaria and over 200 million new infections are
recorded annually, primarily among young children in the developing world. In recent years, malaria
parasites have developed resistance to all inexpensive drugs available in those areas, rendering these
drugs ineffective. In response, the World Health Organization recommended the use of alternative
treatments called artemisinin-based combination therapies (ACTs). This report describes progress
toward the development of a semisynthetic production process whereby an artemisinin precursor is
produced by engineered yeast (Saccharomyces cerevisiae) in large-scale fermentation processes, and
the precursor is then chemically converted to artemisinin.
https://www.pnas.org/content/109/3/E111/1
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Artemisinin biosynthesis in Artemisia annua
Artemisinin biosynthesis
pathway occurs in the
glandular trichomes of
Artemisia annua. The pathway
intermediates are defined as
FPP, farnesyl diphosphate; AD,
amorpha-4,11-diene; AAOH,
artemisinic alcohol; AAA,
artemisinic aldehyde; AA,
artemisinic acid; DHAAA,
dihydroartemisinic aldehyde;
DHAA, dihydroartemisinic acid.
https://www.frontiersin.org/articles/10.3389/fpls.2017.01966/full
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Reconstruction of artemisinin pathway in yeasts
https://www.pnas.org/content/109/3/E111/1
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Artemisinin: a metabolic engineering success story
Production of plant-derived artemisinin compared to semisynthetic artemisinin. Production of plant-derived artemisinin takes
from 14 to 18 months from planting to production. Plant-derived artemisinin requires cultivation of A. annua, followed by
extraction of artemisinin from the leaves and conversion to artemisinin derivatives for incorporation into antimalarial ACT
medication. Semisynthetic artemisinin, by contrast, uses engineered yeast to produce amorphadiene in fermentations. The
amorphadiene extracted from the fermentor is chemically converted to dihydroartemisinic acid and then to artemisinin
derivatives for incorporation into ACT drugs. The entire process could be accomplished in weeks.
https://www.pnas.org/content/109/3/E111/1
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Making artemisinin to fight malaria
As for artemisinin itself, almost 100 tonnes of it are needed each year to treat malarial infections, however, there are still great
challenges in finding cost-effective ways of producing artemisinin. The main way of producing artemisinin used to be by
extraction from Artemisia annua plant itself, however, the costs of growing the plant and the efficiency of the process (5 kg or
artemisinin per 1,000 kg of dried leaves) has never been sufficient to meet the global demands. Therefore, the producers have
now focussed on looking for new synthetic ways of making the drug. With a financial backup from Melinda and Bill Gates
foundation a company called Amyris and its collaborators have created a yeast strain capable of making artemisinic acid (a
precursor for artemisinin) in fermentation reactors at relatively high yields. Even so, the cost of producing artemisinic acid in
yeast is still quite high making it difficult to obtain for the poorer countries in Asia and Africa, which are usually most afflicted by
malaria.
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Engineering plant metabolic pathways
• Plant metabolic pathways can be reconstituted in heterologous hosts.
• Metabolism in crop plants can be engineered to improve the production of biofuels.
• Crops can be engineered to express metabolic pathways that improve human health
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Plant metabolic engineering for chemicals, fuels, and precursors
Many different chemicals and
fuels can be produced from
plants; the photos in the center
are several representative
species. The outer boxes show
various chemicals that can be
produced from plants, including
shikimic acid and morphine.
The inner ring shows different
enabling technologies that
facilitate production routes for
these and other fuels and
chemicals.
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Conversion of sugars to chemicals by means of
microbial catalysts
https://www.ncbi.nlm.nih.gov/books/NBK84444/
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Biological fuel cells (BFCs) and the bio-production
of hydrogen
https://www.fuelcellstore.com
Biological power systems have many advantages over traditional chemical systems due to lowtemperature
operation and non-hazardous materials. These systems have the potential to eliminate the
transport and storage of large quantities of hydrogen because the hydrogen is created in-situ. Also, the
BFC eliminates the occurrence of catalyst poisoning over time due to the use of biological catalysts. These
naturally-occurring systems hold the future of energy production for portable and stationary systems.
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How algae could change our world
https://www.forbes.com/sites/jenniferhicks/2018/06/15/see-how-algae-could-change-our-world/#f6653cc3e466
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Industrial biotechnology and microbial
metabolic engineering are poised to help
meet the growing demand for
sustainable, low-cost commodity
chemicals and natural products, yet the
fraction of biochemicals amenable to
commercial production remains limited.
Common problems afflicting the current
state-of-the-art include low volumetric
productivities, build-up of toxic
intermediates or products, and
byproduct losses via competing
pathways. To overcome these limitations,
cell-free metabolic engineering
(CFME) is expanding the scope of the
traditional bioengineering model by using
in vitro ensembles of catalytic proteins
prepared from purified enzymes or crude
lysates of cells for the production of
target products.
Cell-free metabolic engineering:
biomanufacting beyond the cell
Paradigms for metabolic engineering (A) sample pathway (B) traditional and cell-free approaches.
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New opportunities to use crude cell
lysates for pathway optimization and
debugging.
Cell-free metabolic engineering:
biomanufacturing beyond the cell
http://europepmc.org/articles/pmc4314355
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Metabolomics
Concepts
Methods
Applications
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Flux analysis and metabolomics for metabolic engineering
Metabolomics is the large-scale study of small
molecules, commonly known as metabolites,
within cells, biofluids, tissues or organisms.
Collectively, these small molecules and their
interactions within a biological system are known
as the metabolome.
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Metabolome
• Metabolome refers to the
complete set of small-molecule
(<1.5 kDa) metabolites (such as
metabolic intermediates,
hormones and other signalling
molecules, and secondary
metabolites) to be found within a
biological sample, such as a
single organism.
• Although the metabolome can
be defined readily enough, it is
not currently possible to analyse
the entire range of metabolites
by a single analytical method.
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Metabolomics: methodology
• Gas chromatography-mass spectrometry (GC-MS)
• NMR spectroscopy
41
Understanding cellular metabolism
Applications of various techniques to
understanding and manipulating cellular
metabolism. Solid lines represent widely
used strategies, dashed lines represent
underused strategies. Both metabolomics
and transcriptional profiling provide a
direct readout that helps enable a deeper
understanding of cellular metabolism, but
only transcriptional profiling has seen
widespread application to enhance
standard computational modeling and
metabolic engineering strategies.
Integrating metabolomics data into
metabolic engineering and computational
modeling strategies would help bridge
gaps in biochemical knowledge and
improve our ability to control cellular
metabolism.
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From Metabolite to Metabolite (FMM) is a critical tool for
metabolic engineering
FMM (From Metabolite to Metabolite) is a critical tool for synthetic biology. FMM can reconstruct metabolic
pathways from one metabolite to the other one. The different KEGG maps can be connected by our system. Both
local and global graphical views of the metabolic pathways were designed. Furthermore, metabolic pathways
can be comparative between several species by FMM (Comparative Analysis). In addition, post-translational
modification (PTM) information of enzymes from numerous species is also supplied in FMM.
http://fmm.mbc.nctu.edu.tw/
43
FMM: a software tool for metabolic pathway design
http://fmm.mbc.nctu.edu.tw/
44
Synthetic epigenetics
Concepts
Methods
Applications
45
What is epigenetics?
• Changes in gene expression (turning a gene on) and gene silencing (turning
a gene off), which do not change the underlying DNA sequence, are
collectively referred to as EPIGENETICS.
• Metamorphosis is a perfect example of a power of epigenetic control of gene
expression.
• Organisms have silenced genes and activated genes.
• Genes can be switched on or off rapidly with needs of adaptation.
• These clever mechanisms point to creation and intelligent design.
One genome → multiple structures (phenotypes)
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Epigenetic regulatory mechanisms
47
Chromatin structure: a basic building unit (nucleosome)
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Key epigenetic regulators
49
The histone code
• The histone code is a hypothesis that the transcription of genetic information
encoded in DNA is in part regulated by chemical modifications to histone proteins,
primarily on their unstructured ends.
• Together with similar modifications such as DNA methylation it is part of the
epigenetic code.
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Epigenetic therapy (targeting epigenome)
Epigenetic therapy is the use of
drugs or other epigenome-influencing
techniques to treat medical
conditions. Many diseases, including
cancer, heart disease, diabetes, and
mental illnesses are influenced by
epigenetic mechanisms, and
epigenetic therapy offers a potential
way to influence those pathways
directly.
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Epigenetic therapy (targeting epigenome)
There is a growing awareness that epigenetic
dysregulation plays a significant role in many
types of cancer, and that is fuelling an
increasing number of studies into drugs that
target epigenetic regulators.
Such regulatory proteins fall into three main
categories: writers, readers and erasers. Writers
‘mark’ histones and DNAs by adding chemical
groups, indicating either transcription, replication
or repair. Modifications include acetylation,
phosphorylation and methylation. Readers
recognize and act upon the modifications,
whereas erasers remove them (see ‘The
epigenetic landscape’).
Several drugs that inhibit epigenetic writers and
erasers have been approved by the US Food
and Drug Administration (FDA) for the treatment
of cancer. These include DNA
methyltransferase and histone deacetylase
(HDAC) inhibitors. Further candidates, including
inhibitors of acetyl-lysine readers
(bromodomain-containing proteins), are
undergoing clinical evaluation for efficacy in
different cancer settings.
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Engineering epigenetic regulation using synthetic
read-write modules
• Regulatory networks involving molecular
writers and readers of chromatin marks are
thought to control the epigenetic programs.
• Guided by this common principle, an
orthogonal epigenetic regulatory system in
mammalian cells using N6-methyladenine
(m6A), a DNA modification not commonly
found in metazoan epigenomes, was
established.
• The system utilizes synthetic factors that write
and read m6A and consequently recruit
transcriptional regulators to control reporter
loci.
• Inspired by models of chromatin spreading
and epigenetic inheritance, we used our
system and mathematical models to construct
regulatory circuits that induce m6A-dependent
transcriptional states, promote their spatial
propagation, and maintain epigenetic memory
of the states. These minimal circuits were able
to program epigenetic functions de novo,
conceptually validating “read-write”
architectures.https://www.cell.com/cell/pdf/S0092-8674(18)31461-2.pdf
54
Exploring epigenetic mechanisms and regulations in schistosome parasites
Anti-parasitic drug discovery in epigenetics
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Fine-tuning small-molecule epigenetic inhibitors
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Targeting epigenetic enzymes for drug discovery
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Secondary metabolites are synthesised by biosynthetic
pathways that are encoded by gene clusters
https://academic.oup.com/femsre/advance-article/doi/10.1093/femsre/fuz018/5521207
A) Secondary metabolites (SMs) are
synthesised from few precursors by
biochemical pathways centred around
characteristic core enzymes (red).
Core enzymes often contain multiple
domains or modules that operate
conjointly to support the synthesis of
the precursor. The precursor is usually
further modified by tailoring or
decorating enzymes (orange) to
produce the final active compound,
which is subsequently exported by
transporters (green).
B) In fungi, genes encoding core (red)
and tailoring enzymes (orange) as well
as transporters (green) involved in SM
efflux or self-protection are often
physically inked in the genome,
defining a so-called gene cluster
organization
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Secondary metabolites are synthesised by biosynthetic
pathways that are encoded by gene clusters
https://academic.oup.com/femsre/advance-article/doi/10.1093/femsre/fuz018/5521207
59
Histone post-translational modifications in human, yeast,
and the secondary metabolite producing Aspergillus
https://academic.oup.com/femsre/advance-article/doi/10.1093/femsre/fuz018/5521207
60
How epigenetic machinery influences secondary
metabolism in fungi
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6685777/
Strategies for interfering with chromatin regulation. A) Action of an epigenetic eraser under wild-type
conditions. This enzyme removes the activating modifications represented by the blue dots, which leads to
more condensed, repressed chromatin where BGC are often found. B) Deletion of the eraser prevents the
removal of the activating modifications, and the chromatin remains open and active, allowing for expression
of genes which are typically repressed. C) Adding chemical inhibitors (represented by the light blue
hexagons) which prevent the eraser from removing the activating PTM. This leads to a similar outcome as
deletion of the enzyme, and allows for expression of genes which are typically repressed.
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Synthetic biology in drug discovery
Concepts
Methods
Applications
62
Concepts behind synthetic biology tools in drug discovery
(A) Inducers of gene expression using light or small molecules (nutrient, drugs, cell messengers, etc)
(B) Gene circuit to control expression of specific genes
(C) Reporter genes to control output signals related to a disease phenotype
• With the recent advanced genome editing, molecular biology, and protein engineering
tools, synthetic biology has focused its aim at creating biological devices that can
produce controlled phenotypes from a given input, such as a molecular or light switch.
• The design of genetic circuits in synthetic biology is used in pharmaceutical research
not only for bioproduction of drugs by microorganisms but also to support the different
steps of drug development.
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Synthetic biology tools in various steps of drug discovery
64
Protein engineering for exploring chemical diversity
(A) Modify enzyme specificity by single amino acid mutations in binding site
(B) Use substrate analogs to induce mutation on selected enzyme
(C) Use alternating spliced isoforms to modify arrangement of enzymatic modules
65
Metabolic engineering for drug production
Production of secondary metabolites through heterologous genetic circuits (A) or
by modifying chemical precursor from the milieu with enzyme present in the host
organism (B).
66
Conceptual SB pipeline for drug screening in synthetic cell
(A) A system to induce gene expression of protein target based on small molecule inducer and/or
RNA-based switch for gene expression. (B) A combination of drug target from unique genes. (C) A
genetic oscillator to focus readout on chosen drug target. (D) Generation of diverse drug candidate
libraries from genetic shuffling of enzymatic modules. (E) A logical AND gate that gives output signal
(F) if a drug candidate binds to biological target.
67
Natural products and synthetic biology
Genomic, genetic, and synthetic biology approaches towards
natural product drug discovery
(a) Exploration of bacteria from understudied environments and taxa follows the hypothesis that
different selective pressures may lead to the evolution of distinct chemistry. Genomes are
sequenced and biosynthetic gene clusters (BGCs) are identified using bioinformatic tools
(b) BGCs of interest are selected. Heterologous expression in suitable hosts can be used to streamline
natural product discovery. Genome editing of native producers offers an alternative approach to aid
discovery and to study gene and BGC function
(c) Synthetic biology-driven pathway modification via BGC reprogramming contributes to structure
diversification of natural products
68
With synthetic biology, drug discovery is going virtual
69
Questions
70
Dr. Martin Marek
Loschmidt Laboratories
Faculty of Science, MUNI
Kamenice 5, bld. A13, room 332
martin.marek@recetox.muni.cz
71
Supplementary materials
72
Molecules to metabolism
73
Metabolic engineering approaches for induced
production of L-tryptophan using engineered E. coli
https://www.mdpi.com/2227-9717/7/4/213
74
Different levels of metabolic engineering
• The enzyme solution space describes the number of possible enzymes reactions available for a given
strategy while the pathway solution space corresponds to the number of possible pathways that can be
constructed.
• While level 1, 2 and 3 metabolic engineering efforts do not differ in enzyme solution space, because
they all rely on known enzymes, level 4 and 5 metabolic engineering efforts provide new enzymes
created through enzyme engineering or de novo protein design.
https://www.sciencedirect.com/science/article/pii/S1367593116302071
75
Biological fuel cells (BFCs) and the bio-production
of hydrogen
https://www.fuelcellstore.com
A biological fuel cell (BFC) or microbial fuel cell (MFC) is a type of fuel cell that converts biochemical
energy into electrical energy. Like other types of fuel cells, a biological fuel cell consists of an anode, a
cathode, and a membrane that conducts ions. In the anode compartment, fuel is oxidized by
microorganisms, and the result is protons and electrons. In the cathode compartment, ions are consumed,
and the by-product is water. In BFCs, there is the redox reaction between the carbohydrate substrate (such
as glucose and methanol) and the catalyst - which is a microorganism or enzyme. The biological fuel cell is
illustrated in Figure 1. The main difference between a standard fuel cell and a BFC is the catalyst is a
microorganism or enzyme. Therefore, noble metals are not needed for the catalyst in BFCs. The fuel cell
operates in a liquid media in a near neutral environment and at a low temperature. The potential
applications for biological fuel cells are (1) low power energy sources; (2) sensors based upon direct
electrode interactions; and (3) electrochemical synthesis of chemicals.
76
Metabolomics and systems biology
• Metabolic engineering is a field where the level of a specific metabolite is
quite obviously a very important thing, because that metabolite is the
valuable product that one is trying to produce. If knowing the level of one
metabolite is useful, it stands to reason that knowing the levels of many
metabolites --- immediate precursors and products of the target metabolite,
as well as other related molecules --- should be even more useful.
• Metabolomics is a powerful approach because metabolites and their
concentrations, unlike other "omics" measures, directly reflect the underlying
biochemical activity and state of cells/tissues. Thus metabolomics best
represents the molecular phenotype.
• Metabolomics provides a downstream,
phenotypic snapshot of the cell that can
be critical to understanding metabolism
and metabolic dynamics.
77
Plant metabolic engineering and synthetic biology
Genetic improvement of crops started since the dawn of
agriculture and has continuously evolved in parallel with
emerging technological innovations. The use of genome
engineering in crop improvement has already revolutionised
modern agriculture in less than thirty years. Plant metabolic
engineering is still at a development stage and faces several
challenges, in particular with the time necessary to develop
plant based solutions to bio-industrial demands. However
the recent success of several metabolic engineering
approaches applied to major crops are encouraging and the
emerging field of plant synthetic biology offers new
opportunities. Some pioneering studies have demonstrated
that synthetic genetic circuits or orthogonal metabolic
pathways can be introduced into plants to achieve a desired
function. The combination of metabolic engineering and
synthetic biology is expected to significantly accelerate crop
improvement. A defining aspect of both fields is the
design/build/test/learn cycle, or the use of iterative rounds of
testing modifications to refine hypotheses and develop best
solutions. Several technological and technical improvements
are now available to make a better use of each design,
build, test, and learn components of the cycle. All these
advances should facilitate the rapid development of a wide
variety of bio-products for a world in need of sustainable
solutions.
78
How epigenetic machinery influences secondary
metabolism in fungi
The discovery of chromatin as a central regulator of fungal secondary metabolism (SM)
production significantly impacted on our understanding of the complex transcriptional
regulation of biosynthetic gene clusters. The rapid progress in identifying the genetic
determinants underlying this chromatin-based regulation provided new tools to activate silent
gene clusters. Thus far, most efforts have focused on two well-known chromatin
modifications, histone methylation and histone acetylation. However, most SM gene clusters
remain untouched by any genetic or chemical modification of chromatin, and thus we still lack
a comprehensive understanding on how chromatin modifications regulate SM gene clusters.
Based on examples from other eukaryotes and the availability of novel technologies that allow
to comprehensively study the composition and organisation of chromatin, we are now entering
a new decade during which several outstanding questions can be answered. What is the
contribution of other chromatin modifications to the regulation of SM gene clusters? How do
chromatin modifications interact with each other to provide a tight and subtle regulation of SM
gene clusters? How are chromatin modifications orchestrated in the different genomic subcompartments,
and what is the chromatin dynamic during interactions with other organisms?
Answering these questions is important to advance our basic knowledge on chromatin-based
regulation, but it will also provide the needed tools to successfully activate the wide diversity
of fungal SM gene clusters. Exploitation of the fungal kingdom to the discovery and
development of novel bioactive compounds would then enter a completely new era.
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https://www.ddw-online.com/enabling-technologies/p315003-with-synthetic-biologydrug-discovery-is-going-virtual.html
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How epigenetic machinery influences secondary
metabolism in fungi
Areas of future research. Depiction of a fungal hyphae, which contains two nuclei represented by the ovals.
Each letter represents an area that needs further research relating to chromatin regulation of secondary
metabolism. “a” is labeling the fungus, because despite the extensive research that has occurred within
Aspergillus and Fusarium, very few taxa of fungi have been studied for how their BGC are regulated by
chromatin. “b” marks the bacterial interaction which has been demonstrated to regulate secondary
metabolite production. “c” a zoomed in look in the nucleus, shows chromatin with various purple reading
proteins. This represents an untapped resource of proteins which may be controlling secondary metabolism
through recognition of PTM, and recruitment of writing or erasing enzymes. “d” represents the use of
chemical inhibitors to prevent the actions of the histone modifying enzymes.
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Design of synthetic quorum sensing in microbial consortium
The sender cell synthesizes a messaging molecule
(inducer) that stimulates its receptor synthesized in
the receiver cells. This complex triggers inhibition (or
activation) of a target gene in a cell densitydependent
manner.
Abbreviations: Ind, inducer; Pr, promotor; Precept,
receptor’s promoter.
• Designing of synthetic cell–cell
communication network as a
model to study persistence in
bacteria and how to sensitize
cells or to use it as a
screening platform to target
QS in bacterial communities
• QS is a cell–cell
communication system that
allows bacteria or
microorganisms to
synchronize expression of a
particular gene in a cell
density-dependent manner