Metabolic engineering:

enhanced production
September 4, 2018
 Background
• Men used microbes to produce fermented foods and beverages
• Microbes have been used to produce chemicals
• During World War I, Chaim Weismann developed the acetone-
butanol-ethanol fermentation process
• In the 1920s, fermentation of Aspergillus niger was adapted to
generate citric acid, a food and beverage ingredient.
• During World War II, the same technology was used for industrial
scale production of penicillin, the first pharmaceutical produced by
fermentation
• 1960’s products of pharmaceutical interest: antibiotics, cholesterol
lowering agents, immune suppressants, anti cancer drugs
Metabolic engineering

• Bioinformatics and mathematical models of metabolism

allowed quantitative analysis

• This enabled specific genetic modification altering cellular

metabolism to be introduced, such that fluxes could be directed
towards product of interest.

• Metabolic engineering began in 1990

Metabolic engineering
• Metabolic engineering is defined as the directed improvement
of product formation or cellular properties through the
modifications of specific biochemical reactions or the
introduction of new genes with the help of recombinant DNA
technology.
• It is making cells into efficient factories.
• Challenging because cells have evolved robust metabolic
networks
• Engineering a cell factory involves ‘Design-build-test’ cycle
• Applications: generation of fuels, chemicals, foods, feeds, and
pharmaceuticals
Metabolic engineering

• Two main objectives:

• To increase the titre of the target compound

• To modify the natural product for improved

pharmacological properties
Examples
 Steps involved
• Identification of molecule of interest

• Determination of whether there exists a metabolic pathway in

nature to produce this molecule

• Check if this natural producer cell factory can be used for further
improvement.

• If, on the other hand, the biosynthetic pathway is to be transferred to

a heterologous host, and if all of the enzymes of the biosynthetic
pathway have not yet been identified.

• In some cases, it is difficult to identify all the biosynthetic

enzymes needed to produce a molecule. Eg. Not all the enzymes
involved in biosynthesis of the anti-cancer drug taxol have yet been
identified
E. coli as a model organism for Metabolic
Engineering

• Fast growth rate

• Genetic flexibility
• Metabolism is well understood
• Natural production of organic acids
• Ability to grow on various substrates
• Simple growth media
• Plasticity of the metabolism
• High theoretical yields
Each of the cell factories have specific advantages:

• A. niger and B. subtilis have very efficient protein secretion

and are therefore widely used for production of industrial
enzymes
• CHO (Chinese Hamster Ovary) cells are well suited for
production of glycosylated proteins to be used as
pharmaceuticals
• For fuels and chemicals, there is an increasing focus on use
of S. cerevisiae and E. coli as platform cell factories, with
Corynebacterium glutamicum as an attractive third choice.
Metabolic engineering strategies for
improvement of product titre
• Metabolic engineering for strain improvement
• Increasing the precursor supply
• Biosynthesis in heterologous hosts
• Overexpressing or increasing the efficiency of bottleneck enzymes
• Altering the regulation of gene expression
• Deletion of competing pathways

• Metabolic engineering for structural diversification

• Gene disruption and mutasynthesis
• Pathway engineering and combinatorial biosynthesis
• Protein engineering
Strategies
1. Increasing the precursor supply
• Malonyl CoA important building block for polyketides
biosynthesis
• Its production increased by overexpressing ACCase gene

2. Biosynthesis in heterologous host

• Native producer does not grow well in fermenters
• Long growth periods
• Eg. 70% of microbial antibiotics production in bacteria
Contd…
3. Overexpressing or increasing the efficiency of bottleneck
enzymes
• E.g. glycosylation was found to be rate limiting step in
biosynthesis of anticancer aromatic polyketide doxorubicin.
• Production increased by 6 folds by overexpressing deoxysugar
biosynthesis and glycosyltransfer genes

4. Altering the regulation of gene expression

• Production enhanced by pathways specific regulators
• E.g. streptomyces antibiotic regulatory protein (SARP):
OVEREXPRESSION
Contd…
5. Deletion of competing pathways

• Downregulate or delete competing pathways that may consume

important precursors or intermediates

• Aim is to increase the cell efficiency by directing the cellular

resources towards the desired pathways

• Example: amorphadiene synthase and squalene synthase

compete for precursor farnesyl pyrophosphate (FPP)
Metabolic engineering strategies for
improvement of product titer
Example of Rational metabolic engineering

• Designed overproduction of Phenylalanine

• Key raw material for synthesis of artificial sweetener aspartame
• Chemical synthesis is too expensive
• Overproduction of metabolite possible only by gene knockout
of any feedback inhibition
 Redirecting Metabolite Flow

Directing traffic toward the

desired branch
• Many forks in biochemical
pathways, need to direct flux away
from competing pathways

• Example: Production of threonine

by Brevibacterium lactofermentum.
Cloned homoserine dehydrogenase
(HD), homoserine kinase (HK), and
phosphoenolpyruvate carboxylase
(PEPCase) into a strain lacking
feedback inhibition from threonine.
Redirecting Metabolite Flow
• Reducing competition for a limiting resource
• Cells have a limited number of ribosomes, can limit production of
desired peptides
• A cloned mutant 16S ribosomal RNA makes ribosomes that only
translate mRNA with a certain Shine-Dalgarno sequence mutation.
• This method separates translation of heterologous transcripts from
native transcripts, improving yield of these products.
• Revising metabolic regulation
• Can upregulate biosynthetic genes to improve yields
• Example: yeast with maltose permease and maltase with
constitutively active promoters to overcome glucose repression,
allowing for faster CO2 production in bread baking.
Improving strain performance
• Most challenging part: Moving from a proof-of-principle strain to
a production strain that meets industrial TRY (titre, rate and
yield)
• The main reason for the long development time is the need to go
through many rounds of strain construction and subsequent
phenotypic characterization.
• Most strains used for industrial production require a large number
of genetic modifications, not only in the pathways of interest, but
also in other pathways in order to efficiently redirect metabolic
flux.
Production of most molecules of interest often requires several
enzymes, and the expression of the genes encoding these enzymes must
be coordinated.
There are many ways to coordinate expression of multiple genes:
1. Use different inducible promoters for each gene;
2. Use the same inducible promoter for each gene but vary the
promoter strength;
3. Use a non-native RNA polymerase or transcription factor to
control the expression of more than one gene;
4. Group multiple, related genes into operons (and use internal
ribosomal entry sequences in eukaryotes;
5. Vary the ribosome binding strength for the enzymes encoded in the
gene
 L-Alanine in E. coli
• L-Alanine is produced commertially by an enzymatic decarboxylation of L-
aspartic acid
• World demand is on the order of 500 tons/year
• L-Alanine is used as a nutrition and food additive
• L-Alaninie can be produced from pyruvate by some organisms: A.
oxydans, B. sphaericus, G. stearothermophilus etc.

In this study:
• Lactate overproducer harboring the following mutations (pflB, frdBC,
adhE, and ackA) was used to produce L-Alanine
• Replaced ldhA with the alaD (alanine dehydrogenase) gene from
Geobactillus stearothermophilus.

Appl Microbiol Biotechnol. 2007

Nov;77(2):355-66.
 L-Alanine in E. coli

• Removal of ldhA (lactate

dehydrogenase) resulted in
availability of pyruvate for L-
Alanine production

• alaD (homologous; on a
plasmid) reaction is co-factor
coupled because it uses
NADH

• Knocked out mgsA gene to

eliminate lactate production

• Knocked out dadX to

improve chiral purity of L-
Alanine
Artemisinic acid in E. coli
• Artemisinic acid is a precursor of Artemisinin
• Artemisinin – a drug used to treat malaria
• Isolated from plant: Artemisia annua
• Cost to produce is $2.40/dose – TOO expensive for
developing countries—need $0.25/dose

• In these studies:
• Mevalonate pathway from S. cerevisiae was introduced
in E.coli
• Cytochrome p450 from A. annua was introduced in E.
coli in order to carry out the oxidation to artemisinic
acid in vivo
 Artemisinic acid in E. coli
•Eukaryotic and plant biochemical pathways were introduced
into E. coli
S. A.
cerevisia annua
e

Nat Biotechnol. 2003 Jul;21(7):796-802.

Artemisinic acid in E. coli
• Engineering successful
amorphadiene producing E.
coli took over 3 years
• Over a million fold increase
in production was observed
• High-throughput data
together with traditional
techniques (pathway
overexpression) were used to
successfully engineer this
strain

ACS Chem Biol. 2008 Jan 18;3(1):64-76.

Nat Chem Biol. 2007 May;3(5):274-7