This document discusses metabolic engineering and summarizes key points about manipulating metabolic pathways in organisms. Metabolic engineering involves genetically modifying organisms to modulate their metabolism and produce desired products. It can be done by directly manipulating genes encoding enzymes in pathways or indirectly altering regulatory pathways. The document outlines several approaches to metabolic engineering, including overexpressing rate-limiting enzymes, inhibiting competing pathways, and expressing heterologous genes from other organisms. It also summarizes applications of metabolic engineering for producing amino acids, antibiotics, and manipulating plant metabolism to create foods with improved nutrients or biofuel properties.

1. Dr Soniya Kasliwal

3. INTRODUCTION
Metabolic engineering, as first defined by Baily in 1981 , is “the
improvement of cellular activities by manipulating enzymatic,
regulatory, and transport functions of the cell with the use of
recombinant DNA technology.”
Metabolic engineering is a process for modulating the metabolism of
the organisms so as to produce the required amounts of the
desired metabolite through genetic manipulations.
Metabolic engineering is not only widely applied in industrial
fermentation for strain improvement and metabolite overproduction,
but has also found many important applications in functional
genomics, biological research (e.g., signal transduction),and
medical research (e.g., drug discovery and gene therapy)

4. WAYS TO ENGINEER METABOLIC PATHWAYS:
Directly by manipulating the genes encoding
the enzymes catalyzing the reactions in the
pathways
Indirectly by altering the regulatory
pathways affecting gene expression and
enzyme activity

5. Setting up a
metabolic pathway
for analysis
Analyzing a
metabolic pathway
Determining the
optimal genetic
manipulations
Experimental
measurements
Metabolic
flux analysis

6. Setting up a metabolic pathway for analysis
The first step in the process is to identify a desired goal to achieve through
the improvement or modification of an organism's metabolism. Reference
books and online databases are used to research reactions and
metabolic pathways that are able to produce this product or result.
Analyzing a metabolic pathway
The completed metabolic pathway is modeled mathematically to find the
theoretical yield of the product or the reaction fluxes in the cell. A flux is
the rate at which a given reaction in the network occurs.
Determining the optimal genetic manipulations
After solving for the fluxes of reactions in the network, it is necessary to
determine which reactions may be altered in order to maximize the yield
of the desired product. To determine what specific genetic manipulations
to perform, it is necessary to use computational algorithms, such as
OptGene or OptFlux.
Experimental measurements
In order to create a solvable model, it is often necessary to have certain
fluxes already known or experimentally measured. In addition, in order to
verify the effect of genetic manipulations on the metabolic network (to
ensure they align with the model), it is necessary to experimentally
measure the fluxes in the network. To measure reaction fluxes, carbon
flux measurements are made using carbon-13 isotopic labeling.

7. Fundamental requirements for metabolic
engineering
1. The biosynthetic pathway of the chemical to be produced,
2. Genes encoding the related enzymes,
3. Regulation of such enzymes, with ability to transfer and
express or suppress the required genes in the host
organism.
4. Mutate the gene in vivo and in vitro to be able to alter
properties of the encoded enzyme, and
5. Assemble an array of genes for their expression inside the
host cell.

8. DIFFERENT APPROACHES OF METABOLIC ENGINEERING
1. One of the most obvious approaches is over expressing the gene
encoding the rate-limiting enzyme of the biosynthetic pathway of the
desired end-product.
Example : the vitamin E content of Arabidopsis (a
model plant system) has been increased by over expression of a gene
encoding the enzyme γ tocopherolmethyltransferase.
(2) Another way is to inhibit the competing metabolic reactions which involve
the same substrate. In this way, the substrate is metabolically channeled
specifically towards the desired chemical.
Example: the production of 1,2-propanediol is increased, (which is
mainly used for production of biodegradable polymers) by inhibiting the
lactate dehydrogenase and glyoxylase genes which encode for the
competing enzymes.

10. (3) The third strategy is , the production of the desired biochemical can
be carried out in the non-native organism, i.e., heterologous host. In
this method , a gene can be isolated from the organism which
naturally produces the desired biochemical and can be expressed in
another organism which might be easier to cultivate than the host
organism.
In such a case, an important factor is the availability of the
substrate of the desired pathway. Thus, multiple genes encoding an
array of the enzymes of a pathway can be expressed in the non-
native host.

11. Example: the production of biofuel molecules – fatty acid ethyl
esters – in E. coli by expressing the genes encoding the successive
enzymes of the pathway which are obtained from various sources such as
plants and bacteria.
(4) The fourth approach is to engineer an enzyme which is not found in
nature. This mode relies on creating mutations in the related gene so that
the amino acid composition of the enzyme is altered. This might result in
alteration in the substrate and product specificities of the enzymes, as
they are dependent on the amino acid composition and sequence.
Example : Such a method has been used for the production of a
nonnatural amino acid L-homoalanine, (which is an important precursor of
the many drugs), by creating rational mutations in glutamate
dehydrogenase enzyme in E. coli .

12.  The application of metabolic engineering to industrial biotechnology
has mainly focused on improving cell metabolism to increase
productivity – higher product yield, production rate, and cell growth
efficiency (energy efficiency), and to eliminate or reduce undesirable
byproducts.
 It has also been applied to eliminate or reduce feedback inhibition.
By recruiting heterologous activities ,a partial pathway in an organism
can be completed for the production of the desirable product.
 Also, hybrid or new metabolic pathways can be developed to
produce new products, extend substrate range, or enhance
processing characteristics.
APPLICATIONS :

13. AMINO ACIDS
Amino acids are the basic building blocks of proteins and have many important
applications in food and biomedical industries. The essential amino acids,
such as lysine, methionine, threonine, and phenylalanine are important
nutritional supplement in animal feed. MSG (monosodium glutamate) is
used as a flavoring agent, and tryptophan is an important pharmaceutical
ingredient.
Since the discovery of glutamate-overproducing Corynebacterium glutamicum
in 1957, traditional strain development and, more recently, metabolic
engineering have been widely studied and applied to enhance the
production of various L-amino acids from sugars by fermentation.
Today, except for methionine and a few others, many important amino acids
are commercially produced from sugars by microbial fermentation in large
quantities with high product titers and yields .

16.  To overproduce amino acids in the aspartate group, a change in the
metabolic flux resulting from mutations affecting the repression or
feedback inhibition of the key biosynthetic enzymes is usually
required .
For example, lysine synthesis in C. glutamicum is controlled
by aspartate kinase (AsK), which is allosterically inhibited by lysine
and threonine; this feedback control is eliminated by mutations in the
β-subunit of the kinase or relieved due to a low threonine
concentration resulting from low homoserine dehydrogenase (HDH)
activity.
 Threonine synthesis in corynebacteria is regulated by the activity of
HDH, which is strongly inhibited by threonine. In addition, the genes
coding for homoserine dehydrogenase (hom)and homoserine kinase
(thrB) are on the same operon, which is repressed by methionine .

17.  Because corynebacteria do not convert homoserine to threonine
effectively or secrete threonine well, efforts to breed a
corynebacterial threonine producer have not been assuccessful as
those with E. coli, which has threonine-insensitive AsK and HDH.
 The diversity of the anaplerotic enzymes present in C. glutamicum
exert a complicated control over carbon flux that affects the
biosynthesis of lysine. The biosynthesis of methionine in
microorganisms requires energy (ATP) and is highly regulated.
Consequently, no methionine overproducing microorganism is
available for industrial fermentation, and methionine is currently
produced either by chemical synthesis or by hydrolyzing proteins .

18. Engineering biosynthetic pathways
Amplification of the gene coding for the rate-limiting enzyme in the biosynthetic pathway to
eliminate the bottleneck and thus increase the amino acid production.
 Amplification of the gene coding for the branch-point enzyme to redirect metabolic flow
from the common intermediate to another amino acid.
 Introducing heterologous enzymes with different control architectures allowing them to
bypass the regulatory step (e.g., feedback inhibition or repression) in the biosynthetic
pathway.
 Introducing heterologous enzymes with different catalytic mechanisms that are
functionally or energetically advantageous.
 Amplification of the first enzyme in the pathway diverging from the central metabolism to
increase carbon flow down the biosynthetic pathway.
Engineering central metabolism
Central metabolism supplies precursors and energy that may limit amino acid biosynthesis in
overproducing strains whose bottlenecks in the terminal pathways have been removed.
Engineering the central metabolism is complicated because it is subjected to extensive and
global regulation, which is still not fully understood. Deletion of phosphoenolpyruvate
carboxykinase has been shown to lead to increased threonine productivity by more than
40% in E. coli model strains.

19. Transport engineering
Mutant strains with altered transport systems can maintain a low intracellular level of the
product amino acid and thus are not subject to feedback control; this has been shown to
increase product yield in tryptophan and threonine fermentations with C. glutamicum and
E.coli .
Whole-cell engineering
An effective new metabolic engineering approach is to use data from functional genomics
to carry out whole-cell engineering and genomic breeding, which improves strains by
removing the unwanted mutations acquired during numerous mutation steps .
The minimally mutated strains obtained in “genomic breeding” by only introducing
beneficial point mutations into the wild-type C. glutamicum genome resulted in
remarkable lysine titers and productivity.

20. ANTIBIOTICS
 β-lactam antibiotics such as penicillins are among the oldest and largest
industrial products produced by fermentation .
 Recombinant DNA technology can provide direct and more efficient
approaches to strain improvement, and has recently been extensively studied
and used to further improve the production of β-lactam antibiotics, including
penicillins, cephalosporins, and cephamycins.
 For example, penicillin production in P. chrysogenum was increased
significantly when additional copies of the pcbC (Isopenicillin N synthase) and
penDE genes (Acyl-coenzyme A:6-aminopenicillanic-acid-acyltransferase
PenDE )were introduced.
 cephalosporin C production in A. chrysogenum was increased 2 to 3-fold by
overexpressing the cefG gene alone.

22. METABOLIC ENGINEERING IN PLANTS
PLANTS: THE NATURAL FACTORIES
 Production of fine chemicals, pharmaceuticals, and industrial
products
 Bioremediation
 Man has also known how to grow, harvest, and process plant
tissues since the beginning of modern agricultural practice.
 Plants contain an amazing diversity of genes encoding enzymes
for thousands of distinct biochemical reactions.
 Plant cells are highly compartmentalized, facilitating,

25. Golden Rice: Metabolically engineered plants.
Nutrient content of a crop plant can be improved by metabolic
engineering.Rice, maize, .wheat, tomatoes and other vegetables are
breaded to enhance the level of certain nutrientsAddition of β-
carotene pathway to rice to yield rice having higher levels of Vitamin
A .

26.  Broccoli :
Conventional plant breeding led to the beneforte strain of broccoli.
Higher levels of glucosinolate glucoraphanin. Early studies have
demonstrated that human consumption of high glucoraphanin
broccoli results in improved metabolism and reduced level of fatty
acids. In the lipid compound associated with inflammation.

28. Lechner et al.2017

29. PLANT METABOLIC ENGINEERING
 Identification of a mutant gene
 Knocking out gene function
 Interfering with protein function using specific inhibitors or antibodies.
The expression of genes in plants or plant cells is dependent on several
factors that include:
1. Method to introduce genes into the plant.
2. Promoters
3. Source for the gene encoding the enzyme of interest.
CHOICE OF PLANT SYSTEM FOR METABOLIC ENGINEERING
 Whole Plants
 Plant Cells in Culture Cell
 Using Plant Genes in Microorganisms

30. STRATEGIES FOR INCREASING FLUX OF EXISTING
PATHWAYS
Manipulating the Activity of ‘‘Rate Limiting’’ Steps
Metabolic Flux Analysis and Modeling : Metabolic flux is the rate at
which the material is processed through a specific pathway.
Expression of Multiple Genes in Plants : Progress and Limitations
as exemplified by the attempts to increase the levels of
monoterpenoid alkaloid biosynthesis by coexpressing tryptophan
decarboxylase and strictosidine synthase.
Targeting Entire Pathways with Transcription Factors
Anthocyanin accumulation is regulated in many plant species by the
concerted action of transcription factors corresponding
to the MYB and bHLH families.

31. MAKING NEW COMPOUNDS IN PLANTS
Plants as a Source of Industrial Materials
 Polyhydroxyalkanoates (PHA) in plants. These polyesters of 3-
hydroxyacids have unique biodegradable and elastomeric properties.
 Starch
Plants as Pharmaceutical Factories
Plants as Nutraceutical Factories
1. Levels of provitamin A carotenoids in food staples such as corn,
wheat, and rice by metabolic engineering.
2. A similar success was recently reported in the manipulation of
folates (including tetrahydrofolate) in tomato fruits

32. Heterologous reconstitution of plant metabolic pathways

33.  Opioids such as thebaine, codeine and morphine are widely used around the globe to treat pain . Currently,
farming of opium poppies and isolation of opiates from the poppy latex is the only commercial source of these
compounds. However, in a recent study, yeast (S. cerevi-siea) was engineered to produce the opiates thebaine
and hydrocodone de novo from an exogenous sugarcarbon source . The resulting strains expressed 21 genes
for thebaine production and 23 genes for hydro-codone production. While yields were low (<1 mg/L), this study
provides a dramatic proof-of-principle that complex opiates can be produced in yeast.
HETEROLOGOUS RECONSTITUTION OF PLANT METABOLIC PATHWAYS

34. Triterpenes in N.tabacum plants
Linear, branch-chained triterpenes that are generated by the green alga
Botryococcus braunii are increasingly recognized as important chemical
and biofuel feedstocks. triterpene botryococcene were produced in N.
tabacum plants by the overexpression of an avian farnesyl diphosphate
synthase along with two versions of botryo-coccene synthases in the
chloroplast . High yields of methylated botryococcene derivatives could
also be obtained when triterpene methyltransferases wereexpressed in
the chloroplast.

35. ENGINEERING PLANT PATHWAYS TO CREATE
BETTER BIOFUELS

36.  A key enzyme in lignin biosynthesis, cinammoyl-CoA
reductase(CCR), which catalyzes the conversion of
hydroxycinnamoyl-CoA esters to the corresponding aldehydes.Field
trials on poplar plants have shown that biomass from transgenic
plants with downregulation of CCR is more easily processed to
production of bioethanol
 In another attempt enzyme monolignol ferulate transferase (MFT)
was targeted. The introduction of MTF into transgenic poplar plants
alters the pool of monolignols, with an increase of monolignol
ferulate conjugates. Since the ferulate conjugates are capable of
introducing readily cleavable ester bonds into the lignin backbone
without affecting the plant development lignification process.
 A second strategy to improve access to biofuels is to increase the
content of TAGs in plant vegetative tissues A variety of genes that
enhance TAG accumulation levels have been identified: the
transcription factor WRIN-KLED1, the TAG biosynthetic gene
diacylglycerol acyl-transferase1-2 (DGAT1-2) and a gene encoding
a structural protein oleosin1 (OLE1) that impacts oil body formation.

37. ENGINEERING NEW TRAITS INTO CROPS BY
ENGINEERING PLANT METABOLISM
1. Phenylpropanoids are plant metabolites that act as antioxidant
agents, and therefore have essential health promoting properties
phenylpropanoid production was substantially upregulated in tomato
fruits by introducingfruit-specific expression of the A. thaliana
transcription4 factor AtMYB12 .
2. AtMYB12 increases phenylpropanoid levels by transcriptionally
activating the biosynthetic genes of these pathways.
3. This transcription factor also appears to direct carbon flux towards
aromatic amino acid biosynthesis, which in turn increases the supply
of substrate for phenylpropanoid metabolism.

38. THE NEXT GENERATION OF ENGINEERING
PLANT METABOLIC PATHWAYS
Additionally, plants can be gene-edited with constructs that are
composed exclusively of DNA sequence derived from the same or
similar plant species . These so-called cisgenic plants, which could in
principle be obtained through standard breeding practices, may be
more readily accepted by the public and regulatory bodies.