RECENT ADVANCES IN

PROTEIN AND METABOLIC

ENGINEERING

SUBMITTED BY –
Farheen Khan
Roll no –19MBS007
MSc. Biosciences
1st year (2nd semester)
PROTEIN ENGINEERING
Protein engineering is the design of new enzymes or proteins with new
or desirable functions. It is based on the use of recombinant DNA
technology to change amino acid sequences. Biochemists can now use a
refinement of the genetic modification technique to redesign proteins.
Once the gene is isolated for a particular protein , its code can be altered
so that a change occurs in the protein’s primary structure. Then the
modified gene is incorporated into a microorganism where it is decoded
as before, but this time a new protein appears. There are various
methods involved in protein engineering but the most classical methods
are –
RATIONAL DESIGN – Rational protein design, a scientist uses
detailed knowledge of the structure and function of a protein to make
desired changes. This has the advantage of being inexpensive and
technically easy, since site directed mutagenesis methods are well-
developed but the drawback is that detailed structural knowledge of a
protein is often unavailable .

DIRECTED EVOLUTION – In directed evolution, random

mutagenesis is applied to a protein, and a selection regime is used to
select variants having desired traits . Further rounds of mutation and
selection are then applied. This method mimics natural evolution and,
in general, produces superior results to rational design . No prior
structural knowledge is required however they require very high
throughput screening which is not feasible for all proteins .

RECENT ADVANCES AND APPLICATIONS

Computer-modeling techniques allow protein chemists to make
predictions about how proposed α-amino acid changes might change the
structure and activity of a protein. Genetic and protein modification
have enormous potential. Besides insulin, genetic modification can
produce human growth hormone and the blood clotting factor VIII .
It is possible now to introduce into a plant new genes that enable it to
produce its own protein insecticide or that make it resistant to disease.
Hepatitis B vaccine, oil-digesting bacteria, and bacteria that
produce biodegradable plastic are all recently developed products of
protein engineering and genetic-modification techniques.
There are various other examples of protein design using genetic
engineering techniques in multiple fields :

 DETERGENT INDUSTRY
 Improving the detergent quality of Subtilisin
Subtilisin is a protease enzyme of bacterial origin with a molecular
size of 27.4 kD. The catalytic activity of subtilisin is due to the
presence of a catalytic triad—ser 221, his 64, and asp 32, used in
the manufacturing of washing powder as an ingredient to improve
the quality of detergents; such detergents are called stain cutters or
stain removers. the problem is that the native form of subtilisin
loses its catalytic activity by 90% in the presence of bleach. The
loss of activity is due to the oxidation of methionine at position 222
of the polypeptide chain. By site-directed mutagenesis, the
methionine at the position 222 of subtilisin molecule was
substituted by a variety of other amino acids and the enzyme
activity of the modified subtilisin was studied in the presence of
detergent to come to a conclusion that substitution of Met222 with
Alanine has imparted stability and activity for the enzyme.
 MEDICAL APPLICATIONS
 Creation of novel proteins
There are some amino acid sequences, which are directly involved
in the stimulation of immunity and that part of the protein or
polypeptide, is known as epitopes. A recombinant vaccine can be
designed by involving only the essential epitopes in the immune
response. Such vaccines will be very simple and will be safe
without reducing their effectiveness. These vaccines have the
advantage of reducing deleterious effects on people unlike
traditional ones .
 Cancer treatment
The use of novel antibodies as anticancer agents is also an
important field of application, where the ability of antibodies to
select antigens specifically and with high affinity is exploited, and
protein engineering methods are used to modify antibodies to
target cancer cells for clinical applications . Recently, the term
“modular protein engineering” has been introduced for
emerging cancer therapies. Treatment strategies based on targeted
nanoconjugates to be specifically directed against target cells are
becoming increasingly important.
  FOOD INDUSTRY
 Improving nutritional value of cereals and legumes
The quality of protein can be improved by the modern approach of
protein engineering through recombinant DNA technology. The
storage protein of cereals and legumes can be modified by
introducing essential amino acids. These genes can be transferred
to food plants to be expressed in the respective storage parts of the
plants such as seeds, tubers, or grains. Modifications in proteins
can be made by introducing new amino acids or by substituting the
existing amino acids with new ones .
 Improvement in enzymes
Some large groups of enzymes like proteases, amylases and
lipases are important for food industry as they have a broad
range of applications. Proteases, for example, are used in several
applications of food industry regarding low allergenic infant
formulas, milk clotting and flavors. The improvement of
proteases , for example, high activity at alkaline pH and low
temperatures, or improved stability at high temperatures is a
challenge for protein engineering .Similarly protein engineering
has been used to improve the properties of amalyses .

METABOLIC ENGINEERING
Metabolic Engineering is the practice of optimizing genetic and
regulatory processes within cells to increase the cells’ production of a
certain substance. These processes are chemical networks that use a
series of biochemical reactions and enzymes that allow cells to convert
raw materials into molecules necessary for the cell’s survival.
Advantages of metabolic engineering have been noticed from cloning
and expression of heterologous genes. These can serve several useful
purpose, including extending an existing pathway to obtain a new
product, creating arrays of enzymatic activities that synthesize a novel
structure, shifting metabolic flow towards a desired product, and
accelerating a rate determining step . Metabolic engineering is
basically meant for the production of chemicals, fuels, pharmaceuticals,
and medicine by altering the metabolic pathways .Most of the metabolic
engineering approaches are based on genetic engineering techniques.
Some of the fundamental requirements for metabolic engineering are
knowledge about :
(1) the biosynthetic pathway of the chemical to be produced,
(2) genes encoding the related enzymes,
(3) regulation of such enzymes, with ability to
(4) transfer and express or suppress the required genes in the host
organism,
(5) mutate the gene in vivo and in vitro to be able to alter properties of
the encoded enzyme, and
(6) assemble an array of genes for their expression inside the host cell.

Although bacteria and yeast are the pioneering hosts for metabolic
engineering, other organisms such as fungi, animal as well as plant cells
are also used nowadays for similar experiments. Metabolic engineering
has become a well-recognized field of biotechnology with huge
commercial potential and it can be immensely beneficial to mankind
offering a biological tool for large scale production of useful chemicals
economically .

ADVANCEMENTS AND APPLICATIONS

 METABOLIC ENGINEERING OF THE CENTRAL
METABOLIC PATHWAYS OF E. COLI
 Strategies for acetate reduction to achieve high recombinant
protein production –
Over the past several years the work in our laboratories has been
focusing on the various aspects of manipulating the central
metabolic pathways in E. coli. Specifically, we are interested in
the effect of precise genetic manipulations on the metabolic
activities of E. coli. A potential application is to address the
problem of acetate accumulation in an E. coli culture. One of the
major technical challenges in recombinant protein production
processes is to achieve high expression level of the cloned gene
and high cell density. Unfortunately, under these demanding
conditions, the amount of acetate accumulated in the reactor
increases enormously, often to a level that has a detrimental effect
 on both cell and protein yields.The goal is to reduce the level of
acetate accumulation through the manipulation of cellular
activities. Several potential points of attack leading to a lower rate
of acetate production can be envisioned upon close inspection of
the pathways involved.

PLANT CELL CULTURE

In recent years metabolic engineering has provided a new
promising avenue for enhancing product formation in a plant or
plant cell culture. Metabolic engineering of plant secondary
metabolite pathways in the production of fine chemicals has been
reported to be a possible approach to increase yields. Several ways
of application of metabolic engineering to improve yields have
been reported. Compartmentation strategy used for alkaloid
biosynthesis in Cantharanthus roseus is one of the classical
examples of the same.
PRODUCTION OF DRUGS AND CHEMICALS
Some of the most successful examples of commercial metabolic
engineering strategies include production of drugs such as
artemesinin peritaxel,overproduction of L-valine (a precursor for
many value-added chemicals), production of amino acids including
glutamic acid in Corynebacterium and production of biofuel related
chemicals such as ethanol, alkanes and fatty acid esters . Nobel
Prize in Physiology or Medicine in 2015 was awarded for the
discovery of artemisinin, a natural product, for the treatment of
malaria. This molecule is now produced at large scale by metabolic
engineering in yeast .
 STRUCTURE OF ARTEMESININ

EXAMPLES OF OTHER SUCCESSFUL METABOLIC

ENGINEERING
 Identifcation of constraints to lysine production in corynebactium
glutamicum and insertion of new genes to relieve these constraints
to improve production.
 Engineering of a new fatty acid biosynthesis pathwayy, called
reversed beta oxidation pathway, that is more efficient than the
native pathway in producing fatty acids and alcohols which can
potentially be catalytically converted to chemicals and fuel
 Improved production of DAHP an aromatic metabolite produced
by E. coli that is an intermediate in the production of aromatic
amino acids .

REFERENCES

 INTRODUCTION TO BIOTECHNOLOGY AND

GENETIC ENGINEERING , A.J. NAIR, PH.D.
 https://en.m.wikipedia.org/wiki/Metabolic_engineering
 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3615475/
 https://en.m.wikipedia.org/wiki/Protein_engineering