Introduction

Owing to the recombinant DNA technology, it is now possible to modify the amino
acid sequence of a protein, including substitution, deletion, or insertion of amino
acid residues or to fuse two or more proteins to form hybrid proteins, or even to totally
synthesize genes for new proteins. Thus the crucial question is no longer how
to construct new protein molecules but how to design proteins with desired functions.
What is Protein engineering ?
Protein engineering is the process of developing useful or valuable proteins. Quite
new to the world with much research taking place into the understanding of protein
folding and recognition for protein design principles. Its main objective is to develop
new protein with improved properties.
Strategies
There are two general strategies for protein
engineering rational protein design and directed
evolution. Both of them are not mutually
exclusive . But researchers often apply them
both . With more detailed knowledge of protein
structure and function in the future, the scope
for protein engineering will certainly increase.
Eventually, even unnatural amino acids may be
included, via newer methods, such as expanded
genetic code , that allow encoding novel amino
acids in genetic code.
The two approaches
Rational Design
• In 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,
and, even when available, it can be very difficult
to predict the effects of various mutations since
structural information most often provide a
static picture of a protein structure
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 .
• The drawback is that they require very high
throughput screening which is not feasible for
all proteins .
 Recent applications
• The enzymes used in food industry were
emphasized as an important group of enzymes, the
industrially important properties of which could be
further improved by protein engineering
• An important application area of protein engineering
regarding food industry is the wheat gluten proteins.
Their heterologous expression and protein engineering
has been studied using a variety of expression systems,
such as E.coli, yeasts or cultured insect cells .
• Some enzymes like Proteases, Amylases and Lipases
have wide range of applications in food and detergent
industry .
• Proteases are used in allergenic infant formulas, milk
clotting and flavours and Amalyses are used for
liquefaction and saccharification of starch
• Lipases are used for stability and condition dough and
used in detergent industry to remove lipid stains .
• Medical applications of protein engineering are also
diverse. The use of protein engineering for cancer
treatment studies is a major area of interest. Pretargeted
radioimmunotherapy has been discussed as a potential
cancer treatment advances in protein engineering and
recombinant DNA technology were expected to
increase the use of pretargeted radioimmunotherapy.
• Multifunctional and smart drug vehicles can be
produced at the nanoscale, by protein engineering.
These strategies could be combined to identify and
select targets for protein-based drug delivery
• Protein engineering applications with antibodies are
also diverse. Owing to advances possible recombinant
DNA technology, “antibody engineering” is possible
• Recently, the term “modular protein engineering” has
been introduced for emerging cancer therapies.
Treatment strategies based on targetd nanoconjugatess
to be specifically directed against target cells are
becoming increasingly important.
Other advancements
• Protein engineering applications for biopolymer production are also promising. Particularly, peptides are becoming
increasingly important as biomaterials because of their specific physical, chemical and biological properties. Protein
engineering and macromolecular self-assembly are utilized to produce peptide-based biomaterials, such as elastin-like
polypeptides, silk-like polymers, etc.

• “Virus engineering” is another emerging field, where the virus particles are modified by protein engineering. Viruses have
many promising applications in medicine, biotechnology and nanotechnology. They could be used as delivery vaccines, gene
therapy and targeted drug .Delivery vectors, molecular imaging agents and as building blocks for electronic nanodevices or
nanomaterials construction

• Protein engineering, isolation and study of new extremophilic microorganisms, genetic engineering developments are all
promising advances to develop new biocatalysts for petroleum refining.

• The ability of protein engineering to create and improve protein domains can be utilized for
producing new biomaterials for medical and engineering applications. One such example is
the use of protein engineering to make new protein and peptide domains which enable
advanced functional hydrogel formation .

• Protein Engineering of redox active enzymes pointed out two emerging areas of protein engineering : novel nucleic acid
based catalyst construction and intra molecular electron transfer remodelling
Examples of engineered proteins

• Computing methods have been used to

design a protein with a novel fold,
named Top7 ,and sensors for unnatural
molecules
• Top7 is an artificial 93 residue protein,
classified as a de novo protein since it
was designed  by Brian Kuhlman and
Gautam Dantas to have a unique fold not
found in nature
• The structure consists of two alpha
helices packed on a five-stranded anti-
parallel beta sheet .
•  The engineering of fusion protein has
yielded rilonacept a pharmaceutical that has
secured FDA approval for treating cryopyrin-
associated periodic syndrome .

• Rilonacept is a dimeric fusion protein

consisting of the ligand-binding domains of
the extracellular portions of the human
interleukin-1 receptor component (IL-1R1)
and IL-1 receptor accessory protein (IL-
1RAcP) linked in-line to the fragment-
crystallizable portion of human IgG1 that
binds and neutralizes IL- 1.
Metabolic Engineering
• Metabolic engineering is basically meant for the production of chemicals, fuels,
pharmaceuticals, and medicine by altering the metabolic pathways. This method involves
useful alteration of metabolic pathways to better understand and utilize the cellular
pathways. Metabolic engineering is motivated by commercial applications by which we
can improve the developing strains for production of useful metabolites.
• It 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. Metabolic engineering
specifically seeks to mathematically model these networks, calculate a yield of useful
products, and pin point parts of the network that constrain the production of these
products .
Objective of metabolic engineering
• The goal of metabolic engineering is to be able to use organisms to produce
valuable substances on an industrial scale in a cost-effective manner. Current
examples including producing beer,wine, cheese, pharma, and other
biotechnology  products .
Some of the common strategies used for metabolic engineering are :
• Overexpressing the gene encoding the rate-limiting enzyme of the biosynthetic
pathway
• blocking the competing metabolic pathways,
• heterologous gene expression, and
• enzyme engineering
Recent advancements and applications
• In the past, to increase the productivity of a desired metabolite , a microorganism  was genetically modified by
chemically induced mutation , and the mutant strain that overexpressed the desired metabolite was then chosen. But
the problem with this technique was that the metabolic pathway for the production of that metabolite was not
analyzed, and as a result, the constraints to production and relevant pathway enzymes to be modified were unknown
• Later in the 1990s a new technique called metabolic engineering emerged. This technique analyzes the metabolic
pathway of a microorganism and determines the constraints and their effects on the production of desired
compounds .
• Examples of successful metabolic engineering are the following: (i) Identification of constraints to lysine
production in corynebactium glutamicumm and insertion of new genes to relieve these constraints to improve
production 
• (ii) 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
• (iii) Improved production of DAHP an aromatic metabolite produced by E. coli that is an intermediate in the
production of aromatic amino acids .
• In more 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.
Examples
• 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 .

• The Nobel Prize in Physiology or Medicine in

2015 was awarded for the discovery of
artemisinin, a natural product, for the treat-
ment of malaria. This molecule is now
produced at large scale by metabolic
engineering in yeast .
Future prospects for metabolic engineering
• Manufacturing molecules through metabolic engineering. The potential of metabolic engineering – one of
the principal techniques of modern biotechnology for the microbial production of many of the chemicals
that are currently derived from non-renewable resources or limited natural resources. Examples are the
replacement of gasoline and other transportation fuels with clean, green and renewable biofuels.

• Metabolic engineering continues to evolve in efficiency and processes aided by breakthroughs in the field
of synthetic biology and progress in understanding metabolite damage and repair. Early metabolic
engineering experiments showed that accumulation of reactive intermediates can limit flux in engineered
pathways and be deleterious to host cells if matching damage control systems are missing or
inadequate.Researchers in synthetic biology optimize genetic pathways, which in turn influence cellular
metabolic outputs. Recent decreases in cost of synthesized DNA and developments in genetic circuits help
to influence the ability of metabolic engineering to produce desired outputs.

•
In a nutshell, metabolic engineering can be immensely beneficial to mankind as well as nature by offering
a biological tool for large-scale production of useful chemicals in an economical manner.
Reference
•https://en.m.wikipedia.org/wiki/Protein_engineering

•https://en.m.wikipedia.org/wiki/Metabolic_engineering

•https://www.researchgate.net/publication/221925539_Protein_Engineering_Methods_an
d_Applications
Thank you .