Fermentation
biotechnology
Submitted to:
Sir Tahir A.
baig
Role of
genetic
engineering
in
fermentation
Role of genetic engineering in fermentation
Shizza Fatima
Atta-ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences and Technology
(NUST) H-12 sector, Islamabad
Submitted: 19th November, 2013
Abstract:
The twentieth century is discernible as era of genomics. Modifying the organism at genetic level
commonly known as genetic engineering is being practices everywhere. Recombinant DNA
technology has taken over pharmaceutical, food; chemical etc. industries. In this document
various modified yeast strains with new and modified functions have been elucidated.
Biotechnology:
Biotechnology is exploitation of living
organisms and their components for the
benefit of mankind. It involves the
genetically modified organisms to improve
the
efficacy
of
its
functioning.
Pharmaceutical companies, chemical and
food industries take advantages of
advances in molecular genetics. As microorganisms are involved in fermentation
therefore biotechnology is an important
part of fermentation.
Fermentation:
Recombinant DNA technology is a common
practice nowadays. The gene of interest
unified in a vector is inert itself unless
introduced in a production system. In order
to be over and above a laboratory
application it is made available to the
society at large by incorporating it in a
production system. This process is called
fermentation that is crucial to economic
feat for centuries.
Fermentation industries:
As iterated earlier there are three basic
fermentation processes. Micro-organisms
were for the first time subjugated by food
manufacturers. Middle of 16th century
archives define cultured methods of this
process. In the beginning 20th century,
chemical industry commenced its use to
yield organic products and enzymes e.g.
amylase
Link
between
fermentation:
genetics
and
Genetics is tightly linked to fermentation
technology; subsequent search of a
appropriate species is the leading stage in
evolving this process. Till recently,
geneticists kept a look out for the microorganisms that yielded desired products.
However genetic manipulation involving
recombinant
technology
made
the
production of products, beyond the one
required, feasible. i
Pharmaceutical industries have shown
much progress, where bacteria produce
insulin, growth hormone, interferon,
thymosin a-1, and somatostatin by the
transference of human gene. When the
species have been allocated, they undergo
mutation. Then the most desirable mutants
are selected.ii These approaches usually
include cut and try.
Searching and discovering the new microorganism for desired traits and the genetic
manipulation has gained so importance that
geneticist
are
important
part
of
fermentation industry.
Genetic Engineering Escherichia coli
for Ethanol production:
Sugars are converted into pyruvate, inside
the cell during the process of glycolysis. ATP
and NADH are also produced. Occurrence of
oxidative phosphorylation in cells lacking
electron transport chain, 95% of the
pyruvate is used, for NAD+ production, in
short pathways. This step is important for
continued glycolysis. Yields obtained during
fermentation
are
the
fermentation
iii
products. Under conditions of oxygen
insufficiency, lactic acid is formed in the
muscle tissues of mammals, while in roots
of plants and in certain fishes ethanol is the
main product.
NADH- oxidizing system consists of two
major enzyme components; Pyruvate
decarboxylase that converts pyruvate to
acetaldehyde and carbon dioxide. Two
alcohol
dehydrogenases
convert
acetaldehyde to ethanol in fermentation,
complemented by oxidation of NADH to
NAD+. Zymomonas mobilis consists of this
enzyme complex. So the genes encoding
this complex are transferred from Z. mobilis
to Escherichia coli. E. coli has relatively high
expression level of this genes thereby
resulting in increased yield of ethanol. iv
Metabolic engineering of yests for
better fermentation of pentoses
A small number of yeasts are identified to
convert xylose to ethanol. Yet, amounts and
produces need to be enhanced for
commercialization.
Lignocellulosic
hydrolysates contain large amounts of
glucose that suppress the use of xylose.
Therefore the yeasts should be altered for
glucose regulation and enhanced xylose
uptake. Uptake of xylose requires low
quantity of oxygen for ideal manufacture.
As Respiration decreases ethanol yields,
accordingly the part that oxygen plays need
to be comprehended. Genetic engineering
is in use to increase the yield.v
In lignocellulosic hydrolysate the content of
xylose is second abundant one. Therefore
xylose needs to be fermented if
bioconversion of lignocelluloses to fuels is
required. Only a few yeasts convert xylose
into ethanol. Wild-type Saccharomyces
cerevisiae is not capable of fermenting
xylose to ethanol. So a recombinant variety
was made by shuffling of genome of P.
stipitis with S. cerevisiae.
Genetic
Engineering
and
Fermentation Financial side
In current ages, highly impressive feats have
been achieved utilizing molecular biology.vi
These include prokaryotic and eukaryotic
exchange of genetic substantial, alteration
of cell regulator so as to allow replication
and genetic expression, and ultimately the
proteins produce.
Explicit goals of brewer’s yeast:
Dextrins Fermentation:
Dextrins, which signify about 25% of
molasses, cannot be utilized by yeasts. The
dextrins contribute towards the calorific
content
of
beer.
Expression
of
amyloglucosidases gene that is capable to
hydrolyze α-1, 6 links is taken from A.niger
or A.awamori viithese genes are then
inserted into yeast strain. This procedure
will ultimately result into high production of
enzyme and enhanced degradation of
dextrin. On a trial bases, high quality beer is
produced by enzyme secreted A nigemer.
Removal of Diacetyl:
During the biosynthesis of valine αacetolactate, a transitional product is
released outside the yeast cell where it
undergoes oxidation and fashions diacetyl.
Acetoin and to 2, 3-butanediol are
produced by reduction of diacetyl inside the
yeast cell. Extensive maturation is required
for the removal of diacetylviii (1–3 weeks).
As diacetyl imparts unfavorable aroma to
the beer therefore its removal is necessary.
It is removed by engineering valine
biosynthetic pathway. Unconventionally
acetolactate decarboxylase gene of
Enterobacter aerogenes can be introduced
in brewer’s yeast.
Reduced assembly of hydrogen sulfide
Reduced
sulfates
produced
during
methionine biosynthetic pathway forms
hydrogen sulfide in S. cerevisiae. The
organoleptic characters of beer are
adversely altered by small quantities of
hydrogen sulfide. Hence its elimination
becomes a necessity. The enhanced
expression of cystathionine β-synthase
encoded by NHS5 in brewing yeastix is
elucidated to destroy or lessen its
production, thereby not altering the specific
characters of the beer.
Enhancement of characters of
wine yeast:
Fermentation of Malolactic (MLF):
MLF is
a
method
in winemaking in
which acerbic malic acid; naturally existent
in grape is altered to softer-tasting lactic
acid. Growth of lactic acid producing
bacteria in wine is not very much effective
therefore under these circumstances MLF
sustenance is defective. Malolactic enzyme
forms lactate by malate decarboxylation,
this process known as secondary
fermentation is crucial for the acidification
and maintenance of wine. Wine changes
due to hindered malolactic fermentation
result in varied harms of fermenter.
In order to cope up with these problems
xS.cerevisiae is incorporated with the
malolactic gene derived from Lactococcus
lactis. This strain produces lactic acid and
carbon dioxide by complete degradation of
malolactate.
Lactic acid production:
The precise equilibrium concerning sugar
and acidity is of utmost importance. In
some hot regions, grapes are frequently
inadequately acidic. Conventionally tartaric
acid is added to acidify the wine. However
biological acidification is done by using one
of the two genes of NADH oxidizing
complex (LDH) obtained from Lactobacillus
casei.xi
Thus, S.cerevisiae accomplishes two steps
of fermentation (ethanol and lactate).
Enhanced
break
down
of
polysaccharide:
Wine can be clarified by the usage of
certain enzymes i.e. pectinase, glucanase
etc. moreover yield of wine can also be
enhanced by these enzymes. Enzymes
responsible for polysaccharide break down
might also result in release of trapped
substances from grapes thus refining aroma
and shade of the wine. Commercially
available forms of such enzymes are costly.
Such strains of yeast are being prepared
that produce pectinases, glucanases,
xylanases are being developed.xii
Polypectate degradation was increased by
wine yeast expression of the pectate lysate
gene from Erwinia chrysanthemi pectate
lysate gene and polygalacturonase gene
from E. carotovora result in enhanced break
down of these polypectates.
Precise aims for baker’s yeast:
Yeasts using Melibiose:
8% raffinose is present in molasses and is
involved in production of yeasts. Invertase
converts raffinose and sucrose to fructose
and melibiose. α-galactosidase (melibiase),
is absent in yeasts therefore it is unable to
convert raffinose into disaccharides.
α-galactosidase enzyme is encoded by
MEL1 and occurs xiii bottom fermenting
strains. Thus this gene has been moved to
yeast for better functioning.
CONTEMPORARY
METHODOLOGICAL
EDGES
GENETIC ENGINEERING
IN
Although genes can be altered and
manipulated theoretically but there are
some methodological edges:
 Genetic maps: Locating the position
of preferred genes on different
chromosomes have not been fully
understood
for
important
commercial strains.
 The development and accessibility
of
vectors
has
just
been
commenced, thus is not much
developed.
 Physiological pathways: Series of
enzymatic phases from underdone
material to the looked-for yield have
not been recognized yet. Much
elementary research will be needed
to categorize all the steps.xiv
For a single gene transfer rDNA is quite
beneficial, however when a numbered
genes are to be transferred, this technique
somehow shows failure. In the initial steps
when genes have not been recognized
things become complicated. Even, when the
process has been successfully conducted
the choice of expression system also
presents a difficulty. Therefore much
progress is needed in the field of molecular
biology.xv
As a result of all these edges, for efficient
process, one or two genes are transferred
simultaneously.
Conclusion
During the past few decades considerable
progress has been made in the field of
genetics and fermentation. Different
varieties have been discovered and some
novels have been produced by mutation or
transference of genes. In this entire
scenario public opinion should be
considered. They should be made aware
about the pros and cons. In this era of
knowledge the current available data is not
enough, therefore efforts to gather more
data is needed. Although we have been
through various hurdles and obstacles at
genetic level, but in fact we are still
scrambling in a limbo. A vast increase in the
knowledge is still a necessity.
i
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