Transcript Scientific Writing - University of KwaZulu

MICR 306 Advanced
Applications of Fungi
Prof. J. Lin
University of KwaZulu-Natal
Westville campus
Microbiology Discipline
2014
School of Life Sciences
Theory Test 1
• Venue: Undergraduate lab
• Time: 14h10 – 16h10
Fungi
• Molds consist of long, branching filaments
of cells called hyphae (singular, hypha). A
tangled mass of hyphae visible to the
unaided eye is a mycelium (plural,
mycelia). Avascular -- no specialized
respiratory, digestive or transport systems.
• Yeasts are microscopic, unicellular fungi
with a single nucleus and eukaryotic
organelles. They reproduce asexually by a
process of budding. (some are dimorphic.)
Replication Cycle
Unique Characters
• Fungi grow best where there is a rich supply
of organic matter. (lack photosynthsis)
• Usually found as opportunistic saprophytes
(living on dead organic matter) or in some
parasitic or symbiotic relationship with
plants or other autotroph. (chemoheterotrophic)
Fungal metabolism
(i) Catabolism in which fungi derive energy from
complex molecules.
(ii) Anabolism which energy is used to synthesize
complex molecules from simple molecules;
 Primary metabolism encompasses pathways or
events that are necessary to maintain cell viability.
 Secondary metabolism produces compounds
that are not important for survival of individual cells
but may promote survival of a given population.
Major Roles in Ecology
• Fungi are versatile organisms that feed on
a variety of substrates.
• Along with bacteria, fungi are the major
decomposers in most terrestrial (and some
aquatic) ecosystems, and therefore play a
critical role in biogeochemical cycles and in
many food webs.
• Digest food outside their bodies release
enzymes into the surrounding environment
(exoenzymes).
Biotechnology
• Organisms (bacteria, fungi, viruses) with
desirable properties
• Environments – Conditions
Enrichment process
Competitive exclusive principle
Applications
Agricultural Biotechnology
Industrial Applications
Environmental Biotechnology
– Use of fungi/viruses for insect biological
control
Biotechnological Processes Using Fungi
– Use of Fungi/viruses as Expression Hosts
– Agaric farming (pharming) for production of
human proteins
Food and Feeds Industry?
Agricultural Biotechnology
• A Biotechnological Approach to Plant Protection
with Fungi
 Increase the biomass production, better
health and better resistance
– Fungi as Plant Growth Promoter and Disease
Suppressor
– Challenges and Strategies for Development of
Mycoherbicides / Biofungicides
– The Biological Control Agent Trichoderma:
Fundamentals to Applications
– Control of Nematodes by Fungi
– Insecticides
SYMBIOTIC RELATIONSHIP WITH
PLANTS
Plant growth mechanisms can be grouped
as follows:
1) Direct-like asymbiotic fixation of atmospheric
nitrogen, solubilization of minerals such as
phosphates;
2) Production of plant growth regulators like
auxins, gibberellins, cytokinin and ethylene;
3) Indirect - like HCN production, antibiotics,
siderophores, synthesis of cell wall enzymes,
and
4) Competitions with detrimental
microorganisms for sites on plant roots.
Organic Matters
• Filamentous fungi in soil  degrade organic
matter and help in soil aggregation  Humic
substances. (species of Alternaria, Aspergillus,
Cladosporium, Dematium, Gliocladium,
Helminthosporium, Humicola and Metarhizium)
 maintenance of soil organic matter.
• Producers of organic acids particularly black
Aspergilli and some species of Penicillium.
Acquisition of phosphates
• Fungi capable  forming ectotrophic
associations on the root system of tree
(Boletus and Lactarius)  mobilization of
soil phosphorus and nitrogen into
plants  Biofertilizers
• Fungi have been reported to possess
greater ability to solubilize insoluble
phosphate than bacteria. (Aspergillus spp.,
Penicillium spp., and Fusarium spp.)
Acquisition of Iron
• High affinity iron transport system
termed as Siderophore
• The majority of fungal siderophores are
hydroxamates, apart from the
carboxylate-type siderophore rhizoferrin
produced by Zygomycetes. Most species of
the genus Aspergillus are known to
produce several hydroxamate type
siderophores.
Role of Fungi as PGP
• Some rhizosphere fungi able to promote plant
growth upon root colonization are functionally
designated as 'plant-growth-promoting-fungi’
(PGPF). PGPF belong to genera Penicillium,
Trichoderma, Fusarium and Phoma.
Phytohormones
• Phytohormones such as indole-3-acetic
acid (IAA), cytokines, gibberellins (GA)
and other plant growth promoting
substances  enhance the hosts' uptake
of nutritional elements such as nitrogen
and phosphorous.
• Some to trigger systemic resistance
against various pathogens in cucumber
plants.
Biofertilizers
• Microbes involved in these formulations not only
mobilize N and P but also secrete various growth
promoting and health promoting substances. In
broad sense, the term 'biofertilizer' may be used to
include all organic resources for plant growth,
which are rendered in an available form for
absorption through microorganisms or plant
association or interactions.
• Arbuscular mycorrhizal fungi (AMF) -- alleviate
some nutrient deficiencies, improve drought
tolerance, overcome the detrimental effects of
salinity and enhance tolerance to pollution.
Industrial Applications (1)
• Antibiotics
– Aspergillus, Penicillium and actinomycetes
– Cephalosporin from Cephalosporium spp.
Penicillin and Cephalosporin are antibacterial against Gram (-) bacteria
– Griseofulvin from Penicillium griseofulvum
antifungal antibiotic useful in treating dermatophyte infections
– Lentinan from Lentinus sp.
against Mycobacterium tuberculosis, Listeria sp. and Herpes Simplex Virus
– Schizophyllan from Schizophyllum commune.
antibacterial and antifungal activity  controlling Candida albicans and
Staphylococcus aureus.
-Lactams biosynthetic pathway
Secondary Metabolic Products
Non-antibiotic Therapeutics
• Cyclosporin is an important immunosuppressant
drug that is used in organ transplantation surgery
• Lovastatin from Aspergillus terreus & Pravastatin
from Penicillium citrinum are cholesterol
biosynthesis inhibitors.
• Vitamin B12 (Saccharomyces cerevisiae) and
other vitamins (S. cerevisiae, Ashbya sp.,
Blakeslea sp.), hallucinogens (Psylocybe sp.),
and steroids useful in fertility regulation
(Rhizopus spp.).
Industrial Applications (2)
• Enzymes
Use of Enzymes for different purposes
Food
45 %
Detergent
34 %
Textile
11 %
Leather
3%
Pulp/paper
1%
Others
6%
ENZYME
•Acid, alkaline & neutral proteases
•Cellulase
•Diastase
•Glucoamylase
•Invertase
•Lactase
•Ligninase
•Lipase
•Pectinase (flavours, clear)
Bio-fuel productions
SOURCE
Aspergillus oryzae; A. niger; A. flavus; A. sojae
Trichoderma koningi
Aspergillus oryzae
Aspergillus niger; A. oryzae
Saccharomyces cerevisiae
S. lactis; Rhizopus oryzae
Phanerochaete chrysosporium
Rhizopus spp.
A. niger; Sclerotinia libertina
High-fructose corn syrup
• Cornstarch is treated with α-amylase to produce
shorter chains of sugars called oligosaccharides.
• Glucoamylase - which is produced by
Aspergillus, in a fermentation vat — breaks the
sugar chains down even further to yield the simple
sugar glucose. (Starch  glucose)
• Xylose isomerase (aka glucose isomerase)
converts glucose to a mixture of about 42%
fructose and 50–52% glucose with some other
sugars mixed in.
• Glucose  insulin release; Fructose  X (sweetness)
Organic acids from Fungi
Organic acid
Citric acid
Fumaric acid
Gluconic acid
Source
Aspergillus niger
Rhizopus nigricans
Aspergillus niger
- Food additive, acidity regulator, cleaning products
Itaconic acid
A. terreus
- Paper and architectural coating industry
Kojic acid
A. oryzae
- food and cosmetics (preserve color; anti-bacteria)
Biotechnological Processes
Applications
• Fungi are chemo-organotrophs and
therefore require fixed organic compounds
for their carbon and energy supply.
Carbohydrates  Glucose ( metabolic pathways)
most abundant renewable
 Biomolecules
energy source
 Fermentation
 Sugar (fructose, syrups)
Alkane  acetyl-coenzyme A (Acetyl-coA)
 TCA  Energy/bio-molecules
Plant Tissues
Cellulose  Glucose
• Endoglucanases hydrolyze internal bonds of
amorphous cellulose, cellodextrins and cellulose
derivatives. This releases new terminal ends and
cellobiose which consists of two glucose units.
• Cellobiohydrolases are exo-1,4--glucanases
that act on both amorphous and crystalline
cellulose as well as endoglucanase-generated
chain ends to release cellobiose.
• -Glucosidase breaks down cellobiose into two
glucose molecules that serve as easily
metabolisable carbon source for fungi.
Xylan  Xylose
• Endoxylanases are endo-acting enzymes that
randomly hydrolyze the xylan backbone to
produce a mixture of xylooligosaccharides.
• -Xylosidases are exozymes that liberate single
xylose units from xylooligosaccharides. Xylose is
then catabolized through the xylitol pathway in
filamentous fungi and through the
phosphoketolase pathway in yeasts.
• -L-arabinofuranosidase, -D-glucuronidases,
Acetyl xylan esterases, Feruloyl and coumaroyl
esterases. Thermomyces lanuginosus
Mannans
• The mannans consisting mainly of the
hexose mannose are similarly hydrolyzed
by endo-1,4--mannanases, mannosidase and accessory enzymes
such as -galactosidase and glucosidase, and esterases.
Ligninase
• The chemical structure of lignin makes it resistant
to attack by most microorganisms; however,
several groups of fungi can utilize it as a source of
carbon.
• The major groups of lignicolous fungi are the whiterot and litter-decomposing Basidiomycetes. In
addition, the brown-rot Basidiomycetes and
certain soft-rot Ascomycetes and
Deuteromycetes can partially degraded lignin.
Strategies?
Sources
• Ruminomyces
• White ants ……
Biofuel - Yeast
Leather productions
• the heavy use of polluting chemicals in the tanning
process (chromium)
• air pollution due to the transformation process
(dehairing  H2S and deliming  NH4+).
• Leather biodegrades slowly; it takes 25–40 years
Solutions
• Proteases hydrolyze non-structural proteins
(casein, elastin, albumin and globulin) (bating).
• Lipases  degreasing operation to hydrolyze fat
particles.
• Amylases  to soften skin (strength and flexibility)
Environmental Biotechnology
• Cellulose Degradation by Fungi & Lignocellulose
biodegradation by White Rot Fungi
• The Importance of Wood Decay Fungi in Forest
Ecosystems
• Biomineralization of Heavy Metals  Bioleaching
• Decolouration of Industrial Waste and Degradation
of Dye Water, Azo Dyes & Bioconversion of
Distillery Waste By Fungi
• Degradation of Hydrocarbons by Yeasts and
Filamentous Fungi
• Fungal Degradation of Explosives
• Restoration of Mycorrhizae in Arid Ecosystems
Azo yellow dye
Permethrin
polychlorinated biphenyls (PCB)
TNT
Examples
• Biodegradation of Azo dye and Hydrocarbons
 Peroxidase of Penicillium crysosporium &
Streptomyces sp. The filamentous fungi are also
having role in degradation of toxic hydrocarbons.
• Fungi in Hazardous waste remediation
 by its lignin degrading Enzymes
of Pleurotus ostreatus
• Biomineralization of Heavy Metals in the removal
& recovery of heavy metals from wastewater and
industrial effluents. Hg, Cu, Ni, Pb, Cd are
extracted at pH 2-5 by myceliar beads of
Penicillium. (Bioflocculants, biosorption…)
Metabolism-independent accumulation
The positively charged ions in the solution are
attracted to negatively charged ligands in cell
materials. Biosorption of metal ion occurs on
microbial cell surface. (bioflocculants)
But composition of biomass and other factors affect
biosorption. For example, in Rhizopus arrhizus
adsorption depends on ionic radius of Li3+, Mn2+,
Cu2+, Zn2+, Cd2+, Ba2+, Hg2+ and Pb2+.
However, binding of Hg2+, Ag2+, Cd2+, A13+, Ni2+, Cu2+
and Pb2+ strongly depends on concentration of
yeast cells.
Metabolism-dependent accumulation
In fungi and yeast, heavy metal ions are transported into the
cells through cell membrane. However, as a result of
metabolic processes ions are precipitated around the
cells, and synthesized intracellularly as metal-binding
proteins.
Energy-dependent uptake of Cu2+, Cd2+, Co2+, Ni2+, Zn2+ by
fungi has been demonstrated. Moreover, intracellular
uptake is influenced by certain external factors such as pH,
anions, cations and organic materials, growth phase, etc.
Metal uptake by growing batch culture was found maximum
during lag phase and early log phase in Aspergillus niger,
Penicillium spinulosum and Trichoderma viride.
Limitations and technical
challenges for application
• Sensitivity to biological process operations
• The fungus does not grow well in a suspended
cell system.
• Enzyme induction is negatively affected by
mixing actions and the ability of the fungus to
effectively attach itself to a fixed medium is poor.
• The majority of the research on fungal
performance has been conducted on autoclaved
soil or on synthetic media.  In situ?
low competitive capabilities in the environment
Biological Control Agents
• Biological control is the use of
biological organisms, or their byproducts, to control pests. Biocontrol is
popular in theory, because of its
potential to be host-specific virtually
without non-target effects.
 predators, parasitoids, and pathogens
Understanding and
Preventing Disease
• Holistic
Current
Classical biological control
• Classical biological control is the use of
natural enemies against a host which is
exotic in an area and has established
without its full guild of natural enemies.
This approach has been used successfully
to control a wide variety of pests including
larvae of the gypsy moth, aphids and
rabbits.
Augmentation
• Augmentation is based on the knowledge or
assumption that in some situations there are not
adequate numbers or species of natural
enemies to provide optimal biological control,
but that the numbers can be increased (and
control improved) by releases. This method
involves the addition of in vitro-produced mycelia
or conidia, (genetic modified) viruses in aqueous
suspensions to a field or glass house crop often
in combination with synthetic materials which are
formulation components to enhance persistence
and/or infectivity.
Augmentation
• Augmentation involves the addition of in vitroproduced mycelia or conidia in aqueous
suspensions to a field or glass house crop often
in combination with synthetic materials which are
formulation components to enhance persistence
and/or infectivity.
 Inundation
 Inoculation
Inundation
• Inundation involves the application of a biocontrol
agent in large amounts, so that the pest can be
rapidly removed. However, the biocontrol agent
(fungus) is only active within a short period and
no secondary infection is expected.
• Hyphomycetes have great potential as
inunadative biocontrol agents since they are
relatively easy to mass produce and formulate for
use with conventional spray application
equipment .
Example
• Verticillium lecanii is used as an inundative
mycoinsecticide in glasshouses in Europe for the control
of aphids and related insects.
• V. lecanii blastospores are produced and formulated with
a nutrient source in a wettable powder which can be
applied with a carrier such as aphid alarm pheromone or
an adjuvant based on emulsifiable vegetable oil. The
nutrient formulations allow satellite colonies to grow on
leaf surfaces, increasing effective coverage 40-fold. The
pheromone serves to attract the aphids and therefore
facilitates widespread infections while oil based
adjuvants promote adherence of the spores to the cuticle
and enhance activity at low humidity.
Inoculation
• Inoculative augmentation is the
introduction of the biocontrol agent in
small amounts early in the season of the
crop with the expectation that it will
repeatedly cycle (i.e. establish epizootics)
in pest populations and spread over a
period of time thereby maintaining the pest
populations below the economic threshold.
Examples using fungi
• Insect population control:
Advantages: specificity; natural present
Disadvantages: Ecology?
Example:
• Beauveria brongniartii used against larvae of
cockchafer beetles in Europe
• The conidia of B. brongniartii are cultivated on
barley kernels and applied into sites infested with
cockchafer larvae.  allowing them to produce
mycelia and aerial conidia.  then applied in an
orchard and natural meadow, using a tractor or
through helicopter. This kind of application is
often carried against swarming adult females at
the edges of forests with the aim that they could
carry the fungus into breeding sites
Fungal based
biological control agents
• Their broader spectrum in terms of
disease control and yield
• Trichoderma harzianum
mycoparasitism, competition for space
and nutrients, secretion of antibiotics
and fungal cell wall degrading
enzymes Control of phytopathogens.
• Trichoderma could have a stimulatory
effect on plant growth as a result of
modification of soil conditions.
Obligate pathogenic Fungi
• The fungal order Entomophthorales in the
class Zygomycetes is composed
principally of obligate pathogens that
infect arthropods.
• The >200 species infect a wide variety of
hosts, and nearly every fungal species
or strain is quite host specific.
Examples of entomopathogenic fungi:
• Beauveria bassiana (against white flies, thrips, aphids,
weevils)
• Paecilomyces fumosoroseus (against white flies, thrips and
aphids)
• Metarhizium spp. (against beetles, locusts and
grasshoppers, Hemiptera, spider mites and other pests)
• Lecanicillium spp. (against white flies, thrips and aphids)
• Cordyceps species (includes teleomorphs of the above
genera: that infect a wide spectrum of arthropods)
• Trichoderma species are used to manage certain plant
pathogens. Trichoderma viride has been used against
Dutch Elm disease, and to treat the spread of fungal and
bacterial growth on tree wounds. It may also have potential
as a means of combating silver leaf disease.
Pesticides
• The use of chemical pesticides, has led to
dramatic improvements in the production
of crop plants.
• Environmental impacts??
• Trichoderma harzianum
 reduce the use of fungicides, growth
regulators and labor  lower the
production costs and environmental
impact.
Fungal Interactions with Others
• Fungi commonly attack insects, nematodes and
other invertebrates in natural environments. In
doing so, they act as natural population regulators.
• Yeast has been used for controlling apple
postharvest diseases associated with Penicillium
expansum through antagonistic effects.
• The water mold Coelomomyces is obligate
parasite of the larvae of mosquitoes, black flies,
chironomids, and tabanids.
• Common insect pathogenic fungi include species of
Beauveria, Metarhizium, Lecanicillium,
Entomophthora and Pandora.
Australia January 1955
A load of rabbit skins
The most significant known factor in species loss in Australia
Continues
•
•
•
•
Cats Released to Stop Rabbits
Carp -- to control the plants growing there
Mosquito Fish
Cane Toad -- Sugar Cane growing in Australia was
being attacked by two native beetles, the Greyback
Cane Beetle, Dermolepida albohirtum, and Frenchi’s
Cane Beetle, Lepidiota frenchi.
• Cactoblastis -- A moth, Cactoblastis cactorum, was
introduced in 1926 to control the introduced Prickly Pear.
Failure cases
• When farmers in Australia tried to
eliminate the cane beetle using the South
American cane toad, the results were
catastrophic. The cane toad did not eat the
cane beetle and the toad population
spread rapidly, resulting in the decline of
native species of mammal and reptile.
Examples using viruses
• Weeds control: Araujia mosaic virus vs Moth plant?
• Pathogenic fungi control:
Totiviruses against Fusarium
• Rabbit population control: calicivirus, myxoma
virus
• Insect population control: baculoviruses
• Rat control: synthetic pox viruses
Advantages: specificity; natural present
Disadvantages: Ecology?
Viruses  biological control agents
Baculoviruses (Baculoviridae)
• Baculoviruses (dsDNA) are pathogens that
attack insects and other arthropods.
• They have been shown to have no negative
impacts on plants, mammals, birds, fish, or even
on non-target insects.
X Genetic engineering techniques to expand virus
host ranges to the desired pest species
X Cost  the labour intensive nature in production
i.e in vitro (within cell cultures in the laboratory) or
in vivo (living insects).
Resistance
• In contrast to the resistance to viruses
developed by rabbits, resistance to viral
insecticides in susceptible forms of the host
insect is very rare, which may reflect the
more limited (and typically non-adaptive)
immune system present in insects. Many
insects acquire increasing resistance as
they age, with adult forms often very highly
resistant however it is not transmitted to the
next generation of larvae.
Mosquitoes control
Questions
• Safety and Security
• Social and Ethical
• Scientists are very intelligent people, very
dedicated to their work and their fields of
endeavor. But many times they fail to
communicate to the public policymakers
the importance of the things that they’re
involved in
Heterologous Protein Production
• Protein based therapeutic agents are emerging as
the largest class of new chemicals in drug industry.
• Yeasts and filamentous fungi
– to grow in chemically defined medium in the absence of
animal-derived growth factors
– to secrete large amounts of recombinant protein
– with ease of scale-up and low cost of production
– Post-translational modifications (animal & human)
A good expression host
• It should have the ability to produce the product in
large quantities.
• It should not have proteolytic enzymes which
destroy the product.
• It should be easy to cultivate in large volumes in
fermenters.
• Its biology should be thoroughly understood.
• It should be able to carry out post-translational
modifications such as protein folding, disulfide
bond formation and glycosylation (needed for
human proteins).
Molecular Genetics
Expression systems
Inducer (enhancer) ; Promoter
• E. coli
• Yeasts and filamentous fungi
• Cell tissue culture
– insect cell cultures
Autographic californica using the genome of the
baculovirus (500 mg/L)
– animal cell cultures
Chinese Hamster Ovary (CHO) cell cultures,
Baby Hamster Kidney (BHK) cell cultures.
A good expression host for
recombinant proteins
• It should have the ability to produce the product
in large quantities.
• It should not have proteolytic enzymes which
destroy the product.
• It should be easy to cultivate in large volumes
in fermenters.
• Its biology should be thoroughly understood.
• It should be able to carry out post-translational
modifications such as protein folding, disulfide
bond formation and glycosylation (needed for
human proteins).
Fungi as Expression Hosts
Glycosylation enzymes
HUMAN
α 1,2 Mannosidase-I
Mannosidase-II
β 1,2 N-acetylglucosaminyl transferase –I
β 1,2 N-acetylglucoaminyl transferase-II
β 1,4 galactosyltransferase
Sialyltransferase
YEAST
1,6 Mannosyl transferase
1,2 Mannosyl transferase
Humanizing the Glycosylation
Pathways:
• cloned the human glycosylating gene for β 1,4N-acetylglucoaminyl transferase in the
filamentous fungus Aspergillus nidulans.
• The best therapeutic results by using
the enzyme from the patient’s system.
Yeast (Saccharomyces cerevisiae)
BRAND NAME
•
•
•
•
•
•
•
Actrapid
Ambrix
Elitex
Glucagen
HBVAXPRO
Leukine
Regranex rh
RECOM. PROTEIN
Insulin
Hepatitis-B surface antige
Urate oxidase
Glucagon
Hepatitis-B Surface antigen
Granulocyte macrophage CSF
Platelet-derived Growth factor
Pichia pastoris
Protein
•Angiostatin
•Elastase
•Endostatin
•Epidermal growth factor
•Hepatitis-B surface antigen
•Human serum albumin
•Insulin-like growth factor-1
Indication
Anti-angiogenic
Cystic fibrosis
Anti-angiogenic
Diabetes
Serum hepatitis
Stabilizing blood in burn
Deficiency of the same
Production of Hepatitis-B
Vaccine (Pichia pastoris)
• Reduce the cost from R200 to R20 (1990s)
• The gene for Hepatitis-B Surface Antigen
(HbSAg) was cloned between the promoter
region of the alcohol oxidase gene 1
(AOX1p) and the termination and
polyadenylation signal region of the same
gene (AOX1t).
• Add methanol (Inducer)  as much as
30% of the total cellular protein
Components of P. pastoris
expression vector
→Ori pp→ AOX1p---HBsAg---AOX1t→
↑
↓
↑
↓
←Ampr ← OriE ← 3’AOX1← HIS4 ←←
histidinol dehydrogenase deficient (HIS4-) mutant strains of P. pastoris
P. pastoris expression vector
AOX1p-HbSAg-AOX1t unit
• Origin of replication that functions in P. pastoris (ORI pp)
• Part of the plasmid pBR 322 with the E. coli origin of
replication (ORIE).  enables the vector to be maintained
in E. coli.
• An E. coli selectable ampicillin resistance marker (Ampr).
• A segment of DNA that lies downstream from the AOX1t
sequence (3’-AOX1) that facilitates the inserted gene
(HbSAg) into the specific chromosomal site in P. pastoris.
• A functional histidinol dehydrogernase gene (HIS4) which
encodes an enzyme that is required for the synthesis of
amino acid histidine as a selectable marker in P. pastoris
(histidine non-producing mutants are used transformation).
• Integrate the gene with the host (Gene Stability)
Other applications
• Production of Immunotoxin from yeast
as anti-tumor agent
• Bacillus thuringiensis toxin gene (Pichia
pastoris) -- Cry family, active against
insects
• Phytase (myo-inositol phosphates) from
Aspergillus niger cloned in Pichia pastoris.
 environmental pollution of phosphorus
Protein Expressions using
Virus Vector
• Eschericihia coli expression systems have
been the first choice for the expression of
heterologous proteins
• Lack or inappropriate post-translational
modifications, incorrect folding,
proteolytic degradation, inefficient
secretion, and amino acid
misincorporation
abundant polypeptide (20-30%) of total
cellular protein and to 90 mg/L
• Baculoviruses for protein expression
• Adenoviruses for protein expression
Baculoviruses  protein
expression
•Safe, as baculoviruses will not replicate in mammalian cells
•Wide host range – will infect a broad range of mammalian cell types
•Capable - transient expression allows production of toxic proteins.
•Non-toxic – Greatly decreased cytotoxicity when compared to
traditional transient expression techniques.
•Flexible – Different viruses can be used in the same experiment as
required, to express different proteins to varying concentrations
•Efficient – High transduction efficiency maximises gene delivery into
cells
•Rapid – much faster than generating a stable cell line for protein
production
Adenoviruses protein expression in
mammalian cell lines
a) Broad host range and low pathogenicity in humans.
b) Infection and expression of genes in both replicative and
non-replicative cells.
c) Replicates efficiently to high titers.
d) Helper-independent Ad can accommodate up to 7.5 kb
of foreign DNA.
e) Simultaneous expression of multiple genes.
f) No insertional mutagenesis; remains epichromosomal.
g) Propagation in suspension cultures.
h) Homologous system for human genes.
Agaric farming (pharming)
Production of human proteins (pharmaceuticals)
• The new concept  an agaric farm as a
recombinant protein farm  mushrooms as the
expression system for several recombinant
proteins, including vaccines.
• Basidiomycetes such as Schizophyllum
commune, Agaricus bisporus, Pleurotus sp. and
Phanerochaete chrysosporium are candidates
for protein pharming. However, difficulties are
there in gene manipulation.
Food and Feeds Industry
• Role of Fungi in Fermented Foods
• Production of Edible Fungi (mushroom, SCP)
• Genetic Variability of Yeasts in Wine Fermentation (Brands)
• Yeast in the Dairy Industry (Cheeses; breads)
• Flavours and Aroma
• Vitamins, Carotenoids, amino acids  supplements
• Antifungal Food Additives - sorbic acid, benzoic acid, propionic acid
• Molecular Detection of Fungi in Foods and Feeds
• The Role of Spoilage Fungi in Seed Deterioration (Practical)
Mycotoxin  Aflatoxins, Fumonisins & Ergot alkaloids
• Genetics and Biochemistry of Toxin Synthesis
Food and Feeds Industry
Fungi are used as the rich protein sources of
Single Cell Proteins. Some of the fungi
for SCP are given as
1) Yeast (S. cerevisae)
2) Aspergillus niger
3) Penicillium chrysogenum
4) Fusarium avenacum
5) Neurospora sitoplila
Food products and Fungi
Type of Food
Fungus
Cheese: blue, Limeburger
Penicillium species
Beer, Wine
Saccharomyces carisbergensis
Saccharomyces cerevisae
Soy products: miso (Japan) Aspergillus oryzae, Rhizopus
Soy sauce, tofu (Chinese)
species; Mucor species
Breads
Saccharomyces cerevisae
Nutritional yeast
Saccharomyces species
Flavours
Flavouring
Chemical
Odor
Diacetyl
Buttery
Isoamyl acetate
Banana
Benzaldehyde
Bitter almond
Cinnamic aldehyde
Cinnamon
Ethyl propionate
Fruity
Methyl anthranilate
Grape
Limonene
Orange
Ethyl decadienoate
Pear
Allyl hexanoate
Pineapple
Ethyl maltol
Sugar, Cotton candy
Ethylvanillin
Vanilla
Methyl salicylate
Wintergreen
Tastes
Acid
Description (GRAS)
Acetic acid
Gives vinegar its sour taste and distinctive smell
Ascorbic acid
Found in oranges and green peppers and gives a crisp,
slightly sour taste. Better known as vitamin C.
Citric acid
Found in citrus fruits and gives them their sour taste
Fumaric acid
Not found in fruits, used as a substitute for citric and tartaric
acid
Lactic acid
Found in various milk or fermented products and give them
a rich tartness
Malic acid
Found in apples and gives them their sour/tart taste
Phosphoric acid
Used in all Cola drinks to give an acid taste
Tartaric acid
Found in grapes and wines and gives them a tart taste