PLP 130 MT2

hypotheses to why secondary metabolism has evolved in fungi (and some plants)

1. competition theory
2. inc in pathogenic prowess
3. detoxification
4. maintenance of cell agility
5. evolution of primary metabolism

why are we interested in fungal SMs?

* mycotoxins to humans/animals
* disease on plants (phytotoxins)
* exploited in medicinal drugs eg Abx, anti-infective agents, immunosuppressants, anticancer agents

SMs as drugs

* statins = anti-cholesterol
* penicillin, chephalosporin = Abx
* cyclosporin A = immunosuppressant
* strobilurin = antifungal

Chaga

product of white rot fungus (Basidiomycetes) → makes sclerotia

* dark color bc antioxidant melanin → protects body against uncontrolled oxidation & free radicals
* SMs = betulin, sesquiterpenes, benzoic acid deriv
* used in many cultures

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* contains a lot more antioxidants than acai, pomegranatas, blueberries

Reishi mushrooms (Ganoderma lucidum)

* used for chronic diseases ie arthritis, insomnia, etc
* wood degrading Basidiomycota (Ganoderma)

Otzi the Iceman carried _

* Piptoporus - as medicine to clear parasitic worms via agaric acid → causes diarrhea
* also hoof fungi → start/transport fires

acetate malonate pw

synthesis of polyketides & fatty acids

mevalonic acid pw

aka isoprenoid pw

synthesis of isoprenoids (terpenes, carotenoids, steroids)

shikimic acid & amino-acid derived pw

synthesis of non-ribosomal peptide & aa derivatives

SMs pw’s committing enzymes

need key enzymes that catalyze 1st committed step for each class:

* polyketides synthases (PKS)
* terpene synthase (TS)
* non ribosomal peptide synthetases (NRPS)

complex SMs ex

combination of different pw → complex SMs

* indole alkaloids (ergots)
* derived from L-trypotophan via shikimic acid pw & dimethylalllyl pyroP

SMs production depends on _

* limited (esp nitrogen) nutrients
* idiophase = start where most SMs being made → make idiolites
* trophophase = feeding & growth phase; N is abundant

SM biosynthetic gene clusters

major components

* 1st committed enzyme (PKS, NRPS, TS): make backbone of SM
* additional tailoring enzymes: can be dozens, make specific modifications to backbone SMs
* transcription factors
* transporters: exporters
* other genes: defense

aflatoxin or sterigmatocystin in Aspergilli fungi

* most carcinogenic mycotoxin

aflatoxin biosyn pw intermediates

* polyketide progenitors
* antraquionones
* xanthones
* bisfuranocoumarins (aflatoxins)

1st committed enzyme in Polyketides & Non-ribosomal synthetase pw

PKSs and NRPs are multimodular & multidomain enzymes = make core structure of many SM

polyketide syntheases

essential domains

* acyltransferase = selects extender untis (malonyl-/acetyl-/methylmalonyl-CoA) → transfers to ACP
* acyl carrier protein (ACP) = loads unit to extended product
* ketosynthases = accepts polyketide chain from upstream ACP & catalyzes decarboxylation condensation b/t this substrate & an extender unit attached to the ACP in the same module

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auxiliary domains: optional domains ie enoyl reductases, dehydratase, ketoreductase

starter & termination domains = SAT is the starter ACP transacylase domain & thiolesterase the termination one

reducing vs non-reducing PKS

* when auxiliary domains are present in PKS → reducing PKS
* only essent domains present → non-reducing PKS

Type I, Iterative vs non-iterative (modular) PKS

non-iterative = multiple sequential modules, have extending & tailoring domains → each module does only one round of chain elongation (usually in bacteria)

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iterative = single copy of each domain are made into 1 module, used repeatedly during biosynthesis, (in fungi)

Non-ribosomal peptide synthetases

essential domains

* adenylation domain
* peptidyl carrier protein (PCP)
* condensation domain

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auxiliary domain

* methyltransferase, B-ketoacyl reductases, epimerization domains

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starter & termination domains: PCP can function as starter domain & thiolesterase is termination one

NRPSs Type A, B, C

linear NRPSs (type A): # & sequence of modules in NRPS matches the # and order of aa in the peptide

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iterative NRPSs (type B): modules or domains are used more than once to synthesize the peptide, which consists of repeated sequences

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nonlinear NRPSs (type C): seq of aa in generated peptide does not correlate to arrangement of modules on the template

iterative hybrid PKS-NRPSs (iPKR-NRPSs)

single module of an iterative PKS is followed by a single NRPS module (Fusing the polyketide chain to an aa) and an off-loading domain

transcriptional regulational of SM clusters stimuli

stimuli include

* C & N-sources
* temperature
* light
* pH
* aa in environement

SM physiological roles

assoc;d w/sporulation

* slow down germination spores until more favorable conditions
* toxic metabolites secreted to protect dormant spore from predators
* activate sporulation
* pigments for sporulation structures

most SM clusters are _ under lab conditions that don’t provide appropriate ecological triggers

silent or cryptic

regulation of SM gene clusters

pathway-specific = TFs that usually belong to clusters that factors regulate

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global regulation = globally acting TF which are encoded by genes that don’t belong to any cluster, & which also regulate a # of genes that are not involved in secondary metabolism

transporters for secretion of SMs

efflux system of SMs

* major facilitator superfamily (MFS): use energy from electrochemical gradients across membranes
* ABC transprorters: use ATP hydrolysis

mycotoxins

low MW natural products made as SM by fungi → mycotoxosis

mycotoxin in food chain

contamination of food/feed can occur at any stage during food production

* causes many economic losses
* 56% of rejection to food to EU is due to mycotoxins

factors influencings mycotoxin production

* climatic & environment: high T & humidity
* fungal strain
* plant variety
* damage of crop by other factors eg insects, mechanical
* substrate on which fungus is growing (idiophase)
* xs use of pesticides/fungicides that leads to resistance & induction of stress

mycotoxin exposure sources

* food
* water damaged buildings
* outdoors
* vehicles

health effects of mycotoxins depend on

* animal sp
* type of mycotoxin
* fitness of animal
* dosage & duration
* synergistic effects w/other mycotoxins

acute mycotoxicosis

after a single v severe exposures

* GI disturbances
* abortions
* skin irritation

chronic mycotoxicosis

* after periodic exposure to low doses over a long period of time
* hepatotoxicity (liver cancer is most prominent)
* nephrotoxicity
* genotoxicity
* etc

symptoms of mycotoxin exposure

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same happens to aniamls

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hard to diagnose bc of same symptoms of other diseases

MOA (modes of action) of mycotoxins

* damage membranes in intestines → impair absorption of nutrients & barrier to bloodstream
* target protein synthesis pw esp DNA/RNA structure
* induce immunosuppression
* disrupt microbiota homeostasis
* act as a neurotoxin

mycotoxin in the GIT & leaky gut syndrome

* most mycotoxins absorbed in duodenum
* encounter GITs epithelium
* epithelial cells allow selective absorption of nutrient & are a barrier to pathogens & toxins into the bloodstream

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leaky gut syndrome

* leads to minor: bloating & gas, cramps, fatigue
* severe: autoimmune conditions, depression

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* death or illness can result

mycotoxin amplification in enterohepatic cycle

part of mycotoxins can re-enter GIT via bloodstream & hepatic portal vein → prolonged retention in GIT → amplify damage to host

* can cause oxidative damage to liver
* bloodstream → spread to other organs

mycotoxin & effect on gut microbiota

* can alter microbiota in gut → growth of pathogens in GIT & inflammation

mycotoxin detection

* hard to prevent contamination
* v stable
* can be masked by other nutrients
* can act synergistically to enhance toxicity

mysterious turkey X disease

in UK 1960s; 100K turkey poults died

* all had same liver damage
* all got fed same peanuts from Brazil - contaminated w/Aspergillus flavus→ aflatoxins

aflatoxins

* polyketide mycotoxin; produced by Aspergillus flavus & Aspergillus parasiticus
* common: B1, B2, G1, G2 types
* toxicity: highest AfB1 > G1 > B2 >G2 lowest

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* AfM1 & M2 were 1st isolated from milk of lactating animals fed on aflatoxin grains

aflatoxin & hepatocellular carcinoma (HCC)

* primary disease ass’d w/aflatoxin intake is HCC
* mostly in Asia & Africa
* even 0.04g/kg could be dangerous

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how?

* AfB1 is pro-carcinogen: P450 transforms to AFBO (in humans) → binds to DNA
* DNA mutations
* induces mutations in P53 tumor suppressor gene → liver cancer

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\[insert aflatoxin pic\]

aflatoxins in milk

AfB1 → M1 in milk → Kwashiorkor disease in human babies

Aflatoxin & peanuts & other crops

* 1/2 of peanuts are contaminated by Aflatoxins
* if contaminated → may re-enter as animal feed or local open markets

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* rice, maize

aflatoxin regulation

* food testing set by FDA
* PCR, ELISA,

other common mycotoxins

* Ochratoxin A (in wine) from Aspergillus ochraceus
* patulin (in fruit) A. clavatus
* fumonisin (corn)
* Trichothecenes (corn)

Trichothecenes (TCNs)

* most common in USA
* made by Fusarium mostly
* Type A > Type B in toxicity
* Type A: lead to alimentary toxic aleuka (ATA)
* eg T-2
* TYpe B: cause changes of intestinal immune & nervous systems
* eg DON

TCN: Deoxynivalenol (DON, vomitoxin)

* most common in USA
* Fusarium sp infects corn, wheat, sorghum
* DON can cross the BBB
* in swine: moldy corn toxicosis

TCN: T-2

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Mycotoxins as biowarfare

* Yellow Rain controversy & gulf war syndrome

* powerful inhibitor of protein synthesis & neurotoxin
* made by Fusarium sp
* often in poultry
* inhibits synth of DNA/RNA, induce apoptosis

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* yellow rain: air attacks in Laos by Russian military after WWII
* T-2 was proposed as cause of Gulf War syndrome

Managing at Governmental Level: Hazard Analysis for critical control points (HACCP)

includes streategies for prevention, control, and quality from farm to fork

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Primary prevention: prevent fungal infestations, most important

* use of disease resistant plant strain
* chemical control

Secondary: stop existing infn

* protect stored products from conditions that favor fungus

Tertiary: when heavily infested by toxic fungi, less effective

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mycotoxin exposure routes

consumption of contaminated food: direct & in-direct (animals feed mycotoxins)

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breating in: stachybotrys, toxic black mold in carpets

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skin or eyes (TCNs)

* via pillows?

exposure in houses

* Stachybotrys chartarum - produces TCNs most assoc’d w/houses
* Aspergillus, Penicillium, Trichoderma, Cladosporium

Stachybotrys chartarum (aka S. atra) or Black mold

* causes “sick-building syndrome”
* over 170 mycotoxins, including cytoxic TCN
* chemotype S strain: 30-40%
* chemotype A: do not make TCN, 70-60%
* most common on cellulite (wallpaper, carpets)

health assoc’d risks w/mold in indoors

* allergic rxn: asthma, hay fever, pneumnoitis
* infectious: aspergillosis or histoplasmosis
* toxic: disruption of cellular fxn & interaction w/DNA, cancer

symptoms & mycotoxins in urban environment

emotional changes, respiratory changes, cognitive changes, physical issues

diagnosing a ‘sick building’

* mold growth & condensation
* off smell in particular rooms
* temperature always feels wrong

mycotoxins & human society

* ergot: plant disease, St. Anthony’s fire; cuased by Caviceps purpurea
* grows on rye crops
* infected ovary is replaced by scleortium = ergot → produces toxic alkoids

Ergotism

* convulsive ergostism: nervous dysfxn, tremors, hallucinations
* gangrenous ergostism: victim lose limbs, fingers, toes to dry gangrene

ergot alkaloids

* indole alkaloids
* mode of action: toxicity attributed to interaction of ergot alkaloids w/adrenergic, serotonergic & dopaminergic R in brain
* can be used as med drug in low doses

ergot alkaloids in folklore

* Salem Witch trials (madness like symptoms)
* flying ointments
* dance mania
* 27 y/o Peloponnesian War - contaminated wheat → killed many in Athens
* failed war from Peter the Great

**products of fungi**

food themselves

use as microbial cell factories

agricultural biotechnology & bioremediation

fungal hydrolyzing enzymes

* amylases, glucoamylase, glucose isomerases
* catalases: dyeing process
* cellulaase: digest jean fiber ‘fungal washing”
* xylanasaes, peroxidase, ligninases (paper)

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primary metabolites or secondary metabolites

primary

* citric acid
* gluconic acid
* itaconic acid

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secondary

* terpenoids in fragnance & flor industry, black/brown pigments, food, pharma, textiles
* pharm: antiallergic, antioxidants, antitumor

major pharm drugs of fungal origin

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penicillin

* Beta-lactam Ab are effective against G+ bacteria
* B-lactam ring = responsible for Ab activity via interfering w/bacterial CW biosynthesis by preventing the cross-linking of peptidoglycan
* discovered accidentally by Fleming

penicillin’s discovery & development

1) Fleming discovered accidentally → isolated & classified Penicillium notanum → low yield

2) WWII casualities from bacterial wounds; Chain and Florey

3) Florey; 2nd largest R&D project of WWII after Manhattan project; isolated strain from moldy cantaloupe → NRRL 1951 was a high producer of penicillin

→ induce mutations to get higher production & improved extraction procedures

limitations of natural penicillin

* limited range
* unstable in acidic environment
* painful, but be injected
* allergic
* sensitive to Beta-lactamases

composition of penicillin & improving penicillin

penicillin is composed of

* a thiazolidine ring, a B-lactam ring, free carboxyl acid group, one or more aa side chains
* position 6: variations are limited to acyl aide chain

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improving

* planting & selecting natural mutants

one way to change is vary acyl side chain by adding diff carboyxlic acids to fermentation medium

* corn steep liquior → pen G
* phenoxy-acetic acid → pen V (1st oral form)

classes of drug derivatives

1. natural (penicillin G)
2. biosynthetic: eg fermentation changes
3. semisynthetic (ie ampicillin, amoxycillin): cmpds are further chemically modified in lab after isolated from natural processes
4. synthetic: fully synthesized chemically

immunosuppressant drugs: Cyclosporine A (CsA)

* MOA

CsA is only member used clinically

used in BM transplants

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MOA

* inhibits fxn of several proteins involved in activation of T-cells at transcription level

Cyclosporine MOA

\[insert pic\]

* cyclophylin (CpN) blocks fxn of phosphatase enzyme cacineurin (CaN) → CaN fails to deP TF, NF-ATc ----→ T cells do not produce IL-2 → don’t activate full T-cell activation & immunity

statins

inhibit ergosterol bspw

* interact w/LDL cholesterol (bad - clogs veins) & lower triglycerides in blood
* some can inc also HDL (good) cholesterol

statins development

* change strain, fermentation media
* discovered accidentally in Penicillum

statin controversy

media reports of muscle pain and weakness → the Lancet reported that positives outweigh the negatives

common fungal-dervied enzymes in industry

cellulase:

glucosidase

laccase

amylase

pectinase

lipase

protease

chitinase

xylanase

textile industry

biostoning: use cellulases instead of pumice to degrade the jeans

* cellulase from Trichoderma reesei

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bleach clean up: catalase to get white fabric via splitting H2O2

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bioscouring: uses pectinases to remove impurities ie pectin & wax

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biopolish: cellulase to removes microhairs

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desizing w/amlyases: removing applied size material ie starch that were added to improve strength of yarn for weaving

6 major enzyme classes

oxidoreductases

transferase

hydrolases

lyases

isomerases

ligases

CAZymes structure

1. carbohydrate-binding domain (CDM): keeps CD nearby substrate
2. catalytic (hydrolyzing) domain (CD): does cleavage
3. a linker domain: seq of aa connecting cellulose BD & CD = flexible hinge to allow indep fxn of each domain

fungal isoenzymes of CAZymes

types of CAZymes

* endocellulases: hydrolyzes glycosidic bonds w/in a chain
* exocellulases: ““ from ends of chains or free ends generated by endoglucanases
* beta-glucosidases: cleave cellobiose into glc monomers

research areas on fungal enzymes

enzyme discovery

* exploitation of natural biodiv/screening, genome seq, metagenomics

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enzyme engineering

* rationale design, directed evolution

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selected applications

* industrial, environmental, biomedical

enzyme discovery

conventional culture-dependent & bioactivity-based approach

* enrichments
* disadvantages: screening is against a library of predefined substrates, costly, laborious, limited to only culturable organism

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genomics approach (genome mining)

* sequence, annotate, look for CAZymes
* databases
* dbCAN: for CAZymes
* Gene Ontology
* KEGG
* antiSMASH & SMURF
* disadvantages: need to isolate DNA, loses majority of diversity

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metagenomics

metagenomics

sequences-based screening: uses enzyme-encoding genes based on seq homology (genome-mining)

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activity-based screening (functional metagenomics): req cloning of environmental DNA into vectors to screen clones expressed selected enzymatic activity

enzyme engineering

improve

* rate of catalytic activity
* stability and robustness
* substrate specificity; reduction in side activity
* enantioselectivity

enzyme engineering approaches

rational design: mutants designed based on protein structure, prep by site directed mutagenesis

directed evolution: prep of large library of mutant genes, transformation & expression, screening for mutants w/desired properities is conducted & selected mutants are tested

fungal biotech violins

increase acoustic properties of violin wood by making v small holes

biofuels

1st gen: from edible plants, ethanol/butanol via yeast fermentation

2nd gen: from lignocellulosic material from non-food crops ie wood to feed microbes

3rd gen: from algae, resilient organisms that can be grown from sunlight, CO2, doesn’t use arable land, fastest growing sources, C-neutral

4th gen: genetic engineering

1st gen biofuels

starch (amylose or amylopectin) from corn, sugar cane, cassava →high-yielding ethanol source

* via fermentation w/S. cerevisiae, but lack amylolytic activity → initial degradation of starch into fermentable sugars → ferment

2nd gen biofuels

* need ligninases, cellulases, hemicelluloses to degrade lignin in cobs, stalk, leaves to make ethanol
* lignocellulolytic enzymes are for pretreatment of material or enzymatic hydrolysis to make fermentable sugars

consolidated bioprocessing

CBP-enabling microbe bust be able to solubilize a practical biomass substrate & produce desired products at high yield → need genetic engineering

* native strategy: begin w/microbes w/native ability to use cellulosic biomass
* recombinant strategy: begin w/microbes that do not have ability → require heterologous expression of a saccharolytic enzyme system

Asperigillus niger

produces pectinases → clarified fruit juices

bread w or w/o yeast

unleavened = w/o yeast, flat bread

leavened = w/yeast

chemistry of bread-making

glc → yeast will make CO2 and ethanol

amylopectin & amylose = need amylose to break down starch → maltose → glc

enzymes in baking industry

amylase: alpha & beta, glucoamylases

lipase

protease

xylanase

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externally added bc yeast is a poor producer of amylases

amylases family

alpha

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beta

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glucoamylase: most high demand → get glucose syrup from starch

glucose isomerase

production of high fructose sugar → 2x sweeter than glucose

* come from corn
* fructose → obesity epidemic in US, bc fructose is lipogenic, glc is preferred

fructose negative health effects

* fructose is only metabolized in the liver →
* tooth decay
* leaky gut
* fatty liver
* type II diabetes
* etc

fungal consumption history

fungal consumption is almost 20,000 years ago

Monascus pigments

* makes red fermented rice


* pigments are added into food or textiles
* therapeutic uses, antimicrobial, anticancer

food products from fungi

cheese, salami, red yeast rice, miso, tempeh

edible mushrooms

* B vitamins & D (if exposed to UV)
* Agaricus mushrooms (button mushrooms) are >95% of total US mushrooms