3_Biosynthetic Pathways for Metabolic Products Part 2

(i) has no apparent function in the organism.

(ii) produced in response to a restriction in nutrients.

(iii) restricted to some species of plants and microorganisms.

(iv) usually have ‘bizarre’ and unusual chemical structures and several closely related metabolites may be produced by the same organism in wild-type strains.

(v) The ability to produce a particular secondary metabolite is easily lost. This phenomenon is known as strain degeneration.

(vi) Secondary metabolite production is believed to be controlled by plasmids instead of Chromosomes.

(vii) The factors which trigger secondary metabolism, the inducers, also trigger morphological changes (morphogenesis) in the organism.

Secondary metabolism, which was first observed in higher plants, has the following characteristics

strain degeneration

Secondary metabolism, which was first observed in higher plants, has the following characteristics:

• (v) The ability to produce a particular secondary metabolite is easily lost. This phenomenon is known as ___

plasmids

Secondary metabolism, which was first observed in higher plants, has the following characteristics:

• (vi) Secondary metabolite production is believed to be controlled by ____instead of Chromosomes.

inducers

Secondary metabolism, which was first observed in higher plants, has the following characteristics:

• (vii) The factors which trigger secondary metabolism, the ___, also trigger morphological changes (morphogenesis) in the organism.

• special small molecules that can trigger another metabolite

ɣ-butyrolactones (butanolides)

Inducers of Specialized (Secondary) Metabolites:

• of the actinomycetes

N-acylhomoserine lactones (HSLs or AHLs)

Inducers of Specialized (Secondary) Metabolites:

• of Gram negative bacteria

oligopeptides (AIP)

Inducers of Specialized (Secondary) Metabolites:

• of Gram positive bacteria

B-factor [3’-(1-butylphosphoryl)adenosine]

Inducers of Specialized (Secondary) Metabolites:

• of rifamycin production in Amycolatopsis mediterrane.

Inducers of Specialized (Secondary) Metabolites

They function in development, sporulation, light emission, virulence, production of antibiotics, pigments and cyanide, plasmid-driven conjugation and competence for genetic transformation.

development, sporulation, light emission, virulence, production of antibiotics, pigments and cyanide, plasmid-driven conjugation and competence for genetic transformation

Inducers of Specialized (Secondary) Metabolites function in ___

Actinomycete fermentations

is the inducing effect of endogenous -butyrolactones, e.g. A-factor (2-S isocapryloyl-3R-hydroxymethyl--butyrolactone).

endogenous -butyrolactones, e.g. A-factor (2-S isocapryloyl-3R-hydroxymethyl--butyrolactone)

Actinomycete fermentations is the inducing effect of

A-factor

• induces both morphological and chemical differentiation in Streptomyces griseus and Streptomyces bikiniensis, bringing on formation of aerial mycelia, conidia, streptomycin synthases and streptomycin.

• acts as a barrier to adpA

Streptomyces griseus and Streptomyces bikiniensis

A-factor induces both morphological and chemical differentiation in___ , bringing on formation of aerial mycelia, conidia, streptomycin synthases and streptomycin.

-butyrolactones

Many other actinomycetes produce A-factor, or related butyrolactones, which differ in the length of the side-chain. In those strains which produce antibiotics other than streptomycin, the ____ induce formation of the particular antibiotics that are produced, as well as morphological differentiation.

antibiotics that are produced, as well as morphological differentiation

Many other actinomycetes produce A-factor, or related butyrolactones, which differ in the length of the side-chain. In those strains which produce antibiotics other than streptomycin, the -butyrolactones induce formation of the particular ____.

not permanent (loss of the ability)

In the case of microbes, specialized metabolites are ___

• Nutrient availability

• Nutrient availability

• Growth phase

• pH of the medium

• Temperature

• Oxygen availability

• Carbon and nitrogen sources

• Genetic regulation of the microorganism

• Presence of precursors or inducers

• Inhibitors or feedback regulation

• Triggered by stress-intensity factors

Factors affecting specialized metabolites:

ArpA binds to the adpA promoter and represses its transcription.

Production of Antibacterial Compounds from Actinomycetes:

• In the absence of A factor,

it binds to ArpA that is detached from the adpA promoter, leading to its expression

Production of Antibacterial Compounds from Actinomycetes:

• When A factor accumulates and reaches a threshold level,

• Biosynthetic genes

• Activators

• Repressors

• Resistance genes

Involved in producing secondary metabolism:

Biosynthetic genes

Involved in producing secondary metabolism:

• genes that encode enzymes for making the metabolite.

Activators

Involved in producing secondary metabolism:

• regulatory genes that turn ON metabolite production.

Repressors

Involved in producing secondary metabolism:

• genes that turn OFF or reduce production.

Resistance

Involved in producing secondary metabolism:

• protect the microorganism from the antibiotic it produces.

Primary metabolism

supplies energy and precursors needed to produce secondary metabolites.

Metabolic engineering and Ribosome engineering

Methods used in Primary metabolism:

• modifying metabolic pathways to direct more resources to metabolite production.

Metabolic engineering

Methods used in Primary metabolism:

• altering ribosomes to improve gene expression and metabolite synthesis.

Ribosome engineering

Methods used in Primary metabolism:

• altering ribosomes to improve gene expression and metabolite synthesis.

• Carbon source (e.g., glucose)

• Phosphate

• Nitrogen

• Amino acids

The culture medium strongly affects secondary metabolite production: Important components:

• Genes (secondary metabolism)

• Cell metabolism (primary metabolism)

• Growth medium composition

• Regulatory signaling molecules

To improve antibiotic or secondary metabolite production, scientists modify:

Penicillin

Industrial Products of Microbial Secondary Metabolism:

ANTIBIOTICS:

• produced by Penicillium chrysogenum; used clinically as an antibiotic.

Streptomycin

Industrial Products of Microbial Secondary Metabolism:

ANTIBIOTICS:

• produced by Streptomyces griseus; used clinically to treat bacterial infections.

Actinomycin

Industrial Products of Microbial Secondary Metabolism:

ANTI-TUMOR AGENTS:

• produced by Streptomyces antibioticus; used in clinical cancer treatment.

Bleomycin

Industrial Products of Microbial Secondary Metabolism:

ANTI-TUMOR AGENTS:

• produced by Streptomyces verticillus; used as an anti-cancer drug.

Aflatoxin

Industrial Products of Microbial Secondary Metabolism:

TOXINS:

• produced by Aspergillus flavus; a harmful food toxin.

Amanitine

Industrial Products of Microbial Secondary Metabolism:

TOXINS:

• produced by Amanita species; toxic compound found in poisonous mushrooms.

Ergot alkaloids

Industrial Products of Microbial Secondary Metabolism:

ALKALOIDS:

• produced by Claviceps purpurea; used in pharmaceuticals

Gibberellic acid

Industrial Products of Microbial Secondary Metabolism:

MISCELLANEOUS SECONDARY METABOLITES:

• produced by Gibberella fujikuroi; functions as a plant growth hormone.

Kojic acid

Industrial Products of Microbial Secondary Metabolism:

MISCELLANEOUS SECONDARY METABOLITES:

• produced by Aspergillus flavus; used as a food flavoring agent.

Muscarine

Industrial Products of Microbial Secondary Metabolism:

MISCELLANEOUS SECONDARY METABOLITES:

• produced by Clitocybe rivulosa; has pharmaceutical significance.

Patulin

Industrial Products of Microbial Secondary Metabolism:

MISCELLANEOUS SECONDARY METABOLITES:

• produced by Penicillium urticae; has antimicrobial properties.

health, nutrition, and economics

Microbial specialized (secondary) metabolites have a major effect on the ___of our society.

nutrients, growth rate, feedback control, enzyme inactivation, and enzyme induction

Microbial specialized (secondary) metabolites’ formation is regulated by ___

unique low molecular mass compounds, transfer RNA, sigma factors, and gene products

Microbial specialized (secondary) metabolites’ Regulation is influenced by ___formed during post- exponential development

clustered genes on chromosomal DNA and infrequently on plasmid DNA.

The synthases of secondary metabolism are often coded for by ___

Pleiotropic

global gene regulators are activated to generate random wide effects

Biosynthetic gene cluster specific

focused on desired pathways

Targeted genome-wide approaches

combination of Pleiotropic and Biosynthetic gene cluster specific

Pleiotropic, Biosynthetic gene cluster specific, Targeted genome-wide approaches

Methodologies for the activation of silent gene clusters to obtain new natural products:

trophophase and idiophase

were introduced to distinguish the two phases in the growth of organisms producing secondary metabolites.

trophophase

(Greek, tropho = nutrient) - feeding phase during which primary metabolism occurs. In a batch culture this would be in the logarithmic phase of the growth curve.

idiophase

(Greek, idio = peculiar) - during which secondary metabolites peculiar to a given organism are synthesized. Secondary synthesis occurs in the late logarithmic, and in the stationary phase.

logarithmic phase of the growth curve

trophophase (Greek, tropho = nutrient) - feeding phase during which primary metabolism occurs. In a batch culture this would be in the __

late logarithmic, and in the stationary phase.

idiophase (Greek, idio = peculiar) - during which secondary metabolites peculiar to a given organism are synthesized. Secondary synthesis occurs in the __

‘idiolites’

• It has been suggested that secondary metabolites be described as ___ to distinguish them from primary metabolites.

• specialized secondary metabolites

(1) The competition hypothesis

(2) The maintenance hypothesis

(3) The unbalanced growth hypothesis

(4) The detoxification hypothesis

(5) The regulatory hypothesis

(6) The hypothesis of secondary metabolism as the expression of evolutionary reactions

Role of Specialized (Secondary) Metabolites in the Physiology of Organisms Producing Them:

The competition hypothesis

Role of Specialized (Secondary) Metabolites in the Physiology of Organisms Producing Them:

• refers to antibiotics specifically, secondary metabolites (antibiotics) enable the producing organism to withstand competition for food from other soil organisms.

• Core Driver: Ecological competition

• Central Idea: Secondary metabolites provide a survival advantage by inhibiting competitors.

The maintenance hypothesis

Role of Specialized (Secondary) Metabolites in the Physiology of Organisms Producing Them:

• Secondary metabolism usually occurs with the exhaustion of a vital nutrient such as glucose.

• Its selective advantage is that it serves to maintain mechanisms essential to cell multiplication in operative order when that cell multiplication is no longer possible.

• Thus by forming secondary enzymes, the enzymes of primary metabolism would be destroyed. Therefore, the secondary metabolite itself is not important; what is important is the pathway of producing it.

• Core Driver: Nutrient depletion / stationary phase

• Central Idea: Secondary metabolism maintains metabolic systems when growth stops.

The unbalanced growth hypothesis

Role of Specialized (Secondary) Metabolites in the Physiology of Organisms Producing Them:

• states that control mechanisms in some organisms are too weak to prevent the over synthesis of some primary metabolites.

• primary metabolites are converted into secondary metabolites that are excreted from the cell. If they are not so converted they would lead to the death of the organism.

• Core Driver: Weak metabolic regulation

• Central Idea: Excess primary metabolites are converted into secondary metabolites to prevent harmful accumulation.

The detoxification hypothesis

Role of Specialized (Secondary) Metabolites in the Physiology of Organisms Producing Them:

• states that molecules accumulated in the cell are detoxified to yield antibiotics.

• consistent with the observation that the penicillin precursor penicillanic acid is more toxic to Penicillium chrysogenum than benzyl penicillin.

• Nevertheless not many toxic precursors of antibiotics have been observed.

• Core Driver: Toxic intracellular intermediates

• Central Idea: Harmful compounds are converted into less toxic secondary metabolites.

The regulatory hypothesis

Role of Specialized (Secondary) Metabolites in the Physiology of Organisms Producing Them:

• Secondary metabolite production is known to be associated with morphological differentiation in producing organisms.

• In the fungus Neurospora crassa, carotenoids are produced during sporulation.

• In Cephalospoium acremonium, cephalosporin C is produced during the idiophase when arthrospores are produced.

• In this theory the production of secondary metabolites is necessary to regulate some morphological changes in the organism. It could of course be that some external mechanism triggers off secondary metabolite production as well as the morphological change.

• Core Driver: Morphological differentiation

• Central Idea: Secondary metabolism is linked to and may regulate developmental changes

The hypothesis of secondary metabolism as the expression of evolutionary reactions

Role of Specialized (Secondary) Metabolites in the Physiology of Organisms Producing Them:

• In this hypothesis, it is important to bear in mind that both primary and secondary metabolism are controlled by genes carried by the organism. Any genes not required are lost.

• secondary metabolism is a mixed bag of biochemical reactions, undergoing tests for possible incorporation into the cell’s armory of primary reactions. Any reaction in the mixed bag which favorably affects any one of the primary processes, thereby fitting the organism better to survive in its environment, becomes incorporated as part of primary metabolism.

• the antibiotic properties of some secondary metabolites are incidental and not a design to protect the microorganisms.

• implies that secondary metabolism must occur in all microorganisms since evolution is a continuing process.

• Core Driver: Natural selection

• Central Idea: Secondary metabolism represents biochemical reactions undergoing evolutionary testing

• “abstract”

death of the organism

primary metabolites are converted into secondary metabolites that are excreted from the cell. If they are not so converted they would lead to the____

Neurospora crassa

In the fungus___ carotenoids are produced during sporulation.

cephalosporin C

In Cephalospoium acremonium, ___ is produced during the idiophase when arthrospores are produced.

Cephalospoium acremonium

In____ , cephalosporin C is produced during the idiophase when arthrospores are produced.

neutralized to another product

In some microbes, some primary products are toxic which are ___

Polyketides

specialized metabolites in fungi

Terpenoids/Steroidal mevalonic acid

___(or simply mevalonate) is the precursor responsible for the biosynthesis of terpenoids and steroids.

Psilocybin

“magic mushroom”

incidental and not a design to protect the microorganisms

the antibiotic properties of some secondary metabolites are ___

C. M. UG. D. R. EE

Competition, Maintenance, Unbalanced Growth, Detoxification, Regulatory, and Evolutionary Expression

In short, the Role of Specialized (Secondary) Metabolites in the Physiology of Organisms Producing Them includes the hypotheses:

1. Conversion of a normal substrate into important intermediates of general metabolism;

2. The assembly of these intermediates in an unusual way, by means of a combination of standard general mechanisms with a selection from a relatively small number of special mechanism;

3. These special mechanisms while being peculiar to secondary metabolism are not unrelated to general or primary mechanism;

4. The synthetic activity of secondary metabolism appears in response to conditions favorable for cell multiplication

The unifying features of the synthesis of secondary metabolic products by microorganisms can be summarized thus:

(1) Secondary products derived from the intact glucose skeleton

(2) Secondary products related to nucleosides

(3) Secondary products derived through the Shikimate-Chorismate Pathway

(4) The polyketide pathway

(5) Terpenes and steroids

(6) Compounds derived from amino acids

Specialized (Secondary) metabolites may be classified according to the following six metabolic pathways:

(1) Secondary products derived from the intact glucose skeleton

Specialized (Secondary) metabolites may be classified according to the following six metabolic pathways:

• The carbon skeleton of glucose is incorporated unaltered in many antibiotics and other secondary metabolites. The entire basic structure of the secondary product may be derived from glucose as in streptomycin or it may form the glycoside molecule to be combined with a non-sugar (aglycone portion) from another biosynthetic route.

• The incorporation of the intact glucose molecule is more common among the actinomycetes than among the fungi.

(2) Secondary products related to nucleosides

Specialized (Secondary) metabolites may be classified according to the following six metabolic pathways:

• The pentose phosphate pathway provides ribose (5 carbon) for nucleoside biosynthesis.

• Many secondary metabolites in this group are antibiotics and are produced mainly by actinomycetes and fungi.

• Examples are nucleoside antibiotics such as bleomycin.

(3) Secondary products derived through the Shikimate-Chorismate Pathway

Specialized (Secondary) metabolites may be classified according to the following six metabolic pathways:

• Shikimic acid (C7) is formed by the condensation of erythrose-4- phosphate (C4) obtained from the PP pathway with phosphoenolypyruvate (C3) from the EMP pathway.

• It is converted to chorismic acid which is a key intermediate in the formation of numerous products including aromatic aminoacids, such as phynylalamine, tyrosine and tryptophan.

• Chorismic acid is also a precursor for a number of secondary metabolites including chloramphenicol, p-amino benzoic acid, phenazines and pyocyanin which all have antimicrobial properties.

• The metabolic route leading to the formation of these compounds is therefore referred to as the shikimate pathway. In view of this central role of chorismic acid, however, the route is more widely known as the shikimate-chorismate route. The shikimate chorismate route is an important route for the formation of aromatic secondary products in the bacteria and actinomycetes.

• Examples of such secondary products include chloramphenicol and novobiocin. The route is less used in fungi, where the polyketide pathway is more common for the synthesis of aromatic secondary products.

(4) The polyketide pathway

Specialized (Secondary) metabolites may be classified according to the following six metabolic pathways:

• polyketide biosynthesis is highly characteristic of the fungi, where more secondary metabolites are produced by it than by any other, a much smaller number being obtained from bacteria and higher plants.

• compounds widely used as clinical medicines for the treatment of various acute and chronic diseases.

• Examples include antibacterial (erythromycin and tetracycline), antitumor (anthracycline and doxorubicin), antifungal (amphotericin and griseofulvin), antiparasitic (avermectin) and anti-cholesterol (lovastatin) drugs.

(5) Terpenes and steroids

Specialized (Secondary) metabolites may be classified according to the following six metabolic pathways:

• The second important biosynthetic route from acetate is that leading via mevalonic acid to the terpenes and steroids. Microorganisms especially fungi and bacteria synthesize a large number of terpenes, steroids, carotenoids and other products following the ‘isoprene rule’. The central point of this rule is that these compounds are all derivatives of isoprene, the five carbon compound. Simply put the isoprene rules consist of the following:

• (i) Synthesis of mevalonate from acetate or leucine

• (ii) Dehydration and decarboxylation to give isoprene followed by condensation to give isoprenes of various lengths.

• (iii) Cyclization (ring formation) e.g., to give steroids

• (iv) Further modification of the cyclised structure. The route leads to the formation of essential steroid hormones of mammals and to a variety of secondary metabolites in fungi and plants. it is not used to any extent in the actinomycetes.

(6) Compounds derived from amino acids

Specialized (Secondary) metabolites may be classified according to the following six metabolic pathways:

• The amino acids are derived from various products in the catabolism of glucose.

• A. Serine (C3N) and glycine (C2N) are derived from the triose (C3) formed glucose;

• B. valine (C5N) is derived from acetate (C3);

• C. aspartatic acid (C4N) is derived from oxeloacetic acid (C4)

• D. glutamic acid(C5N) is derived from oxoglutamic acid (C5).

Shikimic acid (C7)

is formed by the condensation of erythrose-4- phosphate (C4) obtained from the PP pathway with phosphoenolypyruvate (C3) from the EMP pathway

chorismic acid

• Shikimic acid is converted to ___ which is a key intermediate in the formation of numerous products including aromatic aminoacids, such as phynylalamine, tyrosine and tryptophan.

• is also a precursor for a number of secondary metabolites including chloramphenicol, p-amino benzoic acid, phenazines and pyocyanin which all have antimicrobial properties

phynylalamine, tyrosine and tryptophan

Shikimic acid is converted to chorismic acid which is a key intermediate in the formation of numerous products including aromatic aminoacids, such as ___which all have antimicrobial properties.

chloramphenicol, p-amino benzoic acid, phenazines and pyocyanin

Chorismic acid is also a precursor for a number of secondary metabolites including __n which all have antimicrobial properties.

shikimate-chorismate route

is an important route for the formation of aromatic secondary products in the bacteria and actinomycetes

chloramphenicol and novobiocin

Examples of shikimate-chorismate route secondary products include:

polyketide biosynthesis

is highly characteristic of the fungi, where more secondary metabolites are produced by it than by any other, a much smaller number being obtained from bacteria and higher plants.

• antibacterial (erythromycin and tetracycline),

• antitumor (anthracycline and doxorubicin),

• antifungal (amphotericin and griseofulvin),

• antiparasitic (avermectin) and

• anti-cholesterol (lovastatin) drugs

polyketide pathway example products:

malonate group

The addition of CO2 to an acetate group gives a ___

mevalonic acid to the terpenes and steroids

The second important biosynthetic route from acetate is that leading via

‘isoprene rule’

Microorganisms especially fungi and bacteria synthesize a large number of terpenes, steroids, carotenoids and other products following the __

(i) Synthesis of mevalonate from acetate or leucine

(ii) Dehydration and decarboxylation to give isoprene followed by condensation to give isoprenes of various lengths.

(iii) Cyclization (ring formation) e.g., to give steroids

(iv) Further modification of the cyclised structure. The route leads to the formation of essential steroid hormones of mammals and to a variety of secondary metabolites in fungi and plants. it is not used to any extent in the actinomycetes.

Simply put the isoprene rules consist of the following:

Mevalonate pathway

• also known as the isoprenoid pathway or HMG-CoA reductase pathway is an essential metabolic pathway present in eukaryotes , archaea , and some bacteria .

• The pathway produces two five-carbon building blocks called isopentenyl pyrophosphate and dimethylallyl pyrophosphate (IPP) (DMAPP), which are used to make isoprenoids , a diverse class of over 30,000 biomolecules such as cholesterol , vitamin K all steroid hormones .[2] , coenzyme Q10

• begins with CoA , and acetyl and ends with the production of IPP and DMAPP. It is best known as the target of statins class of cholesterol lowering drugs. Statins inhibit , a HMG-CoA reductase within the mevalonate pathway

Statins

The mevalonate pathway begins with CoA , and acetyl and ends with the production of IPP and DMAPP. It is best known as the target of ___class of cholesterol lowering drugs. ___inhibit , a HMG-CoA reductase within the mevalonate pathway

isopentenyl pyrophosphate and dimethylallyl pyrophosphate (IPP) (DMAPP)

The mevalonate pathway begins with CoA , and acetyl and ends with the production of __

Serine (C3N) and glycine (C2N)

are derived from the triose (C3) formed glucose

triose (C3) formed glucose

Serine (C3N) and glycine (C2N) are derived from the

acetate (C3)

valine (C5N) is derived from

valine (C5N)

is derived from acetate (C3)