Primary And Secondary Metabolites

Question 1

What is the definition of a primary metabolite?

Answer 1

Substance directly involved in normal growth, development and/or reproduction, usually performing an intrinsic physiological function in the organism

Question 2

What is the structure of a fatty acid?

Answer 2

Hydrocarbon chain ending in a carboxyl group (COOH)
Full of single bonds = saturated
Double bond = unsaturated
Multiple double bonds = polyunsaturated

Question 3

What does the short-hand 18:3,n-3 tell you about the fatty acid?

Answer 3

18 = number of carbons
3 = position of first db relative to end of methyl chain n)

Question 4

What types of enzymes are involved in fatty acid synthesis?

Answer 4

FA Synthase (FAS) makes saturated FA up to 16 carbons long.
Elongases extend 16:0 by adding 2C to chain
Desaturases introduce double bonds at specific points along the carbon chain

Question 5

Why are 18:2,n-6 and 18:2,n-3 FA essential dietary components?

Answer 5

Humans have the pathways to make n-3 and n-6 eicosanoids but lack some important precursor desaturases (delta 12 and 15) so get from diet e.g. oily fish, nuts/seeds

Question 6

Describe manipulation of Mortierella alpina to increase yield of fatty acids.

Answer 6

50% of their FA are 20:4,n-6 oil.
Desaturase and elongase genes have been cloned and separated.
Could disrupt delta 5 through knockout, preventing more production of polyunsaturated FA and get higher accumulation of the ones we want more.

Question 7

Describe manipulation of Saccharomyces cerevisiae to increase yield of fatty acids.

Answer 7

Lacks delta 6/12/15 so can make unsaturated or polyunsaturated FAs.
Can add 2 genes for delta 6 and 12 from M. alpina, introduced on a single plasmid.
Even once manipulated, don’t make as much as M. alpina

Question 8

Why are trans FA worse than Cis FA in membranes?

Answer 8

Cis FA are unsaturated and have a kink due to the double bond - allows fluidity.
Trans FA act as saturated FA and stay in completely straight line without any kinks - no positive effects for membranes

Question 9

What are the industrial considerations for microbial fatty acids?

Answer 9

FA must be easy to extract and purify but not obtained from another source.
Organism should be generally regarded as safe, preferably grown on cheap growth media and produce good yield

Question 10

Describe Citric Acid as an example of a microbially produced FA.

Answer 10

Aspergillus niger - growth at low pH (prevents contamination and inhibits production of unwanted acids), excreting large amounts.
Often uses sucrose rather than glucose substrate.
A. niger has extracellular mycelium-bound invertase active at low pH

Question 11

Why is it difficult to manipulate yield of citric acid and what are the potential solutions?

Answer 11

Citric acid is part of the Krebs/TCA cycle.
1. Could theoretically attempt to KO/block aconitase to prevent citric acid being converted to cis-aconitic acid (in reality, organism won’t be happy about that).
2. Citric acid is a feedback inhibitor, it feedbacks phosphofructokinase - try to block feedback inhibition, so will continue producing more regardless of amount already produced

Question 12

Describe Glutamic Acid as an example of a microbially produced FA.

Answer 12

Used as food additive and flavour enhancer, as monosodium glutamate (MSG).
Fed from krebs cycle (alpha-ketoglutaric acid).
Bacterium Corynebacterium glutamicum used to make 2 million tons per year.

Question 13

Describe L-Lysine as an example of a microbially produced FA.

Answer 13

C. glutamicum also dominates microbial lysine production - 1.5 million tons per year.
Closely linked to metabolism but rare example of successful genetic engineering to improve yield vs 50 years of classical optimisation (allowing to evolve under conditions but no direct manipulation.) - computational design, determining where bottlenecks are and removing them to streamline production.

Question 14

Describe the secretory lifestyle of bacteria and fungi.

Answer 14

Secrete enzymes into surrounding environment which break down environmental products.
Smaller molecules absorbed back into the organism which can pass over the cell membrane and be used for its metabolism.

Question 15

What are the two types of enzymes produced by microorganisms?

Answer 15

Endoenzymes = product and act within cells.
Exoenzymes = exported from cell where they act extracellularly and products are taken up by microbes

Question 16

What are the applications of microbially produced enzymes?

Answer 16

Agriculture: Agro-processing, animal feed additives, agro-chemicals, animal feed from agricultural waste.
Industry: leather tanning, pulp and paper, starch and fuel, chemicals and pharmaceutical, detergent, food and beverage, textile, protein hydrolysis.
Environment: Waste degradation, detection of toxic pollutants, bioremediation

Question 17

What is the difference between batch, fed-batch and continuous fermentation.

Answer 17

Batch: Mix all together and start, let grow then harvest.
Fed-batch: Add ingredients at start, allow microbe to grow to optimal growth state then add a bit more
Continuous: Get microbe growing at a certain level, steadily remove some harvest from the fermenter while adding new nutrients

Question 18

Describe detergents as an example of microbiologically produced enzymes.

Answer 18

Combination of proteases (Bacillus bacteria), lipases (Aspergillus fungi), amylases and cellulases (Trichoderma fungi).
Tend to be non-specific blend of 2-4 enzymes.
Allows reduced temperature washes, better cleaning performance and environmental benefits (degradable).

Question 19

Describe baking as an example of microbiologically produced enzymes.

Answer 19

Amylases from Aspergillus oryzae soften dough by breaking down starch in flour.
Xylanases from Aspergillus degrade hemicelluloses in flour to improve dough texture, crumb structure.
Glucose Oxidase from Penicillium for more elastic dough.

Question 20

Describe drinks and brewing as an example of microbiologically produced enzymes.

Answer 20

Pectinases from Aspergillus niger increases fruit juice yield by breaking down fruit cell wall.
Amylases from Aspergillus species used in brewing for conversion starch both prior and during fermentation to reduce viscosity and increase fermentable sugars (yeast use the sugars and is converted to alcohol resulting in low calorie beers)

Question 21

Describe Confectionary as an example of microbiologically produced enzymes.

Answer 21

Amylases from Aspergillus species used to convert starch to syrups for confectionary manufacture.
Invertases from Aspergillus and Saccharomyces used to convert sucrose to fructose and glucose. Also used to create soft-centred chocolates as sucrose is broken down

Question 22

Describe Biofuels as an example of microbiologically produced enzymes.

Answer 22

Fuels produced from renewable biological sources. So called 2nd gen biofuels produced from plant lignocellulose (plant waste left over from other processes) and other waste.
Use variety of fungal enzymes to release sugars (cellulases, lignases, amylases) - used in fermentations with yeast Saccharomyces to produce bioethanol

Question 23

What is meant by the term Heterologous proteins.

Answer 23

Proteins produced in a different host organism from where the gene and ‘native’ protein were originally identified

Question 24

What are the advantages of heterologous proteins?

Answer 24

Fast growth/host incubation period.
Ability of host to use inexpensive growth sources.
Overall process more economic.
Host is normally GRAS status so minimal risk and avoid legal issues.
Tools available for genetic manipulation of host.
Ease of downstream processing of enzyme.

Question 25

What are the disadvantages of heterologous proteins?

Answer 25

Eukaryotic genes have introns that aren’t dealt with in bacterial expression systems.
Low gene expression and stability.
Desired protein might need glycosylation (sugar side chains) for activity, expression hosts lack ability to add or edit.
Protein needs to be correctly folded in host cell by chaperone proteins.
Host proteases can degrade foreign proteins.
Protein ideally needs exporting but lacks signal.
Even when protein purified, can be toxic contaminants (especially from bacteria)
Time consuming and major set up costs possible

Question 26

Describe microbial biotech solutions for heterologous proteins.

Answer 26

Can use cDNA copy of gene lacking introns (made from mRNA with introns removed).
Exploit aa signal sequences present at start of proteins for cell export.
Genetically manipulate these into start of heterologous proteins - can link yeast S. cerevisiae mating alpha-factor hormone to heterologous protein via Lys-Arg ‘KR’ sequence - allows editing by Kex proteases or fused with carrier protein (approach to improve secreted yield - can trick cell intro producing protein we want.)
Use strong promoters for expression.
Can combine with classical strain improvement by UV mutagenesis.
Generally observe bacterial proteins best in bacterial system and fungal enzymes best in fungal hosts

Question 27

Describe insulin as a heterologous protein.

Answer 27

Pro-insulin (precursor) has A and B chains that need joining by Kex protease to give mature 51aa hormone (with connecting polypeptide between them).
Original pig pancreas supplies insufficient.
Heterologous protein first produced E.coli but low yields.
Now uses S. cerevisiae system - incorporated yeast alpha-factor PrePro export sequence fused to A and B chains with linker 3-5aa C peptide, latter removed after cell export and purification

Question 28

Describe Chymosin (Rennet) as a heterologous protein.

Answer 28

Protease helps produced curds by cleaving casein production causing aggregation of milk proteins.
Traditionally obtained from calf stomach but limited and expensive.
Pro-sequence removed outside cell as chymosin is protease.
Tried production of prochymosin in yeast Kluyveromyces (bovine chymosin cDNA fused to yeast mating alpha-factor PRE leader sequence under control of native strong lactase promoter in genome).
Pro-chymosin activated to chymosin by acidification.

Finally tried expression in A. niger – select high glucoamylase producer strain, remove resident protease by GM. Next insert multiple copies of chymosin gene into genome fused to carrier protein glucoamylase. Classic mutagenesis.

Question 29

What is the definition of secondary metabolites?

Answer 29

A product of secondary metabolism is linked to specialised biochemical pathways particular to certain organisms. They are most often produced in stationary phase of growth after standard growth has finished. Often show biological activity so role in natural environment.

Question 30

What are polyketides and how are they produced?

Answer 30

Produced by both actinomycete bacteria and fungi.
Starting with 2C (acetyl-CoA) or 3C (propionyl-CoA), a series of 3C units are added in succession (malonyl-CoA or methyl malonyl-CoA) and one C is lost as CO2.
Catalysed by Polyketide Synthase (PKS) to form backbone of molecule.
Each elongation step requires action of 3-6 different polypeptide domains with specific enzymatic catalytical ability which form a module.
In bacteria, a growing polyketide then transferred to another module.
Just one module in fungi but growth of chain iterative with same module repeated until final length.

Question 31

What is the difference between the two types of Polyketide Synthase enzymes.

Answer 31

Type 1:
- Large single protein with one or more modules with multiple domains, each catalysing different stage of synthesis, present on same pp chain.
- Form more complex polyketides.
- Eukaryotic or Prokaryotic

Type 2:
- Several smaller pp, each with specific domain, which catalyse successive stages of polyketide synthesis when coming together, normally as quaternary protein.
- Simpler polyketides
- Usually bacterial

Question 32

Describe erythromycin as an example of a polyketide.

Answer 32

broad range antibiotic produced by soil Actinomycete Saccharopolyspora.
Inhibits bacterial protein production.
Useful if allergic to penicillins.
Synthesised by 3 T1PKS which contain 2 modules of 3-6 domains

Question 33

Describe Statins as an example of a polyketide.

Answer 33

Cholesterol lowering drugs from Penicillium and Aspergillus filamentous fungi.
Competitive inhibitors of HMG-CoA reductase, key enzyme in biosynthesis and release of cholesterol.
e.g. Lovastatin

Question 34

Describe Amphotericin as an example of a polyketide.

Answer 34

Antifungal drug used to treat thrush and life-threatening systemic fungal infections.
Originally extracted from soil actinomycete Streptomyces nodosus.
Bind to ergosterol (fungal-specific sterol) in membrane, effecting membrane fluidity and creating pores, resulting in leakage and cell death - one of few examples that doesn’t kill eukaryotic cells too

Question 35

Describe Griseofulvin as an example of a polyketide.

Answer 35

Antifungal drug used to treat scalp, skin (ringworm) and nail infections.
Produced by filamentous fungi Penicillum griseofulvum.
Prevents assembly of fungal microtubules and mitosis.
Made by T1PKS followed by various modifications including chlorine addition

Question 36

Describe Doxorubicin as an example of a polyketide.

Answer 36

Anti-tumout drug used in cancer chemo.
Derived by fermentation of GM engineered strain Actinomycete Streptomyces peucetius.
Intercalates DNA, stopping replication (goes between layers of DNA helix).
Made by series of T2PKS enzymes, followed by export of specific transporter giving self-resistance.

Question 37

Describe Avermectnis as an example of a polyketide.

Answer 37

Used to treat nematode and arthropod parasitic infections.
Produced by fermentation of soil Actinomycete Streptomyces avermitilis.
Blocks nerve transmission to inhibit growth of parasites, don’t know exact mechanism.
Made by 4 PKS enzymes, followed by various modifications including glycosylation

Question 38

What are Non-Ribosomal Peptides and how are they produced?

Answer 38

Organic compounds which all feature polypeptide chain but not made by classical ribosomal pathway.
Synthesised by Non-Ribosomal Peptide Synthetases (NRPs).
PP backbone produced using individual aa as monomers then chemically modified to produce final product - 10-12aa

Question 39

What is the structure of NRPs?

Answer 39

Large proteins composed of a series of modules, each with 2-4 specific domains with particular catalytic activity.
Similar organisation to T1PKS, each module acts a specific aa monomer.
Different NRPs can act together to produce final product, usually found clustered together in genome.

Question 40

Describe beta-lactam antibiotics as an example of a non-ribosomal peptide.

Answer 40

e.g. penicillin produced from filamentous fungus Penicilium chrysogenum.
Interferes with bacterial cell wall formation.
NRPS ACV synthetase (combines first 3aa L-amino-adipic acid, cysteine, valine) before further enzymatic modifications.
Gene acvA and next gene in pathway (isopenicillin synthase) clustered.

Each module of ACVsynth. binds one building block; combined activity allows 3aa to be combined together to get final beta-lactam product; condensation and translocation to pass aa along chain to get 3aa product; no free intermediate products.

Question 41

Describe Cyclosporins as an example of a NRP.

Answer 41

Powerful immunosuppressants to counter organ rejection in transplants.
Methylated 11aa cyclic peptide produced from filamentous fungus Tolypocladium.
Prevents T cell activation.
Synthesised by cyclosporin synthetase - 11mod
Shows anti-insect activity, pathogen of beetle larvae.
Includes unusual D-alanine instead of L-alanine

Question 42

Describe Echinocandins as an example of a NRP.

Answer 42

Antifungul drug used to treat system fungal infections.
Cyclic 6aa peptide core linked to lipid side chain, produced by various filamentous fungi including Aspergillus.
Bnid to glucan synthase (making part of cell wall) resulting in cell lysis and death.

Produced semi-synthetically. Echinocandin B synthesised by NRPS gene ecdA (protein has 6 modules each with 3 domains; uses standard and non-standard aa building blocks; clustered in genome region with 12 other genes involved in echinocandin biosynthesis) e.g. modifying enzymes and efflux pump (constantly pumping it out to increase its own growth while inhibiting other fungi)

Question 43

Describe Bleomycin as a NRP.

Answer 43

Antibiotic and anticancer tumour treatment.
Produced by soil actinomycete Streptomyces verticullus.
Mechanism of action involves induction of DNA strand breaks.
Interesting hybrid peptide-polyketide structure, produced by joint action of NRPS and PKS enzymes