Primary And Secondary Metabolites Flashcards
What is the definition of a primary metabolite?
Substance directly involved in normal growth, development and/or reproduction, usually performing an intrinsic physiological function in the organism
What is the structure of a fatty acid?
Hydrocarbon chain ending in a carboxyl group (COOH)
Full of single bonds = saturated
Double bond = unsaturated
Multiple double bonds = polyunsaturated
What does the short-hand 18:3,n-3 tell you about the fatty acid?
18 = number of carbons
3 = position of first db relative to end of methyl chain n)
What types of enzymes are involved in fatty acid synthesis?
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
Why are 18:2,n-6 and 18:2,n-3 FA essential dietary components?
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
Describe manipulation of Mortierella alpina to increase yield of fatty acids.
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.
Describe manipulation of Saccharomyces cerevisiae to increase yield of fatty acids.
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
Why are trans FA worse than Cis FA in membranes?
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
What are the industrial considerations for microbial fatty acids?
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
Describe Citric Acid as an example of a microbially produced FA.
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
Why is it difficult to manipulate yield of citric acid and what are the potential solutions?
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
Describe Glutamic Acid as an example of a microbially produced FA.
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.
Describe L-Lysine as an example of a microbially produced FA.
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.
Describe the secretory lifestyle of bacteria and fungi.
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.
What are the two types of enzymes produced by microorganisms?
Endoenzymes = product and act within cells.
Exoenzymes = exported from cell where they act extracellularly and products are taken up by microbes
What are the applications of microbially produced enzymes?
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
What is the difference between batch, fed-batch and continuous fermentation.
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
Describe detergents as an example of microbiologically produced enzymes.
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).
Describe baking as an example of microbiologically produced enzymes.
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.
Describe drinks and brewing as an example of microbiologically produced enzymes.
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)
Describe Confectionary as an example of microbiologically produced enzymes.
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
Describe Biofuels as an example of microbiologically produced enzymes.
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
What is meant by the term Heterologous proteins.
Proteins produced in a different host organism from where the gene and ‘native’ protein were originally identified
What are the advantages of heterologous proteins?
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.
What are the disadvantages of heterologous proteins?
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
Describe microbial biotech solutions for heterologous proteins.
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
Describe insulin as a heterologous protein.
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
Describe Chymosin (Rennet) as a heterologous protein.
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.
What is the definition of secondary metabolites?
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.
What are polyketides and how are they produced?
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.
What is the difference between the two types of Polyketide Synthase enzymes.
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
Describe erythromycin as an example of a polyketide.
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
Describe Statins as an example of a polyketide.
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
Describe Amphotericin as an example of a polyketide.
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
Describe Griseofulvin as an example of a polyketide.
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
Describe Doxorubicin as an example of a polyketide.
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.
Describe Avermectnis as an example of a polyketide.
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
What are Non-Ribosomal Peptides and how are they produced?
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
What is the structure of NRPs?
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.
Describe beta-lactam antibiotics as an example of a non-ribosomal peptide.
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.
Describe Cyclosporins as an example of a NRP.
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
Describe Echinocandins as an example of a NRP.
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)
Describe Bleomycin as a NRP.
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
