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.