Term
| What are the three principles of antibiotic biosynthesis of bioactive secondary metabolites? |
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Definition
1. supply of basic building blocks
2. Connection of building blocks
3. Tailoring of the precursor molecule |
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Term
| Tailoring of the precursor molecule |
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Definition
a. methylations
b. hydroxylations
c. glycosylations
d. ring formations
e. halogenations |
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Term
| Supply of basic building block |
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Definition
from primary metabolism
by biosynthesis pathways
feeding |
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Term
| connection of building block |
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Definition
by non-ribosomal peptide synthtases
by polyketide synthases
by glycosyl transferases |
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Term
| describe part A of precursor directed biosynthesis |
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Definition
| Natural product biosynthesis by the wild-type strain |
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Term
| describe part b of precursor directed biosynthesis |
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Definition
| biosynthesis is abolished by mutating the pathway at a crucial step |
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Term
| describe part c of precursor directed biosyntehsis |
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Definition
| precursor directed biosynthesis- the culture medium is supplemented with an analogue of the natural building block which competes for incorporation into the natural product. both native and modified products usually result |
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Term
| describe part D of precursor directed biosynthesis |
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Definition
| mutasyntehsis: biosynthesis by the nonproducing mutant strain is reactivated by addition of a mutasynthon only novel analogues are produced |
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Term
Complex media design is critical for ____ of antibiotic production
provide 6 reasons why the above statement is accurate |
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Definition
optimization
1. high productivity than defined media
2. lower cost, better availability, higher titers
3. production regulated by rapidly utilized carbon, nitrogen, phosphate sources- insoluble nutrients are more desirable (slow release rates)
4. carbon nitrogen pshophate repression reduce yield
5. can lead to variable results
6. chemically defined media used to determine the effects of various nutrients present in complex media |
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Term
| Why do microorganisms produce antibiotic exometabolites (extrolites)? |
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Definition
natural products of secondary metabolism
may be expressed during slow growth, stationary phase or in response to developmental signals
may provide growth, competitive advantage to producing organism in nature
natural product may serve as precursor for further chemical or enzymatic modification (semi-synthetic antibiotics)
producing organism must be resistant to antibiotic
produced as ":families" of related chemical structures |
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Term
| when do microorganisms produce antibiotics? |
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Definition
| during stationary, slow growth or in response to a developmental change |
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Term
| what are 6 major applications for antibiotics? |
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Definition
1. therapeutics
2. Animal growth promotion
3. Agricultural
4. Antitumor chemotherapy
5. food preservation
6. Research tool in biochemistry and molecular biology |
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Term
| Why is lactose and corn steep liquor used in penicillin media modification engineering? |
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Definition
lactose relieves glucose inhibition
corn steep liquor is found to provide phenyl acetate which increases yield |
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Term
| What enzyme is used to catalyze the biosynthesis of Isopenicillin N in Penicillium chrysogenum? |
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Definition
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Term
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Definition
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Term
| draw the basic structure of phenoxymethylpenicillin |
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Definition
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Term
| draw the basic structure of ampicillin |
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Definition
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Term
| draw the basic structure of ampicillin |
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Definition
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Term
| Briefly explain 4 ways that pathogenic microbes develop resistance to beta lactam antibiotics and what does this mean for reasearchers? |
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Definition
1. produce beta lactamase
2. produce mutated penicillin bidnign proteins to decrease their affinity for beta lactam binding
3. gram negative pathogens down regulate porin channels to limit entry of antibiotic into the periplasm
4. pathogenic strains force efflux of antibiotic from the cytosol
results in need to develop 2, 3, 4 generation cephalosporins, monobactams, carbapenams using chemoenzymatic synthesis methods using aylases, expandases, ester hydrolases |
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Term
| ___ are synthesized by a diverse group of microorganisms |
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Definition
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Term
| Name three parts of the cephalosporin sturucture? |
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Definition
1. D amnino adipic acid
2. cephem nucleus
3. 7 aminocephalosporanic acid |
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Term
| What are 5 enzymes used to modify the side chains of B Lactam antibiotics? |
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Definition
1. Beta Lactamaase
2. Methoxylase
3. Esterase
4. Acylase
5. D amino Acid oxidase |
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Term
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Definition
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Term
| What is used to open the thiazolidine ring during the synthesis of cephalosporins? |
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Definition
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Term
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Definition
Adiptic acid added
cefEF added
cefEF added
CefG added
psuedomonas amidase |
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Term
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Definition
Part A. 7- ACA biosynthesis by a recombinant strain of A. chrysogenum expressing the genes encoding DAO and GLA from F. solani and P. diminuta
Part B. Strategy for 7-ADCA production by a recombinant strain of A. chrysogenum the cefEF distrupted strain accumulates large amounts of penicillin N, which is in vivo expanded to DAOC by the DAOcs encoded by the cefE gene of S. clavuligerus, DAOC is finally bioconverted into 7-ADCA by two enzymatic steps involving DAO and GLA
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Term
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Definition
Part C. DAOC biosynthesis by a recombinant strain of P. chrysogenum expressing the genes enconging IPNE (cefD) and DAOCs (cefE) from S. lipmanii and S. clavuligerus, respectively
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Term
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Definition
| PArt D. 7-ADCAproduction by a recombinant strain of P. chrysogenum expressing the cefE gene (encoding DAOCS) from S. clavuligerus. In adipate -fed fermentations, IPNA biosynthesizes adipoyl-6-APA which is used as a substrate by DAOCS to produce adipoyl-7-ADCA |
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Term
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Definition
Part E. DAC (extracellular) and cephalosporin C (intracellular production by a recombinant strain of P. chrysogen lacking IPNA activity and expressing the cefD1, defD2, cefEF, and cefG genes from A. chrysogenum.
genetic modifications are boxed with solid lines whereas enzymatic bioconversions are boxed with discontinuous lines
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Term
| Briefly explain the biomanufucturing process of Penicillin G? how does nitrogen affect the production of penicillin? |
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Definition
biomanufacturing process is fed-batch: cleavage of pen G by penicillin acylase to 6-APA.
Nitrogen represses penicillin biosynthesis; availability of lysine, methionine, glutamate, valine all affect biosynthesis |
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Term
| ______ are beta lactamase inhibitors. Provide an example and indicate type of antibiotic |
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Definition
APIs
clavulanic acid, carbapen antibiotics |
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Term
| List three techniques used to engineer clavulonic acid biosynthesis. |
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Definition
1. classical strain improvement (CSI)-repetitive mutation and selection (DSM, panlabs)
2. Mutant selection for: increased precursor availability, relief of CNP repression, resistance to CU, NI
End product inhibition
high glycerol tolerance
altered morphology
antibiotic resistance
bioassay larger growht inhibition zones
3. genomic approaches (after 2006) |
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Term
| What are 5 reasons to select a mutant as it relates to engineering clavulanic acid biosynthesis |
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Definition
1. increased precursor availability
2. relief of CNP repression
3. resistance to CU, Ni
4. end product inhibition
5. high glycerol tolerance
6. altered morphology
7. antibiotic resistance
8. bioassay-larger growth ihibition zones |
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Term
| What are the characteristics of selective agents employed in the classical strain improvement of multiple phenotypes to increase clavulonic acid production? |
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Definition
1. C5 precursor availability
2. C3 precursor availability
3. relief of catabolite repression
4. antibiotic resistances |
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Term
| Provide three examples of genomic approaches to increase clavulanic acid production. |
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Definition
1. identify biosynthetic gene cluster in S. clavuligerus
2. identiy key enzymes: b-lactam synthase, clavaminante synthase
3. sequence genome: 6.7 mb, 1.8 Mb linear plasmid
4. clavulanic acid and cephamycin C super cluster on chromosome, alanylclavam cluster on megaplamid
5. microarrays used to determine transcript levels-up regulation of primary, secondary metabolism found
6. modeled pathway flux balances, evaluate up regulation of transporters glutamate/glutamine synthetases |
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Term
| Actinomycetes, such as streptomycess and ___ produce aminoglycoside antibiotics such as streptomycin, ___, ____ and ___. All aminoglycosides contain streptamine or ____ |
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Definition
micromonospora
neomycin
kanamycin
gentamicin
2-deoxystreptamine sugars |
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Term
| Aminoglycosides ___ protein synthesis in gram negative bacteria |
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Definition
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