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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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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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Supply of basic building block |
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Definition
from primary metabolism by biosynthesis pathways feeding |
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connection of building block |
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Definition
by non-ribosomal peptide synthtases by polyketide synthases by glycosyl transferases |
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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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Definition
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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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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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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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