lecture 9 – Chemistry – Flashcards

1. supply of basic building blocks 2. Connection of building blocks 3. Tailoring of the precursor molecule

a. methylations b. hydroxylations c. glycosylations d. ring formations e. halogenations

from primary metabolism by biosynthesis pathways feeding

by non-ribosomal peptide synthtases by polyketide synthases by glycosyl transferases

Natural product biosynthesis by the wild-type strain

biosynthesis is abolished by mutating the pathway at a crucial step

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

mutasyntehsis: biosynthesis by the nonproducing mutant strain is reactivated by addition of a mutasynthon only novel analogues are produced

1. therapeutics 2. Animal growth promotion 3. Agricultural 4. Antitumor chemotherapy 5. food preservation 6. Research tool in biochemistry and molecular biology

lactose relieves glucose inhibition corn steep liquor is found to provide phenyl acetate which increases yield

ACV synthetase

asf

draw the basic structure of phenoxymethylpenicillin

dar

dar

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

beta lactams

1. D amnino adipic acid 2. cephem nucleus 3. 7 aminocephalosporanic acid

1. Beta Lactamaase 2. Methoxylase 3. Esterase 4. Acylase 5. D amino Acid oxidase

Name the 5 enzymes

CDAOC synthase

Adiptic acid added cefEF added cefEF added CefG added psuedomonas amidase

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

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

Tailoring of the precursor molecule a. methylations b. hydroxylations c. glycosylations d. ring formations e. halogenations Supply of basic building block from primary metabolism by biosynthesis pathways feeding connection of building block by non-ribosomal peptide synthtases by polyketide synthases by glycosyl transferases describe part A of precursor directed biosynthesis Natural product biosynthesis by the wild-type strain describe part b of precursor directed biosynthesis biosynthesis is abolished by mutating the pathway at a crucial step describe part c of precursor directed biosyntehsis 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 describe part D of precursor directed biosynthesis mutasyntehsis: biosynthesis by the nonproducing mutant strain is reactivated by addition of a mutasynthon only novel analogues are produced

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

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 when do microorganisms produce antibiotics? during stationary, slow growth or in response to a developmental change

1. therapeutics 2. Animal growth promotion 3. Agricultural 4. Antitumor chemotherapy 5. food preservation 6. Research tool in biochemistry and molecular biology

lactose relieves glucose inhibition corn steep liquor is found to provide phenyl acetate which increases yield

Текст ответа

ACV synthetase Draw a benzylpenicillin asf draw the basic structure of phenoxymethylpenicillin draw draw the basic structure of ampicillin dar draw the basic structure of ampicillin dar

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

1. D amnino adipic acid 2. cephem nucleus 3. 7 aminocephalosporanic acid

1. Beta Lactamaase 2. Methoxylase 3. Esterase 4. Acylase 5. D amino Acid oxidase [image]

CDAOC synthase [image] Adiptic acid added cefEF added cefEF added CefG added psuedomonas amidase [image] 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 [image] 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 [image]

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

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

APIs clavulanic acid, carbapen antibiotics

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)

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

. C5 precursor availability 2. C3 precursor availability 3. relief of catabolite repression 4. antibiotic resistances

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

micromonospora neomycin kanamycin gentamicin 2-deoxystreptamine sugars

inhibit