1.Explain About the Antibiotic Production with Use of R-DNA Tech

1.Explain about the antibiotic production with use of r-DNA tech-:

2.How do you synthesis the novel antibiotics?

CLONING:

Selection of transformed cells

Complements a mutant strains

Identification of enzymes in a biosynthetic pathway

Selection of vector for cloning

SYNTHESIS OF NOVEL ANTIBIOTICS:

S.No. / Strain/Plasmid / Color of culture
Acidic Alkaline / Antibiotic(s)
1 / S.coelicolor / Red Blue / Actinorhodine
2 / Streptomyces sp. / Yellow Brown / Medermycin
3 / Streptomyces sp./pIJ2303 / Red Blue / Medermycin, actinorhodine
4 / Streptomyces sp./pIJ2315 / Red Purple / Mederrhodine A, medermycin
5 / S.violaceoruber B1140 / Red Blue-purple / Grantacin, Dihydrograntacin
6 / S.violaceoruber B1140/pIj2303 / Red Blue-purple / Grantacin, Dihydrograntacin, actinorhodine
7 / S.violaceoruber Tu22 / Red Blue-purple / Grantacin, Dihydrograntacin
8 / S.violaceoruber Tu22/pIJ2303 / Red Blue-purple / Dihydrogranatirhodine, actinorhodine

3.Explain the construction of polyketide antibiotics-:

ENGINEERING OF POLYKETIDE ANTIBIOTICS:

• Synthesis with enzymatic condensation of small carboxylic acids.
• Complex enzymatic mechanisms involved.
• Enzymes are of two types.

Erythromycin derivatives produced by genetic mutation

A. Enoylreductase gene mutation. B. β- Ketoreductase deletion.

ct-actinorhodine

tcm-tetracinomycin

frn-frenolicin

gris-grisusin.

KR (β-ketoreductase)

ARO (aromatase)

CYC (cyclase)

4.How does improve the antibiotic production?

5.Give detail an account on cephalosporin synthsis-:

6.How do you construct the pencillin gene?

IMPROVING ANTIBIOTIC PRODUCTION:

• Yield and rate of production increased.

1. Oxygen-Streptomycesspp, Vitreoscilla- homodimeric heme.

2. Acremonium chrysogenum

Fusarium solani-D-Amino acid oxidase (cDNA)

Pseudomonas diminuta-Cephalosporin acylase (genomic DNA)

Cephalosporin C

D-Amino acid

oxidase

Keto-AD-7ACA

[7-β-cephalosporanic acid (5-carboxy-5-oxopentanamido)]

H2O2

GL-7-ACA

[7-β-cephalosporanic acid (4-carboxy-butanamide)]

Cephalosporin

acylase

7-ACA

[7-amnocephalosporanic acid]

3. Acremonium crysogenum-cef EF

Streptomyces clavuligerus-cef E

4. Polyketine antibiotics by E.coli: S.erythreae-polyketine synthase, B.subtilis- co-factors, S.coelicolor-propionyl-coA carboxylase.

5. Peptide antibiotics: obtaines from frog skin, insects, and bacteria.

Lactobacillus lactis-nicin

cef EF

Cef E

7.Explain about the biopolymer -:

8. Explain the uses of biopolymer-:

9. Write about the Xanthan gum polymer properties -:

Large multiunit macromolecules

Uses:

Food processing industries, food manufacturing industries.

Pharmaceutical industries.

Xanthan gum:

Synthesized as exopolysaccharide by Xanthomonas campestris – naturally very small quantities.

Organism:

Gram-negative, obligate aerobic soil bacterium.

Utilizes Glucose, Sucrose & Starch but not lactose.

Properties of Xanthan gum:

Linear chain of Glucose molecule with one glucouronic acid and two mannose side chain in every second Glucose linear chain.

Similar to plastic.

Uses of Xanthan gum:

Emulsifying agent, Stabilizing and thickening agent.

Genetic Engineering of Xanthan gum Production:

Inexpensive medium – reduce the production cost.

Whey-By product of cheese making industry- Can be use.

Whey contains Lactose (3.5-4%), Used as filling agent but intolerant.

Disposing of whey creates pollution problem in river as well as in soil.

Cloning of Lac Z, Y gene from E.coli to Xanthomonas campestris would increase the synthesis. Xanthan gum is a polysaccharide used as a food additive and rheology modifier. It is produced by a process involving fermentation of glucose or sucrose by the Xanthomonas campestrisbacterium.

Chemical structure

The backbone of the polysaccharide chain consists of two β-D-glucose units linked through the 1 and 4 positions. The side chain consists of two mannose and one glucuronic acid, so the chain consists of repeating modules of five sugar units. The side chain is linked to every other glucose of the backbone at the 3 position. About half of the terminal mannose units have a pyruvic acid group linked as a ketal to its 4 and 6 positions. The other mannose unit has an acetyl group at the 6 positions. Two of these chains may be aligned to form a double helix, giving a rather rigid rod configuration that accounts for its high efficiency as a viscosifier of water. The molecular weight of xanthan varies from about one million to 50 million depending upon how it is prepared.

Biosynthesis

Synthesis originates from glucose as substrate for synthesis of the sugar nucleotides precursors UDP-glucose, UDP-glucuronate, and GDP-mannose that are required for building the pentasaccharide repeat unit. This links the synthesis of xanthan to the central carbohydrate metabolism. The repeat units are built up at undecaprenylphosphate lipid carriers that are anchored in the cytoplasmic membrane. Specific glycosyltransferases sequentially transfer the sugar moieties of the nucleotide sugar xanthan precursors to the lipid carriers. Acetyl and pyruvyl residues are added as non-carbohydrate decorations. Mature repeat units are polymerized and exported in a way resembling the Wzy-dependent polysaccharide synthesis mechanism of Enterobacteriaceae. Products of the gum gene cluster drive synthesis, polymerization, and export of the repeat unit.

Preparation

The polysaccharide is prepared by inoculating a sterile aqueous solution of carbohydrate(s), a source of nitrogen, di-potassium monohydrogen phosphate, and some trace elements. The medium is well-aerated and stirred, and the polymer is produced extracellularly into the medium. The final concentration of xanthan produced is about three to five percent by weight. After fermentation over about four days, the polymer is precipitated from the medium by the addition of isopropyl alcohol and dried and milled to give a powder that is readily soluble in water or brine.

10.Describes the construction of recombinant melanin production-:

11.Explain the uses of melanin-:

Melanin by Streptomyces antibioticus:

Irregular, Random, Polymers.

Composed of indoles, benzthiazoles, and amino acids.

Used in topical sunscreen lotion, sunlight protective coatings in plastics, and additives in cosmetic products.

Biochemical Pathway:

Tyrosine Dihydroxy phenyl alanine Black, Brown, Yellow, Red or violet melanin.

The Gene:

2 ORF – 1.Tyrosinase (30,600), and 2. Unknown (14,800) from ORF438.

E.coli expression vector + Tyrosinase gene-more tyrosinase but no melanin.

 E.coli expression vector + Tyrosinase gene + ORF 438 gene-Melanin synthesized.

ORF 438 may be act as a donor for copper to tyrosinase.

Providing a variety of low mol.wt. non-quinone molecules after getting dihydroxy phenyl alanine leads to production of novel melanin by E.coli

12.Explain about the adhesive biopolymer construction-:

13.Give detail an account on Animal adhesive biopolymer-:

Animal adhesive Biopolymer:

Highly (randomly) cross linked macromolecules.

Properties:

Mytilus edulis- Adhesive biopolymer-Microbial cells.

Strong, water proof, Mussel attach very tightly.

Uses:

Medicine and dentistry.

The Gene:

130 kDa precursor protein-Analyzed biochemically.

130 kDa composed of serine, threonine, lysine, praline & tyrosine (60-70% hydroxylated)

Proline-3-or4-Hyp, tyrosine-DOPA.

Genetic Engineering:

cDNA precursor gene makes unstable.

Chemically synthesized 600bp (25kDa), T7promoter & E.coli).

Hydroxylation by tyrosinase with ascorbic acid in invitro.

TyrosinaseCatechol oxidase

Tyrosine DOPA o-Quinone

½ O2 ½ O2

Ascorbic acid

5’ CCA ACC TAC AAA GCT AAG CCG TCT TAT CCG 3’

3’ TTT CGA TTC GGC AGA ATA GGC GGT TGG ATG 5’

Pro- Thr- Tyr- Lys- Ala- Lys- Pro- Ser- Tyr- Pro- Pro- Thr- Tyr

14.Explain the Hevea brasiliensis to production of rubber-:

15.How does the engineering the PHA gene to production of polymer?

16.Explain the pathway of PHA production-:

Rubber:

cis 1, 4-polyisoperine- Hevea brasiliensis.

17 enzyme catalyzed step, rubber polymerase-polymerize isopentyl pyrophosphate to allylic pyrophosphate (final step).

cDNA- expressed rubber polymerase.

cDNA clone- now used to express rubber.

Polyhydroxy alkonates:

Organism: Alkaligenes eutrophus. (Grow very slowly in expensive medium).

Polyhydroxy alkonates, Poly 3-hydroxybutyrate, Poly 3-hydroxybutyrate-co-3-hydroxyvalerate.

Thermoplastic, elastic properties.

5.2 kb length can be cloned in E.coli. (unstable & loss after 50 generation)

parB+ 5.3 kb Stable and synthesize constitutively.

PHB extracted by alkaline hypochlorite.

PHB crystalline in E.coli, amorphous inAlkaligenes eutrophus.

E.colifad R and ato C loci mutated and clone copolymer gene (ato A & D activate).

Ralstonia eutropha- short chain (4 or 5 monomers),Pseudomonas oleovorans-medium chain (6 to 14 monomers).

Propionate

Propionate

Propionyl-CoA

Acetyl CoA 3-Ketothiolase

3-Ketothiolase3-Ketovaleryl-CoA

Acetoacetyl CoA Acetoacetyl CoA reductase

Acetoacetyl CoA reductase3-Hydroxyvaleryl-CoA

3-Hydroxybutyryl-CoA Poly (3-hydroxybutyric acid)

Poly (3-hydroxybutyric acid) synthase synthase

Poly (3-hydroxybutyric acid) Poly (3-hydroxybutyrate-co-3-hydroxyvalerate.

17.Give one example for construction protein gene in commercial level-:

18.Explain about the recombinant insulin production-:

19.Describes the Human Growth Hormone -:

• Insulin is the peptide homone.
• Secreted from Islet of Langerhans of pancreas.
• It catabolizes the glugose in blood.
• Insulin is a boon for the diabetics.
• It consists of two polypeptide chains 1.chain A-21 a as 2.chain B-30 a as
• Its precursor is proinsulin which consists of two polypeptideA,B is contected by C chain(35 a as).
• Proinsulin-109A as long.

• NH2-(peptide)-β-chain-(peptide)-A chain-COOH.

• First Itakura et al.(1977) chemically synthesis DNA sequences.

Protocol-:

Two chain synthesis separately by using two fresh PBR322 vector .

By the side of β-galactosidase gene

The recombinant plasmids were transferred into E.coli.

Which secreted fused β-galactosidase-A & B chain separately.

This fusion protein cleaved by the Cyanogens bromide to cleave at methyl group.

Make link both of the chains by making the sulfide bond using the Sodium sulphonate.

 Than used for treatment of diabetics.

Human Growth Hormone;

Somatotropin.-secreted by the anterior lobe of pitutery glands which consists if 109 A as.

Construction –DS cDNA from mRNA precursor of hGH gene.