Microbiological routes for the production

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Microbiological Routes For The Production Of Pharmaceutical Products.

Introduction

Microbiological Aspects Of Penicillin

Recent publications and primary literature suggest that penicillin is an excellent therapeutic agent which increases its demand for clinical application and chemical studies. Microbiological production, chemical purification and isolation are major problems associated with its use. Penicillin is produced by penicillium notatum in very minute quantities and its cultures were investigated for large scale production of penicillin. Cultures of P.notatum tend to spontaneously lose their penicillin-producing ability due to physiological or biochemical degeneration. Penicillin activity directly depends upon the nature of strain. It is vital to select strains of highest potency for maximum penicillin activity. All the strains are different from each other in their penicillin-forming ability.

1. Penicillin production in Penicillium Chrysogenum.

Biosynthesis of lysine and Penicillin in penicillium chrysogenum is regulated by branching point intermediate α-aminoadipate. In penicillin pathway, it is condensed with L-valine and L-cysteine to form tripeptide by ACV synthetase. The internal α-aminoadipate pool plays a vital role in lysine and penicillin biosynthesis. The disruption of lys2 gene directs the pool towards penicillin biosynthesis resulting in penicillin over-production. The targeted disruption was carried out by using two different techniques and their effect on penicillin production was studied. P.chrysogenum Wis 54-1255 (low-level penicillin-producing strain) and P.chrysogenum pyrG1 mutant strains were used in transformation experiments. P. Chrysogenum L2, a lysine auxotroph was used as a control. Spores of P.chrysogenum were collected from plates of power medium after having grown for 5 days at 28°C. Two plasmids pDL1 and pDL7 which differed in selectable marker and size of DNA region homologous to target were selected for disruption of lys2 by a single crossing over. pDL1 includes ble (phleomycin resistance) gene while pDL7 contains pyrG gene as a selectable marker. Out of 495 transformants tested, 2 lysine auxotrophs clones (TD7-88 and TD7-115) were obtained. Both were unable to grow in Czapek medium supplemented with α-aminoadipic acid while P.chrysogenum L2 (control strain) grew. These results proved that lys2 gene is disrupted in TD7-88 and TD7-115 strains. Two plasmids pDL2 and pDL10 were constructed for disruption of lys2 gene by double recombination. Out of 964 transformants tested, only one lysine auxotroph (TD10-195) was obtained which was unable to grow in α-aminoadipic acid supplemented Czapek medium. Transformants TD10-195 and TD7-115 were more stable without any reversion rate as compared to TD7-88 with very low level of stability. Although the growth of transformants were slower than parental strain in defined medium containing 4.0 Mm lysine, the penicillin levels were double from those observed in parental strain at 96, 120, and 144 h and threefold higher at 168 h.

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2. Penicillin Production by Fungi growing on food products.

Many fungal species widely used as commercial starters such as P.nalgiovense, P.chrysogenum, P. Verrucosum, P.griseofulvum and P.roqueforti were used to analyze the production of penicillin while growing on food products and presence of penicillin biosynthetic genes in fungi of genus penicillium. The synthesis of toxic compounds might also be secreted to food products. All the strains were screened using bioassays in both solid and liquid medium with micrococcus luteus ATCC 9341 as a test strain to detect penicillin production. Antibacterial activity was found in P.chrysogenum, P.griseofulvum NRRl 2300 and P.nalgiovense. β-lactamase form Bacillus cereus UL1 was used to test whether the antibacterial activity was due to penicillin or other substance. Results indicate that antibacterial activity can be attributed to β-lactam antibiotic penicillin. Fermentation of liquid submerged cultures of P.griseofulvam was carried out to confirm penicillin production which proved that P.griseofulvam strain NRRL 2300 had highest production level. Southern blot analysis was used to analyse the presence of penicillin biosynthetic genes. The presence of penicillin gene cluster in P.griseofulvum proved that the antibacterial activity observed is due to penicillin. P.griseofulvum which is a potential source of penicillin in food products can be frequently isolated from corn, barley, wheat, flour etc. In case of P. Verrucosum, the antibacterial activity observed was due to either patulin or penicilic acid as it contains only one gene of penicillin gene cluster. Both patulin or penicilic acid are secondary metabolites produced by different strains of fungi. In order to obtain safe and high quality food products, antibiotic and toxin production by food microorganisms should be analysed and studied in detail. It also helps to produce modified strains in which the synthesis of toxic compounds can be eliminated without losing their food-ripening and flavouring properties.

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3. Production of penicillin in methylotropic yeast Hansenula polymorpha.

β-lactam antibiotics such as penicillin and cephalosporins are largest selling antibiotics against bacterial infections. Industrially, penicillin and cephalosporins are produced by filamentous fungi Penicillium.chrysogenum and Acremonium chrysogenum respectively. The main aim was to introduce penicillin biosynthesis pathway into yeast Hansenula polymorpha as it is more versatile, easy to handle and cultivate with superior fermentation properties as compared to filamentous fungi. In penicillin pathway, peptide synthetase δ-L-cysteinyl-D-valine synthetase forms tripeptide ACV. It is converted into isopenicillin N (IPN) by enzyme isopenicillin N synthase (IPNS). The pcbC gene which encodes IPNS was cloned in H.polymorpha alcohol oxidase promoter in pHIPX4 and integrated at PAOX locus in H.polymorpha genome. A strain (IPNS 4.2) containing pcbC expression cassette was cultivated at 37°C on methanol medium to induce PAOX. Wild type H.polymorpha serves as control strain. Aliquots were taken at regular time intervals to analyse IPNS protein. Western blots prepared using crude extracts of H.polymorpha and P.chrysogenum revealed that strain IPNS 4.2 produces an α-IPNS specific protein. IPNS was produced at all growth temperatures in strain IPNS 4.2. In stationary growth phase, relatively low IPNS protein levels were observed in cells grown at 37°C. It was assumed that poor or slow folding of protein at high temperature can be an intrinsic factor. However, IPNS protein was fully stable and its level was significantly enhanced at 25°C if compared to its production at 37°C and 30°C.The amount is comparable to highest penicillin producing strains of P.Chrysogenum. Results indicate that penicillin production in heterologus yeast was only successful at reduced growth temperatures. As yeast genome does not encode non - ribosomal peptide synthetases such as ACVs, the next step will be to insert functionally active ACVS in heterologus host in order to introduce entire penicillin biosynthesis pathway in H.polymorpha.

4.  Penicillin production in surface cultures of P.notatum.

Two-liter Erlrnmeyer flasks containing czapek-Dox medium were inoculated with P.notatum and incubated at 25°C. It was tested for antibacterial activity against Staphylococcus aureus in nutrient broth by ordinary dilution methods. The pH of the medium fell from 6.5 to 3.0-4.0 and remained low throughout. Neutralized samples showed high antibacterial activity and batches ranging from 10 to 100 litres were extracted with amyl acetate at pH 2. It was assumed that penicillin in these cultures existed in non-extractable form. The pellicles were thin, without wrinkles or spores, and liquid with faint yellow tinge. The antibacterial activity was extractable at pH 2 with organic solvents when the medium was supplemented with yeast extract, brain-heart infusion. In brown sugar medium (dark brown sugar, 2 per cent; NaNO3, 0.35; MgSO4 .7 H20, 0.05; KCI, 0.05, KH2PO4, 0.15; FeSO4 .7H20, 0.015) growth is more rapid and abundant accompanied by intense yellow pigmentation (chrysogenin) as compared to czapek-Dox medium. The acidity falls in early stages from pH 5.5 to 4.5 and then rises to 8.0. Penicillin started accumulating on fifth day with maximum on the eleventh to the thirteenth day. Maximum penicillin activity appeared after maximum growth was attained. All the sugar was consumed before maximum penicillin was accumulated.

5. Penicillin production in submerged cultures of P.notatum.

Experiments with submerged cultures were conducted on machines shaking at rate of 60 to 90rpm, with maximum growth observed over a period of 6 to 10 days at 25°C. Brown sugar medium was distributed in 80-ml quantities in 250-ml Erlenmeyer flasks and inoculated with a spore suspension of P.notatum, strain 832. The flasks were incubated at 25°C on a shaking machine. Growth commenced on the second day with maximum growth appeared as small round pellets of mycelium. Penicillin started accumulating on 3rd or 4th day and reached its maximum value (20 to 30 oxford units per ml) on 7th or 8th day. Organic fraction in brown sugar is responsible for penicillin-promoting capacity. Zinc also had a catalyzing effect. The shake or submerged culture technique provides better aeration as compared to surface culture. By growing P.notatum strains in shake culture, variable factors of diffusion and pellicle formation are eliminated,while growth and metabolic processes are accelerated. Maintenance of an adequate oxygen supply is essential in the penicillin production in submerged cultures of P.notatum.

6. Aculeacin A Acylase as an industrial biocatalyst for production of penicillin

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Aculeacin A Acylase (AuAAC) from Actinoplanes utahensis NRRL 12052 catalyzes hydrolyses of acyl moieties of antifungal antibiotics. As AuAAC was similar to β -lactam acylase, it was investigated whether AuAAC would behave as new β -lactam acylase. An engineered aac gene was made and cloned into expression vector pEM4. The gene was amplified by PCR using chromosomal DNA from A. utahensis NRRL 12052. Primers were designed according to the DNA sequence of aac gene. Purified PCR products were digested with XbaI and EcoRI endonuclease and cloned into pEM4 vector. The resulting plasmid was then introduced in S.lividans 1326. The AuAAC yield was 21-fold higher in recombinant strain produced by S. Lividans (pEAAC) as compared to A.utahensis. Its purity was determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The thermal stability was studied by pre-incubating AAC for 20 min at different temperatures. It was stable up to 50°C and then reduced at higher temperature. It was determined that purified recombinant AuAAC is able to hydrolyze penicillin V. The kinetic parameters for hydrolyses of different natural β-lactam antibiotics were determined to examine hydrolytic specificity of recombinant AuAAC. The pure recombinant enzyme was incubated with increasing concentrations of penicillins (V, K, F, dihydroF, and G) in 100 mM potassium phosphate buffer, pH 8.0 at 45°C for 15 min in 100 µl. AuAAC shows the same substrate specificity if compared to penicillin V acylase on natural penicillins. Penicillin K was best substrate for which AuAAC shows highest biomolecular constant value of 34.79 mM-1 s-1. Results suggest that AuAAC from A.utahensis should be considered as new subfamily of β-lactam acylases and it should also be considered as an industrial biocatalyst for production of semisynthetic penicillins.

References

1. Finch, R. G., Greenwood, D., Norrby, S. R. & Whitley, R. J. (2003). Antibiotic and Chemotherapy. Anti-infective agents and their use in therapy. (8th ed.). New York: Churchill Livingstone.

2. Hutter, R., Leisinger, T., Nuesch, J. & Wehrli, W. (1978). Antibiotics and Other Secondary Metabolites: Biosynthesis and Production. New York: Academic Press.

3. Flynn, E. H. (1972). Cephalosporins and Penicillins.Chemistry and Biology. New York: Academic Press.

4. Casqueiro, J., Gutierrez, S., Banuelos, O., Hijarrubia, M. J. & Martin, J. F. (1999). Gene Targeting in Penicillium chrysogenum: Disruption of the lys 2 Gene Leads to Penicillin Overproduction. Journal of Bacteriology, 181(4), 1181-1188.

5. Laich, F., Fierro, F. & Martin, J. F. (2002). Production of Penicillin by Fungi Growing on Food Products: Identification of a complete Penicillin Gene Cluster in Penicillium griseofulvum and a Truncated Cluster in Penicillium verrucosum. Applied and Environmental Microbiology, 68(3), 1211-1219.

6. Gidijala, L., Bovenberg R., Klaassen, P., Van der Klei, I. J., Veenhuis, M. & Kiel, J.A. (2008). Production of functionally active Penicillium chrysogenum isopenicillin N synthase in the yeast Hansenula polymorpha. BMC Biotechnology,29(8), 1472-6750.

7. Torres-Bacete, J., Hormigo, D., Stuart, M., Arroyo, M., Torres, P., Castillon, M.P., et al. (2007). Newly Discovered Penicillin Acylase Activity of Aculeacin A Acylase from Actinoplanes utahensis. Applied and Environmental Microbiology,73(16) 5378-5381.

8. Sprote, P., Brakhage, A. A. & Hynes, M. J. (2009). Contribution of Peroxisomes to Penicillin Biosynthesis in Aspergillus nidulans. Eukaryotic Cell, 8(3), 421-423.